Abstracts details 1

Message

We are delighted that this year's annual meeting is being hosted by the Sri Venkateswara University. Tirupati is not only a town dedicated to Lord Venkateswara, but also to Goddess Saraswathy. Apart from SVU, the town also hosts two other universities, namely the Sri Venkateswara Institute of Medical Sciences (SVIMS) and the Sri Padmavathi Mahila University (SPMU). While SVIMS has active researchers in areas of biochemistry, cell biology, immunology and cardiology, SPMU has specialized courses in sericulture, computer applications, psychology. In addition, there is a Herbal Folklore Research Centre, supported by the IDRC Medicinal Plants Network, and a few biotech companies.

Though the programme put together by the organizers is packed, I am sure several of you would find it worthwhile to stay and extra day or so, in order to interact with colleagues in these educational, research and technological centres in Tirupati. The idea of SBCI planning meetings in venues like this is to provide opportunities for such interactions, and hopefully collaboration in areas of common interest. After all, modern biological research has increasingly become a multi-author affair, with partners from more than one institution. So, let us make the most of our stay in Tirupati- both in the SBCI meeting and outside it.

With best wishes and greetings,

D. Balasubramanian

President, SBCI 2007

Professor D. Balasubramanian

Director of Research

L. V. Prasad Eye Institute

Kallam Anji Reddy Campus

Road No. 2, Banjara Hills

Hyderabad 500034, India

Phone: +91-40-2354 3652; Fax: 2354 8271

Email: dbala@lvpei.org

KEY NOTE SPEECH

Redox regulation of the NF-κB pathway by selenium in macrophages

Hema Vunta, Umamaheshwari D. Palempalli, Pradeep Bodumalla, Parisa Kalantari, Ryan J. Arner, K. Sandeep Prabhu and C. Channa Reddy, Department of Veterinary and Biomedical Sciences, 115 Henning Building, The Pennsylvania State University, University Park, PA 16802, USA

ccr1@psu.edu

Inflammation is an integral part of tumorigenesis, which contributes to all the stages of cancer development: initiation, progression, and metastasis. Inflammation contributes to initiation by inducing the release of a variety of pro-inflammatory enzymes, cytokines, and chemokines that alert the vasculature to release inflammatory cells and factors into the tissue milieu, thereby causing oxidative damage, DNA mutations, and other changes in the microenvironment. Such changes in the environment make it more conducive to cell transformation and increased survival and proliferation. Selenium is an anti-oxidant that functions in the form of selenoproteins to mitigate the effects of oxidative stress. However, the underlying mechanism of anti-inflammatory action of selenoproteins is not well understood. Our research is based on the premise that selenoproteins affect a switch in the production of pro-inflammatory and pro-angiogenic prostaglandin (PG) E₂ towards anti-inflammatory 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂). 15d-PGJ₂ is an endogenous prostanoid that targets the inflammatory signaling axis involving the pathway of activation of redox-sensitive transcription factor, NF-B, via two major pathways. 15d-PGJ₂ inhibits the catalytic activity of IKK by alkylating the redox status of the thiol group in Cys179 in IKK leading to a subsequent decrease in the NF-B-dependent pro-inflammatory gene expression. Here we describe the selenoprotein-dependent synthesis of 15d-PGJ₂ in a macrophage cell system and its regulation of the NF-kB pathway. Our long-term objective is to understand the precise molecular mechanism of action of selenium as a nutraceutical.

Plenary talk I

Developmental Paradigm for the Silk Glands in the Mulberry Silkworm: Molecular and Genetic Aspects

K P GOPINATHAN

Microbiology & Cell Biology Department

Indian Institute of Science

Bangalore 560 012

The mulberry silkworm Bombyx mori has long been used as a lepidopteran model system for basic studies on gene expression and its regulation. The entire genome of B.mori comprising of about 500 million base pairs has been recently sequenced. However, the developmental studies at the molecular and genetic levels on this economically important organism has lagged considerably behind Drosophila, which has served as the paradigm for development. We have been exploiting B.mori as a model organism for development for the past several years. There are substantial differences in the developmental programmes between Drosophila and Bombyx. Although the overall programming of appendage development in both the organisms follow common principles, the timings of operation of the patterning mechanisms appear to be significantly different. The silk proteins are produced in the silk glands of B.mori, a pair of tubular structures arising from the labial segment and extending in length all the way up to the caudal region. The silk glands are functionally divided into three distinct compartments, the anterior (ASG), middle (MSG) and posterior (PSG) silk glands. PSG synthesizes the silk fibre proteins and MSG synthesizes the silk glue proteins, the sericins. We have identified the operation of the canonical Wnt signaling pathway in the sub-compartment specification within the MSG to result in the territorial regulation of the different sericin proteins. On the other hand, the PSG specification is through the Ubx signaling pathways. The silk glands are often considered as modified salivary glands because of their labial origin, but unlike B.mori Drosphila lacks the silk glands. B. mori additionally has a pair of salivary glands originating from the mandibular segment. However, there are no subcompartments within the salivary glands. The substantial differences in specification of these two organs have been elucidated.

Plenary Lecture II:

Structural basis for editing/proofreading during translation of the genetic code, Rajan Sankarnarayanan, CCMB, Hyderabad

Plenary Lecture III:

NMR AND STRUCTURAL BIOLOGY, Ramakrishna Hosur, Mumbai

SBCI AWARD LECTURES

1. PA Kurup Endowment Award Lecture: Hasi Das, IGIB, NEW DELHI

2. PS Sharma Memorial Award Lecture: Dulal Panda, IITB, Mimbai

3. M Sadakshara Swamy Endowment Lecture: Sivakumar, N, UOH, Hyderabad

2007 BURMA MEMORIALAWARD by PROF GP TALWAR

Animal Physiology

Sri Venkateswara PG & UG Colleges, Kadapa and Tirupati.

AP Invited talk

Insights in to the hormonal regulation of epididymal
function: A role for estrogen.

A.Jagannadha Rao, Department of Biochemistry, Indian
Institute of Science, Bangalore, 560012.

As an organ designed for the nurture,modification and protection of sperms, the epididymis performs several functions that culminate to produce mature and fertilization competent sperms. A highly specialized environment is maintained in the epidydimis which is formed by the region specific gene and protein expression patterns within the different segments of the epidydimis i.e the caput,corpus and cauda. It is well established that the expression of these genes is tightly regulated by androgens. However, results of studies using the estrogen receptor alpha knock out mice have established an important role for the estrogen in the regulation of epidydimal function. A detailed analysis of the Estrogen receptor sub types
alpha and beta at the mRNA and protein level revealed a strong presence of the receptors along the three
regions of the epididymis in the rat and monkey. Role of estrogen in the regulation of epidydimal function
in the two species was examined by administering the estrogen receptor antagonist ICI 182780 (ICI). It was
found that a the level of expression of aquaporin, a water channel protein was reduced both in the rat and
monkey caput following ICI treatment. Results also indicated a role for estrogen in regulating the fluid
absorption in ret tubules of the bonnet monkey. DDRTPCR analysis of RNA from the control and ICI
treated monkey caput led to the identification of keratin-19 as an estrogen regulated transcript. The presence of LH receptor in the cauda region of epididymis and independent estrogen sinthesizing system
in the epididymis was also demonstrated. These results high light the importance of estrogen in regulation of epididymal function. (Supported by the Ramanna Fellowship from DST, Govt. of India).

AP Invited talk 2: Role of Cyclooxygenase-2 (COX-2) in Male Reproduction

Prof. P. Reddanna, Centre For Biotechnology

School of Life Sciences, University of Hyderabad

Hyderabad- 500 046, India

(E-mail:preddanna@yahoo.com)

Arachidonic acid, the major polyunsaturated fatty acid (PUFA) in cell membrane phospholipids of mammalian systems, is mainly oxygenated by two important pathways, the cyclooxygenase (COX) pathway that generates prostaglandins, thromboxanes and prostacyclin and the lipoxygenase (LOX) pathway that generates leukotrienes and lipoxins. These oxygenated metabolites, collectively termed as eicosanoids, are extremely potent biologically active molecules with bewildering variety of actions in various physiological and pathological processes. The present study is an attempt to understand the role of cyclooxygenase-2 in male reproduction.

Cyclooxgenase, the rate limiting enzyme in the production of prostaglandins, exists in two isoforms, the constitutive cyclooxygenase-1 (COX-1) and the inducible cyclooxygenase-2 (COX-2). Even though COX-2 is known broadly as the inducible isoform expressed in response to inflammatory and mitogenic stimuli, we report, for the first time, the constitutive expression of COX-2 in rat testis. The COX-2 mRNA in the testis is smaller than that of the inducible COX-2. Hormone treatment (Testosterone/follicle stimulating hormone) regimes increased the levels of COX-2 protein suggesting that sustained levels of COX-2 protein in testis can be influenced by gonadotrophins and androgens. Further in vitro studies revealed a novel functional association between testicular membrane associated cytosolic glutathione S-transferases (mac GSTs) and cyclooxygenases in vitro, with possible interactions between them at GSH binding site. These interactions were shown to regulate the production of prostaglandins in testis. Based on the analysis of prostaglandins formed under physiological conditions, it is proposed that the constitutive COX-2 may be involved in spermaogoneal renewal and/or sperm transport in the testis. Endotoxin-induced acute inflammation, however, induced COX-2, iNOS and oxidative stress in male rats. This inflammation-induced COX-2 was shown to decrease steroidogenic acute regulatory (StAR) protein levels and thus affect testosterone biosynthesis and stermatogenesis. These studies thus reveal a role for COX-2 in male reproduction under physiological and pathological conditions.

IT AP: Molecular insight into male factor infertility

Dr. Pradeep Kumar G, Scientist-F, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Poojappura, Trivandrum-14.

Approximately 11% of the men of fertile age group present with male-factor infertility disorder. The cause of male infertility is classified under four general causes: spermatogenic disorder, obstruction of the seminal tract, inflammation, sexual disorders. Idiopathic spermatogenic disorder accounts for more than 50% of all of them. The cause of spermatogenic disorder is not yet identified. Growing literature has shown that spermatogenesis failure or reproductive failure (pregnancy wastages) occurred in male carriers of chromosomal distortion/aberration. But the mechanisms remain largely unsolved.

In this talk, I would outline our efforts towards identifying molecular markers of human spermatogenic disorder through a global Reprome Profiling to define a set of qualifying candidates as diagnostic markers. These qualifying criteria include the predicted or experimentally proven involvement of a specific marker in spermatogenesis, meiosis, post-testicular sperm development and activation, gamete interaction, early oocyte development and implantation. I would outline the leads obtained in identifying molecules that might regulate meiosis and gamete interaction. I would also highlight our projections to define the molecular causes of spermatogenic failure by performing high-throughput screening of the germ cell transcriptome and proteome from fertile males and males with spermatogenic failure. This approach is anticipated to help us generate a network map in which identified errors could be placed in a hierarchical order.

AP 1

COMPARATIVE DIGESTIBILITY OF SOME EDIBLE AROIDS OF NORTH EAST INDIA

Jyoti Prasad Saikia*, B.K.Konwar.

Department of Molecular Biology and Biotechnology, Tezpur University, Napaam-784028, Assam.

*email- jyotizone@gmail.com

Three edible aroids belonging to Colocasia, Xanthosoma and Amorphophallus species of Northeast India were selected. The corms were homogenized using blender and sieves and then dried to get fine powder. Starch estimation of the powdered corms was done by perchloric acid method. In vitro digestibility test with the dry powdered corms was done using salivary amylase collected from the mouth of healthy individuals. The total soluble carbohydrate and reducing sugar content generated by the digestion was estimated by anthrone method and DNS method respectively along with the control in which distilled water was used in place of enzyme solution. The results show difference in digestibility between the three aroid species. This study has great potential with regard to their edibility. Further studies will be done with the purified starch of the three aroid corms.

AP 2

ELECTROPHORETIC DETECTION OF GELATINASES AND ANTI-TRYPSIN ACTIVITIES IN BUFFALO (BUBALUS BUBALIS) UTERINE SECRETIONS DURING ESTROUS CYCLE AND EARLY PREGNANCY

Sudhir C. Roy and Jyotirmoy Ghosh

Molecular Biology Laboratory, National Institute of Animal Nutrition and Physiology,

Adugodi, Bangalore-560 030, Karnataka, India

E-mail: sudhircroy@rediffmail.com

The role of secretory proteases and their inhibitors in uterine tissue remodeling during estrous cycle and early pregnancy have been reported in human and rodents but not studied in buffaloes. In the present study attempts were made to detect and partially characterize the gelatinases and anti-trypsin activities (ATA) in uterine secretions of cycling and early pregnant buffaloes and compared with serum and follicular fluid. The whole female genitalia were collected from the local abattoir and categorized in to early-, mid-, and late-luteal and follicular stages of the estrous cycle based on the corpus luteal morphology. Uterine tract of one early pregnant animal (approx. 40-45 days) was also collected. The uterine secretions from the reproductive tracts were flushed out using 0.33 M NaCl and processed. Uterine samples of same stage of estrous cycle were pooled and proteins in the samples were quantified. The samples were subjected to detection of gelatinase and ATA by zymographic techniques. Three protein bands (91, 80 and 72 kDa) in early-luteal, and four in each of mid-luteal, late-luteal and follicular stage uterine secretions (91, 80, 72, and 48 kDa) displayed gelatinase activities. Of these, the 72-kDa-gelatinase band was found in early- and mid-luteal uterine secretions but was absent in late-luteal and follicular stage uterine secretions. However, in mid- and late-luteal uterine secretions, one additional gelatinase band of 48 kDa was also detected. Surprisingly, the 91-kDa gelatinase band of luteal stage was absent in uterine secretions of pregnant animal. Rather, pregnant uterine secretions displayed two new gelatinase bands of 106 and 100 kDa that were absent in all of the non-pregnant uterine secretions. Both serum and follicular fluid displayed two gelatinase bands of 80 and 103 kDa. Protein bands with higher ATA were detected in follicular stage and pregnant uterine secretions compared to a low activity in uterine secretions of other stages, serum and follicular fluid. The molecular weight of the major anti-trypsin protein band was 76 kDa. Thus, the study revealed that the gelatinases and anti-trypsin activities of buffalo uterine secretions varied according to the stage of the estrous cycle and these activities were higher in uterine secretions of pregnant buffalo compared to non-pregnant.

AP 3

LIPID PEROXIDATION MODULATES ENZYME ACTIVITIES OF SURFACTANT LIKE PARTICLES IN GROUNDNUT OIL FED RAT SMALL INTESTINE

Aasma Turan and Akhtar Mahmood

Department of Biochemistry, Panjab University, Chandigarh India.

aasma.turan@gmail.com

Abstract

Intestinal epithelium secretes novel unilamellar membranes having characteristics similar to lung surfactants and thus has been named “Surfactant like particles” (SLP). The production of SLP is stimulated in response to fat feeding, but the efficiency of feeding different dietary lipids on the secretion characteristics of SLP is unknown. Thus in the present study, the role of feeding polyunsaturated fatty acid (n-9) groundnut oil on the characteristics of SLP has been investigated in rat small intestine. Surfactant like particles were isolated and purified using sepharose 6B column and 10 mM Tris-HCl buffer, pH 7.4.The lipid particles were identified by assay AP activity as a marker of SLP.SLP were subjected to lipid peroxidation under in vitro conditions using Fe²⁺/ascorbate system. There was 42% decrease in AP activity in induced SLP compared to control. Analyzing AP activity by staining with BCIP of acrylamide non denatured gel and further confirmed by corroborated the enzyme activity data in these conditions. Brush border dissacharidases, such as sucrase, lactase, maltase and trehalase also showed 63%,32%,33% and 24% decrease respectively under these condtions as compared to control. Although, they present in low amount in SLP. There was no change in the activities of leucine aminopeptidase and gamma glutamyl transpeptidase in SLP under these conditions. These observations indicate that lipid microenvironment is modulated in SLP, this may be responsible for low enzyme activities upon lipid peroxidation.

AP 4

Prenatal exposure to lindane and adult responsiveness of cerebral and hepatic cytochrome P450s

Johri, A., Dhawan, A., Singh, R.L.¹, Parmar, D.

Developmental Toxicology Division, Industrial Toxicology Research Centre, Lucknow, India; ¹Dr. RML Avadh University, Faizabad, India

Email: ashu_itrc@rediffmail.com

The consequences of prenatal exposure to environmental chemicals could be enormous owing to the limited xenobiotic metabolizing capabilities of the developing fetus and neonate. Studies were initiated to ascertain the effects of prenatal exposure to low-levels of pesticides, such as lindane, an organochlorine insecticide on the responsiveness of xenobiotic metabolizing cytochrome P450s (CYPs) in rat offspring to a later exposure to the CYP inducers, namely phenobarbital (PB) and 3-methylcholanthrene (MC). Prenatal exposure to 0.0625-, or 0.125- or 0.25 mg/kg (1/1400^th or 1/700^th or 1/350^th of LD₅₀) of lindane was found to produce dose dependent alterations in the ontogeny of CYP1A1, 1A2, 2B1, 2B2 and 2E1 in brain and liver of the offspring, as determined by RT-PCR, western blotting and enzymatic studies. Interestingly, the effects were found to persist upto pnd 60. That the imprinted overexpression of cerebral and hepatic CYPs in the prenatally exposed offspring may modulate the inductive responsiveness of these CYPs to a challenge by the CYP inducers of the offspring when young at age was demonstrated by an enhanced responsiveness of cerebral and hepatic CYPs in offspring prenatally exposed to lindane. As demonstrated by RT-PCR, western immunoblot studies and enzymatic assays, a much higher mRNA and protein expression of CYP1A and 2B isoenzymes was observed in animals pretreated with lindane (0.25 mg/kg b. wt., p. o. to mother) during gestation and challenged with MC (30 mg/kg b. wt., i. p. x 5 days) or PB (80 mg/kg b. wt., i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC (30 mg/kg b. wt., i. p. x 5 days) or PB (80 mg/kg b. wt., i. p. x 5 days) alone. Furthermore, when the offspring prenatally exposed to lindane were subsequently challenged with a single dose (30 mg/kg b. wt.) of lindane, it lead to the worsening of the toxicity symptoms. While the onset of convulsions was decreased, the incidence was increased indicating higher vulnerability of the offspring in the prenatally lindane exposed group. Our data indicating the enhanced responsiveness of CYPs during adulthood may assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring could modify the adult response to a later exposure to xenobiotics including drugs many of which are metabolized by this enzyme system.

AP 5

Screening of Antioxidant properties of Tridax procumbens Linn.(Asteraceae) in paracetamol induced hepatotoxicity in male albino rats.

Shardul S. Wagh¹, G.B. Shinde².

1.Dept. of Biotechnology and Microbiology, G. H. Raisoni Institute of Interdisciplinary Sciences,B-33, M.I.D.C., Hingna, Nagpur – 16, (M.S.), India.

2.Post Graduate Teaching Department of Biochemistry, L.I.T. Premises, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India.

E-mail: - Shardulwagh@rediffmail.com / gbshinde@rediffmail.com

There is an increasing demand for natural drugs, as continuous consumptions of various allopathic drugs can culminate in many side effects and toxicity. The present study was an attempt to evaluate the antioxidant potential of ethanolic extract of Tridax procumbens Linn. against paracetamol induced liver damage.

The oxidative stress in paracetamol induced hepatic damage in rats was determined by estimating the levels of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalse (CAT), glutathione peroxidase (GPX) and glutathione s transferase (GST), were assessed in rats of induced hepatic damage, and confirmation of generation of oxidative stress was confirmed by comparing levels of these parameters with control group.

Generation of oxidative stress was confirmed by enhanced level of TBARS in toxic group. Reduction in GSH content due to toxicity was nullified by subsequent treatment of rats with Tridax procumbens Linn extract.

The decrease in levels of antioxidant enzymes in toxic group thus revealed the damaging effects of free radicals generated due to paracetamol (2g/kg body wt) administration. The activities of these enzymes returned to normal in Tridax procumbens Linn. extract administered (300mg/kg body wt) rats indicating the antioxidant efficacy of the Tridax procumbens extract in relieving oxidative stress.

AP 6

Nitric oxide and proteolysis in dystrophic muscle – a look at limb girdle muscular dystrophy

R. Dhanarajan, M. Alexander and A. Oommen

Dept. of Neurological Sciences, Christian Medical College, Vellore- 632 004,India. (dhanurajan@hotmail.com)

Muscular Dystrophies are a common, heterogeneous group of inherited neuromuscular disorders characterized by progressive skeletal muscle weakness and wasting. They include Duchenne, Limb Girdle, Becker, Emery-Dreifuss and Myotonic dystrophies. Limb Girdle Muscular Dystrophies are the second most common form of these dystrophies, with a prevalence of 1:100,000. Sixteen subtypes of Limb Girdle Muscular Dystrophies have been identified of which Type 2A with Calpain-3 absent or deficient and Type 2B where dysferlin is absent are the most common and form <30%>

Currently there are no effective treatments for these debilitating muscle diseases. Elucidating pathogenic mechanisms that lead to dystrophy would enable development of therapeutic strategies. The activation of proteolytic systems and increased inflammation and oxidative stress are suggested as possible pathogenic pathways while cytoprotection may occur with the induction of molecular chaperones.

The Limb Girdle Muscular Dystrophies are the focus of our interest and the aim of this study was to evaluate molecular pathogenesis in these dystrophic muscles by examining : 1. Protein status, 2. Nitric oxide signaling and 3. HSP-70 induction. Protein levels would indicate upregulation of proteolytic pathways, NO signaling would give an indication of inflammation and oxidative stress, while HSP-70 would indicate cytoprotection induced in response to dystrophic stress.

Non collagen protein profiles on SDS-PAGE, NO (as nitrite) levels determined by the Greiss reaction, protein thiol blots as a measure of NO signaling and immune blots of inducible HSP 70 were performed, with informed consent, on muscle biopsies received for routine diagnostic immunochemistry. Muscle from 6 patients with Limb Girdle Muscular Dystrophy 2A and 2B, determined clinically and with immunochemistry, and 6 patients with other neuromuscular disorders were studied and compared.

Non collagen protein was significantly reduced in Limb Girdle Muscular Dystrophic muscle compared to other neuromuscular disorders (147 ± 18mg/g tissue and 216 ± 57mg/g tissue, P<0.01)>

Dystrophic stress appears to induce cytoprotective responses in muscle as seen by the induction of HSP 70. Pathology may manifest when pathogenic mechanisms as proteolysis and NO signaling noted in this study predominate. This idea will be discussed.

AP 7

Studies on dielectric properties of bovine cartilage

R. Jeevan Kumar¹, D. Saira Khanam¹, S. MD. Shoaib¹ and Adeel Ahmad².

¹ Molecular Biophysics Laboratories, S.K.University, Anantapur-515003, A.P, India.

E-mail: smdshoaib@yahoo.com.

² Biophysics Unit, Department of Physics, Nizam College (Autonomous), Osmania University,

Hyderabad-500 001, A.P, India.

Advanced knowledge of biological chemistry is very essential to understand the electrical behaviour of bovine cartilage. Interrelationships of electrical and dielectric properties of bovine cartilage with water content and mineral content are poorly known. In the body cartilage occurs in three forms: hyaline cartilage, elastic cartilage, and fibrocartilage. All of these forms consists of specialized cells called chondrocytes, embedded in a dense mucoprotien matrix, with different dielectric behavior. In mammals cartilage is gristly, flexible tissue usually found attached to, or associated with, the joint surfaces of bones or forming attachments between certain bones. It is the substance from which the majority of the bones of the skeleton develop.

The biochemistry and physiology of bovine cartilage can be easily understood through dielectric studies. In the present investigation the dielectric properties such as dielectric constant (e^¢), dielectric loss (e^²) and dissipation factor (tand) of bovine cartilages are calculated as a function of temperature and frequency in different physiological conditions. Significant variation is observed. This variation may be attributed to the inhomogeneous deposition of chondrocytes, and water content of the cartilage. The measurements of dielectric parameters may provide quantitative information that is related to quantity and quality of cartilage due to the heterogeneity of molecular composition in it. The three parameters namely water content, mineral content and orientation of the chondrocytes embedded in a dense mucoprotien matrix, with respect to the applied electric field play an important role in influencing the dielectric properties of the cartilage tissues when measured at bulk level. This paper constitutes a step towards the application of dielectric measurements for medical uses and in vivo cartilage diagnosis.

Key words: dielectric properties, dielectric constant (e^¢), dielectric loss (e^²) dissipation factor (tand), water content, mineral content and chondrocytes.

AP 8

Cardiotonic property of Aegle marmelos Linn

on Isoproterenol induced myocardial infarcted Rats- An Electrocardiographic, Biochemical and Histopathological Evidences

G. SAAYI KRUSHNA, M. ABDUL KAREEM, SAILAJA, KV AND K. LAKSHMI DEVI^*

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, A.P. INDIA.

The present study was undertaken to find out the cardiotonic property of Aegle marmelos unripe fruit aqueous extract (AMUFAE) on the basis of electrocardiographic, biochemical and histopathological evidences in the isoproterenol (IPL) induced myocardial infarcted rats. Wister albino male rats weighing 180-250 g were randomly divided in 4 groups of 6 each. The AMUFAE suspended in water was administered orally to rats for 6 weeks at the dose of 200mg/kg/day followed by subcutaneous administration of IPL (85mg/kg/day for 2 consecutive days). IPL, a synthetic catecholamine and β-adrenergic agonist has reported to cause oxidative stress with subsequent elevation in serum lipid profile, ST segment in ECG pattern and adverse histopathological changes. Pretreatment with AMUFAE in IPL administered rats protected the above mentioned parameters from alterations as compared with myocardial infracted rats. This confirmed the cardiotonic property of AMUFAE on IPL induced myocardial infarcted rats.

AP 9

Affect of Acute Organophosphate Poisoning on the Temporal profile of Acetylcholinesterase in Rat brain.

Amajad Iqbal Kazi¹, Anand Zachariah², Anna Oommen¹.

Neurochemistry Laboratory¹, Medicine Unit 1², Christian Medical College, Vellore.

E mail: amjadkazi@rediffmail.com

Organophosphate pesticides are known to influence several physiological responses. The primary effect is due to inhibition of the enzyme acetylcholinesterase which plays a role in terminating neurotransmission of acetylcholine. The systemic toxicity of organophosphates reflect the symptoms related to cholinergic hyperstimulation consequent to the loss of acetylcholinesterase catalytic activity.

In the present study we investigated the effects of acute organophosphate poisoning on the protein levels of acetylcholinesterase in four brain regions viz, straitum since the majority of the enzyme is synthesized in the straitum, hippocampus for its cholinergic role in memory, cortex and cerebellum to assess cholinergic activity. Rats (female wistar) were administered with a single dose of the organophosphate monocrotophos (80% LD₅₀) by gavage and sacrificed 2.5 hours, 7 days, 15 days, 30 days later. The four regions of brain were assayed for acetylcholinesterase activity and protein levels of the enzyme quantitated by western blotting using antibodies to acetylcholinesterase raised in rabbits.

Results indicate that acetylcholinesterase activity was significantly inhibited early in poisoning in all the brain regions, which is due to mocrotophos inhibition of the enzyme.

Acetylcholinesterase protein levels were decreased in all the brain regions and significantly (p<.05) in the hippocampus and straitum one week after poisoning. One month after poisoning the enzymatic activity and proteins levels of acetylcholinesterase were restored in all the regions but the recovery was lowest in the hippocampus (p < .05).

These results indicate that monocrotophos not only inhibits acetylcholinesterase enzyme activity but also affects the synthesis of the protein. This may be possibly due to an adaptive process to restore physiological function in the brain following chemical stress of organophosphate poisoning. Possible mechanisms involved in the observed effect of acetylcholinesterase will be discussed.

AP 10

Involvement of adrenals in Acephate-induced hyperglycemia in rats

Apurva Kumar R. Joshi and Rajini P.S.

Food Protectants & Infestation Control, Central Food Technological Research Institute, Mysore-570020

E-mail: apurvakmr@gmail.com

Scientific body of information implicating the role of environmental factors in health hazards is steadily growing. Organophosphorus insecticides (OPI) constitute one of the most widely used classes of pesticides being employed for both agricultural and landscape pest control. In addition to symptoms of cholinergic activation resulting from inhibition of acetylcholinesterase, hyperglycemia has been reported as one of the major adverse effects of exposure to OPI. Proposed mechanisms for OPI-induced hyperglycemia include cholinesterase inhibition, oxidative stress mediated damages in pancreas, pancreatitis and increased hepatic glucose output. Although it is known that OPI increase flux through gluconeogenesis in liver, the causative mechanisms are ill defined. In this abstract we discuss hyperglycemia caused by Acephate, an OPI and possible role of adrenal functions in its etiology. Following administration of a single oral dose of Acephate (10% LD₅₀) to overnight fasted rats, blood glucose increased to peak levels (87.1% above control) at two hours and there after tended to revert to euglycemic conditions since at 8h after administration, the blood glucose was only 15.9% higher as compared to control. To understand the mechanisms underlying the reversible hyperglycemia caused by Acephate, over night fasted rats were administered single oral dose of Acephate (10% LD₅₀) and sacrificed either two hours or six hours after administration. Acephate administration was associated with drastic increase in plasma corticosterone (78.8% increase compared to control) in rats exposed for two hours while in the third group (six hours exposure) the corticosterone level was 42.33% above control. Adrenal cholesterol levels decreased in both the treatment groups, and the decrease was proportional to time of exposure (a decrease of 41.1 and 52.9% as compared to control in two hour- and six hour-exposure groups). As was the case with blood glucose, activities of gluconeogenetic enzymes in liver viz., glucose-6-phosphatase^a, fructose-1,6-diphosphatase^b and tyrosine aminotransferase^c increased significantly in rats exposed to Acephate for two hours and decreased in the last group to values which were comparatively higher than control (a: 90.7 & 25%; b: 49.3 & 16.3%; c; 84.3 % 66% increase in two- and six-hour exposure groups respectively as compared to control). Data from these experiments suggests that adrenal glands play an important role in the reversible hyperglycemia caused by Acephate via increased corticosterone secretion, eliciting increased hepatic glucose output through gluconeogenesis.

AP 11

Effect of prenatal exposure to lindane an expression of AhR and ARNT in brain and liver of offspring.

Shikha Srivatava, Ashu Johri, Alok Dhawan, Devendra Parmar.

Previous studies from our laboratory have shown that prenatal exposure to low doses of lindane, an organochlorine insecticide, modulates the ontogeny of xenobiotic metabolizing cytochrome P450s (CYPs) in rat brain and liver. As the regulation of CYPs depends on the interaction of xenobiotics with the specific receptors, attempts were made to investigate the effects of prenatal exposure to low dose of lindane (0.0625 or 0.125 or 0.25 mg/kg) from gestation day 5-21 on the expression of aryl hydrocarbon receptor (AhR) and ARNT transcriptionally induces the expression of CYP1A genes Using semi quantitative RT-PCR, our studies revealed a dose dependent increase in the mRNA expression of AhR and ARNT in the brain and liver of offspring from postnatal day 0 to postnatal day 90. The increase in the mRNA expression of AhR and ARNT were found to persist in brain and liver of offspring upto adulthood. Our studies assume significance in view of the fact that the AhR-ARNT heterodimer initiates the transcription of a battery of AhR regulated genes which include not only the genes encoding for xenobiotic metabolizing enzymes but the genes involved in growth, differentiation and cellular homeostasis as well, thus affecting a wide spectrum of biological responses of the offspring.

AP 12

Antioxidant and antigenotoxic effects of aqueous extract of Phyllanthus niruri in rats

R. Karuna, S. Sreenivasa Reddy, B.Ramesh and D. Saralakumari ^*

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur,

Andhra Pradesh, India

Abstract

Increased levels of oxidative stress may be implicated in the etiology of many pathological conditions. The antioxidant protective effect of natural plants is promising therapeutic drugs for free radical pathologies. Phyllanthus niruri (P.niruri) is a tropical plant traditionally used in treatment of renal and hepatic disorders and viral infections. It was also reported to have a protective ability against chemically induced cytotoxicity. In order to assess the antioxidant potential of Phyllanthus niruri aqueous extract (PNAEt), in the present study, we assessed plasma lipid peroxidation (LPO) and plasma antioxidant levels i.e. vitamin C, uric acid and reduced glutathione (GSH) and activities of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) in PNAEt treated rats (oral dose of 200 mg/kg body wt/day for 8 weeks) by comparing with controls. Genotoxicity of PNAEt was assessed by single cell gel electrophoresis (SCGE) of lymphocytes under both in vitro and in vivo conditions. Protective role of PNAEt against hydrogen peroxide (H₂O₂), streptozotocin (STZ) and nitric oxide generating system induced lymphocyte DNA damage was also assessed by SCGE. PNAEt treated rats showed a significant decrease in plasma LPO and significant increase in plasma vitamin C, uric acid and GSH levels and GPx, CAT and SOD activities. SCGE experiment revealed that PNAEt was devoid of genotoxicity and had a significant protective effect against H₂O₂, STZ and nitric oxide (NO)- induced lymphocyte DNA damage. The results suggest the nontoxic nature of PNAEt and consumption of PNAEt can be linked to improved antioxidant status and reduction in the risk of oxidative stress and genotoxicity

AP 13

KINETIC PARAMETERS OF ACETYLCHOLINE ESTERASE IN THE HEART OF STREPTOZOTOCIN INDUCED DIABETIC AND INSULIN INDUCED HYPOGLYCAEMIC RATS

Sherin Antony, Anu Joseph, C.S.Paulose

Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience,

Cochin University of Science and Technology, Cochin- 682 022, Kerala, India cspaulose@cusat.ac.in

Diabetes mellitus is a major global health problem, characterized by hyperglycaemia. Hypoglycaemia is an obstacle to optimal blood glucose control in diabetic patients. Acetylcholine, the principal parasympathetic neurotransmitter is rapidly hydrolyzed to inactive metabolites, choline and acetate; by the enzyme acetylcholine esterase. The effect of streptozotocin induced diabetes and insulin induced hypoglycaemia on the activity of Acetylcholine Esterase (AChE) in the heart of experimental rats were studied. The animals were grouped into Control (C), Diabetic (D) and insulin induced hypoglycemia in Control (C+IIH) and Diabetic (D+IIH) groups. Kinetic studies of AChE revealed a significant decrease in the V_max (p<0.01);>m (p<0.05); (p<0.001)>max (p<0.01)>m of D+IIH group showed a significant increase (p<0.05)>max (p<0.05)>

Supported by grants from DST, DBT, ICMR Govt. of India, KSCSTE, Govt. of Kerala. Sherin Antony thanks CSIR for JRF.

AP 14

KINETIC PARAMETERS OF GLUTAMATE DEHYDROGENASE IN THE KIDNEY OF STREPTOZOTOCIN INDUCED AND INSULIN TREATED DIABETIC RATS AS A FUNCTION OF AGE.

Pretty Mary Abraham, Savitha Balakrishnan & C.S.Paulose

Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience,

Department of Biotechnology, Cochin University of Science andTechnology, Cochin- 682 022, Kerala, India.

cspaulose@cusat.ac.in

Aging is the progressive deterioration of body functions. Neurotransmitters and their functional regulation have an important role in various metabolic activities. The enzyme glutamate dehydrogenase catalyzes reductive inter-conversion of α-ketoglutarate to L-glutamate with NADP⁺ as a cofactor. The aim of the study was to investigate the role of Glutamate dehydrogenase activity in the kidney of 7weeks and 90weeks old Control (C), streptozotocin (STZ) induced diabetic (D) and insulin treated diabetic rats (D+I). Kinetic studies revealed that V_max significantly increased in 7weeks (p<0.05) style=""> (p<0.05) style="color: red;"> In 90weeks old diabetic rats, Vmax of the enzyme increased (p<0.001) style="color: red;"> Increased GDH activity observed during diabetes leads to an increased conversion of α-ketoglutarate to L-glutamate, altering the carbohydrate metabolism and allied changes in protein and fat metabolism. Glutamate is a putative excitotoxic neurotransmitter and a precursor of GABA, a major inhibitory neurotransmitter. Increased glutamate production due to increased GDH activity in control and diabetic 90weeks old rats causes subsequent toxicity leading to neuropathy and nephropathy. Our results suggest that there is increased glutamate dehydrogenase activity observed in 90weeks old diabetic rats contributes to the early symptoms of physiological and cognitive disorders, which has immense clinical significance in the management of diabetes.

Acknowledgement:

Supported by grants from DBT, ICMR Govt. of India, KSCSTE, Govt. of Kerala. Pretty Mary Abraham thanks DST for JRF.

AP 15

Protective Effect of Polymyxin B on Pregnancy in the Mothers infected with Gram-negative bacteria

M.K. Jaiswal¹, Varkha Agrawal¹, M.M. Chaturvedi² and Y.K. Jaiswal¹

¹Molecular Biology and Reproductive Immunology Laboratory

School of Studies in Biochemistry, Jiwaji University, Gwalior – 474 011 (MP)

²Department of Zoology, Delhi University, New Delhi – 110 007

(E mail Id: jaiswalyk@gmail.com )

Many sterility outcomes are associated with the Gram-negative bacterial infection that may enable the endometrium to support implantation on one hand and may not maintain the viable embryos on other. We have established a mouse model (Park strain) in our laboratory where the systemic administration of ‘minimum dose’ (MD) of LPS (i.e., 250 µg/kg body weight) results in complete failure of implantation of developing embryos. Polymyxin B is a naturally occurring cationic cyclic decapeptide which is highly bactericidal to Gram-negative bacteria and considered to be one of the most efficient cell-permeabilizing compounds. This capacity is due to its high-affinity binding capacity to lipid A molecule of lipopolysaccharide. However, the therapeutic applications of Polymyxin B are very limited because of its relatively high toxicity. The aim of the present work is to investigate the effect of LPS on the viability of developing embryos during pregnancy and to verify the protective effect of Polymyxin B in modulating the embryotoxic effect of LPS on the viability of developing embryos during pregnancy. The ‘MD’ of LPS, Polymyxin B (5-100µg/animal) and ‘MD’ of LPS + Polymyxin B (5-100µg/animal) was injected intraperitoneally to pregnant females (6-7 week; weighing ≈ 20-21 g) separately on day 0.5 of pregnancy. The control animals received 100 µl of sterile normal saline in a similar manner. The viability of embryos was accessed by the percentage of normal gestational sacs present in both of the uterus on day 14.5 of gestational period. We have observed that the treatment of pregnant females with Polymyxin B disturbs the pregnancy in a dose dependent manner and also increases the substantial risk of congenital abnormalities in the developing embryos in the mother. However, it does not show any adverse effect on the implantation of embryos. The embryotoxic effect of ‘MD’ of LPS can completely be prevented by 25µg/animal of Polymyxin B whereas other lower and higher doses of Polymyxin B are not able to protect the embryotoxic effect of ‘MD’ of LPS on pregnancy in a similar manner. Our results clearly demonstrate the embryotoxic effect of Polymyxin B on pregnancy and its ability to protect the pregnancy in a dose dependent manner in the mothers infected with Gram-negative bacteria. However, further studies are being carried out in our laboratory to establish the use of Polymyxin B as a safe antibiotic drug during pregnancy.

AP 16

Analysis of Cyp19 gene expression in different stages of follicular development and differentiation

Swati Jindal, Deepti, Dheer Singh and M.K.Sharma

Molecular Endocrinology Lab, Animal Biochemistry Division,

National Dairy Research Institute, Karnal-132001, India

drdheer.singh@gmail.com

Infertility problems in buffalo breeding, like late maturity, silent heat, prolonged calving intervals and repeat breeding etc are responsible for a short “stayability” or working life (involving premature culling) resulting in great economic losses. A key to efficient female reproductive performance is the proper function of the ovarian cycle that is characterized by growth, selection, ovulation and luteinization of healthy follicles. All these processes but also placentation, pregnancy and parturition are essentially regulated by the steroid hormone oestradiol. The Cyp19 gene encodes the key enzyme of estrogen biosynthesis, aromatase cytochrome P450 (P450_arom). In cattle and sheep the Cyp19 gene and its tissue-specific regulation is well characterized. However our knowledge of the buffalo Cyp19 gene is still insufficient. In the present study tissue-specific expression of aromatase and its transcript variants in different stages of follicular development and differentiation were analyzed using RT-PCR and nested PCR. Total RNA was isolated from ovarian follicular cells and other tissues. There was high aromatase mRNA expression in granulosa cells from large ovarian follicle. Nested PCR approach showed that granulosa cells from ovarian large follicles exhibit significant higher expression of aromatase mRNA as compare to that in other tissues. Its expression declined in corpus luteum. The Cyp19 gene transcript in granulosa cells of large follicles was driven by promoter II whereas these transcripts were chiefly driven by promoter I.1 in corpus luteum. Promoter I.1 can be regarded as placenta specific. In conclusion, tissue specific promoters control the expression of cytochrome P450 aromatase mRNA during follicular development and differentiation in buffalo ovary

AP 17

Lens Aldose reductase,Sorbitol dehydrogenase and Ascorbic acid activities in diabetic cataract

K.Haritha, D.Saimangala^*, D.Saralakumari

Department of Biochemistry, Sri krishnadevaraya university, Anantapur,India-515003.

*Sri satya sai institute of higher medical sciences, Prasantigram,Puttapurthy

Email: bioharil97@rediffmail.com

The polyol pathway of glucose metabolism becomes active when intracellular glucose levels are elevated and it can generate cellular stress through several mechanisms. Aldose reductase (AR) the first and rate limiting enzyme in the pathway reduces glucose to sorbitol further sorbitol dehydrogenase (SDH) metabolize sorbitol to fructose. In order to understand the role of polyol pathway and antioxidants, the activity of AR, SDH and vitamin c levels were estimated in diabetic and nondiabetic cataractous human lenses. Diabetic cataract lens showed a significantly increased AR activity without alternation in SDH activity when compared to nondiabetic cataract lenses, indicating the more accumulation of sorbitol in diabetic cataract lenses compared to nondiabetic cataract lenses. The lens damage and cataract formation appears due to in situ generation of reactive oxygen species. Scavengers of active oxygen species have been found to protect against this damage. Enhanced production of reactive oxygen species was well documented under diabetic conditions. The activity of vitamin c was decreased in diabetic cataractous lenses when compared to nondiabetic cataractous lenses, indicating decreased antioxidant condition of diabetic lenses. Thus the present study leads to the conclusion of decreased antioxidants and accumulation of sorbitol may be one of the reasons for early onset of cataract in diabetes.

AP 18

Gallic acid interactions with brush border peptidases in rat intestine

Nidhi Mahajan and Akhtar Mahmood

Department of Biochemistry, Panjab University, Chandigarh, 160014, India.

Polyphenols constitute a great diversity of compounds with a wide spectrum of pharmacological and biological properties. Gallic acid (3, 4, 5 trihydroxybenzoic acid), a naturally occurring polyphenol, is abundantly present in edible foods viz: fruits, vegetables, nuts and beverages like tea and red wine. Thus intestinal tissue compromises the primary target for the interaction with dietary micronutrients. In this study we investigated the effect of gallic acid on brush border leucine aminopeptidase and g-Glutamyl transpeptidase activities in rat intestine. Gallic acid at low concentrations (0mM-0.5mM) inhibited leucine aminopeptidase and g-Glutamyl transpeptidase activities in rat intestine. Optimum inhibition of leucine aminopeptidase and g-Glutamyl transpeptidase activities was observed at 0.5mM gallic acid concentration while their activities were reduced to 50% at 0.20mM and 0.13mM gallic acid respectively. The enzyme inhibition monitored was reversible in nature as exhaustive dialysis restored the activities to control values. Kinetic analysis revealed that there was essentially no change in the V_max while K_m was increased several folds, thus revealing the competitive mode of inhibition by gallic acid. Furthermore, the effect of gallic acid along with various –SH reacting compounds showed that observed inhibition was additive in nature. These findings therefore indicate that gallic acid interferes with the digestive activities of intestine related to amino acid absorption and transport.

AP 19

PROTEIN PROFILE OF POULTRY KIDNEY

Dr. Parvathala Kalyani, DR M.Raga Sudha and Dr. Vasili Ashok

Department of Veterinary Biochemistry, C.V.Sc., Rajendranagar,

Hyderabad, A.P., India.

E-mail: pkalyani_26@yahoo.co.in.

The extensive prevalence and escalating incidence rates of Chronic Renal Failure (CRF) and End Stage Renal Disease (ESRD) worldwide necessitates renal tissue engineering as a practical solution to meet the organ demand for renal transplantation therapy (RRT). Renal tissue engineering is a multidisciplinary field, which is supported on three pillars- cells, scaffolds and signaling biomolecules. A critical understanding of the role of signaling biomolecules viz., protein inducing factors, growth factors, transcription factors, protooncogene receptors and growth factor receptors, ECM components, intracellular cytoplasmic proteins etc implicated in kidney development makes possible the dream of renal tissue engineering a reality. These signaling biomolecules are differentially expressed with specific spatiotemporal distribution patterns to sculpt the well-defined architecture of kidney tissue. Signalling biomolecules and signal transduction pathways are highly conserved between species during millions of years of evolution, which engenders the scope for probing into mammalian kidney biology by using lower organisms as models. Gallus gallus (chicken) is a suitable model organism to study protein role in kidney organogenesis because of the extensive research done on chick embryogenesis and ease of embryo collection. Metanephric kidneys were collected from different developmental stages of Vanaraja breed chick embryos viz., embryonic days 7,9,11,13,15,17 & 19; day old chick and adult cock in ice cold PBS. Embryonic kidneys & kidneys of day old chick were dissected under diascopic stereo zoom microscope. The kidney tissues were homogenized, centrifuged and supernatants were collected. The supernatants were analyzed by SDS PAGE. Prominent differential protein expression was evident upon comparing protein bands of supernatants of kidney tissue extracts. A single high molecular weight protein band was seen only in the adult sample. Number of proteins are differentially expressed during kidney organogenesis implicating the role of different proteins at different stages of development.

AP 20

ISOLATION OF BUFFALO URINARY PROTEINS

Dr. Raga Sudha Medicherla, Dr. Parvathala Kalyani and Dr. Vasili Ashok

Department of Veterinary Biochemistry, C.V.Sc., Rajendranagar,

Hyderabad, A.P., India.

E-mail: sudha.medicherla@gmail.com

It is an amazing fact to note that normal urine of healthy animals contain certain proteins viz., uroplakins and uromodulins in minute quantities which are elaborated by urinary bladder and thick ascending limb of henle’s loop respectively. The tissue specific expression of these proteins in the urinary system is the basis for transforming the urinary system into a bioreactor to produce proteins of economic importance in the urine. This requires information about the gene sequence of these proteins so that promoter sequences of these genes can be used to express a foreign gene. Gene sequence can be deciphered from the protein sequence and for that isolation of these normal proteins in urine is the hallmark in the transgenic research to convert urinary system into a bioreactor. Buffaloes are the model organisms owing to the advantages in terms of large volumes of urine produced and large buffalo population in India, which enables the harvest of large quantities of desired economically valuable protein. The objective of the present study is to isolate and quantify normal urinary proteins of buffaloes. Urine samples from 10 different apparently healthy lactating buffaloes were precipitated by using acetone, ammonium sulphate and sodium chloride. All these samples were analyzed by SDS PAGE. Coomasie blue stained gels revealed a prominent 85 KDa protein band in all the precipitated samples, which could be uromodulin. Silver stained gels revealed low molecular weight bands (15 & 27 KDa) in addition to the 85 KDa band in all precipitated samples, which could be uroplakins. Though protein presence in urine normally is pathological, there are still certain proteins viz., uromodulins and uroplakins in minute quantities in the normal urine of healthy animals. Sodium chloride is the salt of choice because it precipitates almost a single protein and is required in small quantities when compared to ammonium sulphate. Acetone precipitation yielded more precipitate but the bands were not prominent may be because acetone also precipitates urinary salts and metabolic wastes.

AP 21

Ethion induced toxicity in rat erythrocytes and its amelioration with vitamin E

Gujit Kaur Bhatti, Rajat Sandhir, Ravi Kiran*

Department of Biochemistry, Panjab University, Chandigarh -160014 (INDIA).

e-mail: bhattibiochem@yahoo.com

Easy availability and indiscriminate use of organophosphate pesticides has resulted in hazards to human health, bioaccumulation in non target species, phytotoxicity, pesticide persistence in the environment and the adverse effects on beneficial organisms and wildlife. As erythrocytes are excellent models to measure the toxic effects of xenobiotics and to study the mechanisms of their toxicity, present investigation was designed to study the toxic effects of administration of ethion on rat erythrocytes and their modulation with vitamin E supplementation. Studies were carried out for 15 days. Erythrocytes were separated by centrifugation. Erythrocyte membranes were prepared by the method of Rhoda B (1968) using Tris-HCl buffer containing EDTA (pH 7.4). Ethion administration resulted in changes in the protein, cholesterol and phospholipid contents of erythrocyte membranes and decrease in the activities of membrane bound enzymes - Na⁺ K⁺-, Mg²⁺ - and Ca²⁺ -ATPases. These biochemical changes resulted in changes in the permeability of the erythrocyte membrane. An increase in the ⁴⁵Ca²⁺ uptake by the erythrocytes was observed. Administration of ethion also resulted in morphological alterations in the erythrocytes as evidenced by scanning electron microscopy (SEM). Supplementation of vitamin E along with ethion had beneficial effect.

AP 22

Comparative inhibition of acetylcholinesterase by dichlorvos in Caenorhabditis elegans and rat brain in vitro and kinetics of inhibition

Kisan B. Jadhav and Rajini P.S.

Food Protectants & Infestation Control, Central Food Technological Research Institute,

Mysore-570020

E-mail: kisanb1@gmail.com

Organophosphorus insecticides (OPI) have been used extensively in pest control. OPI are known to cause acute toxicity through irreversible inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). AChE plays an important role in neurotransmission at cholinergic synapses by rapid hydrolysis of neurotransmitter, acetylcholine to choline and acetate. The resultant accumulation of acetylcholine following inhibition of AChE by OPI at the cholinergic synapse leads to a range of neurotoxic effects. Recently, the nematode, Caenorhabditis elegans, which has been extensively used in genetic studies, has emerged as a popular model in toxicological studies. Genetic analysis of Caenorhabditis elegans reveals presence of 4 forms of acetylcholinesterase gene. This compelled us to explore the possibility of employing Caenorhabditis elegans for detecting the presence of AChE inhibitors. We also made an attempt to validate Caenorhabditis elegans as a model by in vitro comparison with rat brain AChE of kinetic parameters viz., substrate inhibition by acetylthiocholine, sensitivity to inhibition by DDVP (OPI), and nature of Lineveawer-Burk plot in the presence of DDVP. In vitro AChE assay was carried out employing homogenates prepared from whole worm and rat brain, in the presence of increasing concentrations of acetylthiocholine iodide (ATCI) to establish the Michaelis-Menten graph which showed the pattern of substrate inhibition in both the systems. Enzymatic hydrolysis of the substrate (ATCI) by the AChE in rat brain homogenate showed a typical saturation curve with K_m of 77 mM and V_max 12 nmol/min/mg protein and substrate (ATCI) concentration beyond 1.5Mm inhibited enzyme. Similarly enzymatic hydrolysis of the substrate (ATCI) by the AChE in C.elegans homogenate showed a typical saturation curve with K_m of 725 mM and V_max 33 nmol/min/mg protein. However only a higher substrate (ATCI) concentration (>5mM) inhibited C. elegans AChE. To determine median inhibitory concentration (IC₅₀) of DDVP on AChE activity in vitro, the assay was carried out after the enzyme source was incubated with different concentrations of DDVP. Regression analysis of ‘% inhibition v/s concentration of DDVP’ plot revealed IC₅₀ as 110 and 41.6 nM in the case rat brain and C.elegans AChE respectively. To understand the nature of AChE inhibition by DDVP, enzyme assay was carried out in the presence of increasing concentrations of ATCI after the enzyme source was incubated with different concentrations ((IC₂₅, IC₅₀, and IC₇₅) of DDVP. The Lineweaver-Burk plot revealed non-competitive type of inhibition of both rat brain and C. elegans AChE. Data from these experiments suggest similar nature of C. elegans AChE inhibition by DDVP when compared to rat brain AChE, as well as higher sensitivity of C. elegans AChE to DDVP. It is evident from these data C.elegans is a reliable sensitive model for prescreening/determining the presence of irreversible AChE inhibitors.

AP 23

REGULATION OF LACTATE DEHYDROGENASE BY EXTRACELLULAR MATRIX COMPONENTS

V.B. Sameer Kumar and P.R. Sudhakaran

Department of Biochemistry,University of Kerala,

Thiruvananthapuram, Kerala, India- 695 581.

sameerkumarvb@gmail.com

The pattern of expression of lactate dehydrogenase (LDH) isoenzymes and the levels of its metabolites varies under several pathological conditions, some of which involve parallel changes in the extra cellular matrix (ECM) composition. To analyse if the ECM components can influence on the pattern of expression of LDH isoenzymes, the effect of various matrix components such as laminin (Ln), an important glycoprotein of the basement membrane (BM) and collagen I (Col I), an important component of the interstitial matrix on the LDH isoenzymic pattern in endothelial cells was analysed. Biochemical assay, analysis of isozymic pattern by zymography and the levels of LDH mRNA by RT-PCR, revealed that, in ECs maintained on Ln matrix substratum, unlike in ECs maintained on Col I matrix substratum, a shift in the isoenzymic pattern of LDH from ‘A’ rich forms to ‘B’ rich forms occur, suggesting that ECM components can differentially regulate LDH isoenzymic pattern in ECs.

AP 24

ANGIOGENIC RESPONSE OF ENDOTHELIAL CELLS TO FIBRONECTIN

R.I.Viji, P.R. Sudhakaran

Department of Biochemistry, University of Kerala,

Kariavattom, Thiruvananthapuram, Kerala, India – 695 581.

vijiraveendran@gmail.com

Endothelial cells (ECs), the key players of angiogenesis interact with their surrounding extracellular matrix components during the process of angiogenesis. Fibronectin (FN), an interstitital matrix component has been shown to be involved in the proliferative and invasive phases of angiogenesis. FN, being a multifunctional multidomain protein, the diverse biological functions attributed to FN has been localized to specific regions of the molecule. The interaction of the central cell binding domain of FN with transmembrane integrin receptors has been shown to contribute to angiogenesis while the role of interaction of heparin binding domain of FN with the cell surface heparan sulfate proteoglycans in the process of angiogenesis is not clearly understood. The present study was designed to examine the role of heparin binding domain (HBD) of FN in angiogenesis using in vitro culture of human umbilical vein endothelial cells (HUVECs). ECs exposed to HBD of FN showed both morphological and biochemical characteristics of angiogenic phenotype. Further, chick chorioallantoic membrane assay also revealed the proangiogenic nature of HBD. Studies using inhibitors of intracellular signaling pathways suggested that the HBD of FN by itself can promote angiogenesis in HUVECs possibly by interaction with cell surface heparan sulphate proteoglycans involving PKC dependent signaling pathways.

AP 25

Lectin induced activation of monocytes leads to the up regulation of MMP-9 through PG/cAMP pathway

K. Saja and P.R Sudhakaran

Department of Biochemistry, University of Kerala

Kariavattom Campus, Kariavattom.

sajadharmajan@yahoo.co.uk

Inflammation involves complex set of interactions among soluble factors and cellular components, often accompanied by destruction of connective tissue. The major soluble factors include cytokines, interleukines, reactive oxygen species, proteases mainly matrix metalloproteinases (MMPs) and arachidonate metabolites. Among the cellular components blood borne monocyte/ macrophages are integral part of inflammation. During inflammation these cells are recruited to the site of inflammation, by the destruction of extracellular matrix barrier. Their transendothelial migration is attributed in part to the production of matrix metalloproteinases (MMPs) .The major MMPs produced by monocyte/ macrophages are MMP-2 and MMP-9. Among these MMP-9 is an inducible enzyme, which is upregulated in response to various inflammatory signals. In this context we have developed an in vitro model system, whch mimic inflammatory monocyte, using a lectin, namely artocarpus lakoocha agglutinin (ALA). Studies using indomethacin and inhibitors of signaling pathway showed that ALA activated monocytes upregulated MMP-9 through PG/cAMP mediated pathway. Detailed investigations showed that ALA mediated upregulation of MMP-9 involve multiple signaling pathways.

AP 26

UPREGULATION OF MMP-9 PRODUCTION IN MONOCYTES BY OXIDIZED LDL

A. RADHIKA, P.R. SUDHAKARAN

DEPARTMENT OF BIOCHEMISTRY

KARIAVATTOM CAMPUS, THIRUVANANTHAPURAM-695 581

KERALA, INDIA

radhikanair02@yahoo.co.uk

Monocyte-macrophage systems are the chief cellular constituents which have numerous physiological and pathological implications to the development of atherosclerotic vascular lesions. Matrix metalloproteinases (MMPs) are a group of cation dependent neutral matrix metalloproteinases which contribute to the development of vascular complications of atherosclerosis particularly the rupture of fibrous plaques. Macrophage derived foam cells appear to be a major cellular source of MMPs in atherosclerotic lesions. Hyperlipoproteinemia leading to the accumulation of oxidized LDL is a major risk factor in the development of atherosclerosis and oxidized LDL is known to influence vascular cell functions. To study whether pre-exposure of monocytes to oxidatively modified LDL influences the production of MMPs, an in vitro model system using isolated human peripheral blood mononuclear cells (PBMC) in culture was used. The production and activity of MMPs were studied using ELISA and gelatin zymography. Results indicate that pre-exposure of monocytes to oxidized LDL (oxidation of lipid moiety by CuSO₄ and protein part by HOCl) upregulated MMP-9 production. Use of antagonists of nuclear receptor pparg and inhibitors of intracellular signaling pathways showed that CuSO₄ oxidized LDL caused upregulation of MMP-9 through a pparg dependent mechanism whereas HOCl oxidized LDL caused MMP-9 production through a pathway independent of pparg.

AP 27

TOLL-LIKE RECEPTOR EXPRESSION IN THE MALE REPRODUCTIVE TRACT OF RODENTS

Barnali Biswas¹, Frank S French², Susan H Hall² and Suresh Yenugu¹

¹Department of Animal Sciences, University of Hyderabad, Hyderabad, India.,²Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

The male reproductive tract is an important organ system in the body, which primarily functions to produce spermatozoa and the secretion of male sex hormones. Spermatozoa that leave the testis mature in the epididymis to acquire forward motility and fertilization capacity. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Proper protection of the reproductive tract against pathogens is very essential and has recently become an active area of investigation to combat sexually transmitted diseases including AIDS. The innate immune mechanisms that exist in the male reproductive tract are not well understood. Toll-like receptors (TLRs) are a broad family of innate immune receptors that play critical roles in detecting and responding to invading pathogens and about thirteen (TLR 1-13) of them have been characterized till date in different species. In this study we attempt to determine the expression pattern of TLRs in the male reproductive tract of rats and the factors that govern them. To accomplish this, RNA was isolated from adult male reproductive tract tissues (caput, corpus and cauda, testis and seminal vesicle) and RT-PCR performed using gene specific primers for Tlr 1-13. Developmental regulation of Tlr expression was analysed using RT-PCR in the RNA samples of male reproductive tract tissues obtained from 10-60 day old rats. To study the effect of androgens on Tlr expression, epididymides were obtained from rats divided into three groups viz control, castrated and castrated + testosterone supplemented. Majority of the TLRs were abundantly expressed in all the three regions of the epididymis, testis and seminal vesicle suggesting the existence of robust innate immune machinery in the male reproductive tract. No age specific pattern of expression of TLRs was observed in 10-60 day old rat epididymides, testes and seminal vesicle. Since androgens regulate gene expression in the male reproductive tract, TLR expression was studied in androgen ablated rats. No appreciable change in TLR expression was observed in castrated rats when compared to the control and testosterone replaced animals. Results of this study demonstrate that Tlr expression in the male reproductive tract is abundant and is not under androgen regulation suggesting the constitutive expression of these receptors, thereby providing this organ system the ability to initiate immediate immune response to an microbial attack.

AP 28: Effect of Cypermerithrin on Protein metabolism in selected fish, Heteropneuses fossils

Y Savithri, L Madhavi Latha, ISR Rajeswari, P Sreelatha and P Jacob Doss, Dept of Zoology, SV University, Tirupati 517 502, AP.

Effect of cypermethrin on total protein content shows decrement in all the tissues of fish, suggesting that existence of high protein hydrolysis. Protease levels in all the tissues of experimental fish have shown uniform increase envisaging the deragement of structural integrity of the tissues. Free amino acid content in the tissues increase of significantly demonstrating elevated hydrolysis of proteins. Ammonia levels elevated significantly in all tissues indicating catabolism of proteins other nitrogen compounds. The increase of urea corroborates effective detoxification of ammonia by converting highly toxic ammonia to less toxic urea.

AP 29

The enzyme pteridine reductase 1 (PTR1) as drug target for antileishmanial drug screening

Neeloo Singh,

DTDD Division, Central Drug Research Institute, Lucknow.

Email: Neeloo888@vahoo.com

The chemotherapy for control of the parasitic disease, leishmaniasis, is limited. The parasite has developed resistance to the previously cost effective pentavalent antimonial drug. The new drugs in the market are expensive and have side effects. Therefore, investigators are still in progress to discover new antileishmanial compounds. The enzyme pteridine reductase 1 (PTR1) of Leishmania donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR), therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant PTR1 isolated from a clinical isolate of Leishmania donovani, we carried out molecular modeling and docking studies to identify inhibitors for antileishmanial drug screening. A Green Screen Assay developed by us enabled the identification of inhibitors showing promising antileishmanial activity both in vitro and in vivo. The mechanism of leishmaniacidal activity of these inhibitors was also determined.

The traditional chemistry based approach of finding new candidate molecules from known active compounds or natural products has driven antiparasitic drug discovery in the past. Recently, the drug discovery strategy has employed the seeking of specific inhibitors for known biological drug targets with the aid of protein crystal structures and/or high throughput screening assays. The high throughput screening assay for antileishmanials along with the molecular modeling and docking details provided in this work identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value for this parasitic disease.

AP 30

EFFECT OF ZINC ON AMINERGIC SYSTEM IN RAT BRAIN

K.Yellamma, M. Vijaya Kumar and B.Nirmala Kumari**

Department of Zoology, S.V.University College of Biological & Earth Sciences, Tirupati, Chitto or (Dt), Andhra Pradesh, India

• *Email: nirmala_svu@yahoo.co.in

Zinc is a heavy metal which belongs to IIb, IV group in the periodic table. Zinc is known to act as neuro-protective agent and also as a modulator of the excitatory and inhibitory transmitters. Zinc has also been closely associated with various neurological diseases such as Epilepsy, Pick's disease, Alzheimer's diseases etc. The present study was aimed to examine the phenomenon of heavy metal (ZnCh) toxicity and the accompanying changes in aminergic system and the associated enzyme (MAO) in different areas of the rat brain. Male albino rat, Rattus norvegicus was selected as the experimental model and the heavy metal salt, Zinc chloride (ZnCh) was chosen as the toxic chemical. Distilled water is used as the Vehicle for administering Zinc chloride. The LDso was determined as per probit method. For acute dose studies, rats were administered with lethal dose of Zinc chloride only one time and for chronic dose studies, the rats were administered with sub lethal doses of Znch once in a day for 90 day. The animals were sacrificed after treatment and different brain regions such as Olfactory lobe, Hippocampus, Cerebellum and Ponsmedulla of the rat were isolated and used to estimate the activity levels of monoamines such as Epinephrine (EP), Norepinephrine (NEP), Dopamine (DA), Serotonin (5-HT) and Monoamine oxidase (MAO) as per standard methods.

The observations in this present study revealed that the activity levels of all monoamine neurotransmitters were increased during acute and chronic dose exposures to Zinc chloride. But they were elevated differentially in different brain areas showing the area specificity effect of Zinc chloride. The rats treated with acute dose of Zinc chloride for 24h, showed significant accumulation of Epinephrine, Norepinephrine, Dopamine and Serotonin was observed from 3h onwards. In all brain areas onset of recovery process at the end of 24h was evident. In the contrary, inhibition of MAO was observed from 3h onwards with maximum inhibition at 24h in all brain arias. In chronic dose exposures, a progressive increase in the levels of EP, NEP, DA and 5-HT was noticed till 30^th day in all the areas. But in case of Serotonin, a slight depletion was noticed on 10^th day following Zinc chloride treatment and thereafter its level was elevated in a progressive manner till 30^th day. From 60^th day onwards a slight recovery was observed and it was more pronounced on 90^th day. Similarly, MAG registered insignificant inhibition under acute exposures and significant inhibition under chronic exposures in all regions of the brain at all time intervals. The results were discussed in the light of recent literature.

AP 31

ALTERATIONS OF DETOXIFICATION MECHANISM UNDER THE SUBLETHAL EXPOSURE OF CHLORPYRIFOS IN ALBINO RATS

S. Rajendra Prasad, P. Ravi Sekhar, S. Siraj Mohiyuddin, M .Siva Prasad,

K. Ramesh Babu and P. Jacob Doss

Department of Zoology, S.V.University, Tirupati-517 502, A.P., India.

rajendraprasad@gmail.com

The activity levels of XOD (Xanthine Oxidase), SOD (Super oxide dismutase) and catalase were assayed in different brain regions (cerebral cortex, hippocampus, cerebellum and medulla oblongata) of albino rat, during the exposure of sub lethal concentration of chlorpyrifos
(20 mg/kg) as single, double and multiple doses with an interval of 48 hrs. The results indicate a steady decrease in SOD and catalase activity with concomitant increase in XOD activity. The increase XOD activity indicated the over production of super oxide anions () in the brain regions of albino rat in response to chlorpyrifos, decreased SOD and catalase activity levels indicate their depleted activities under toxic stress condition in different brain regions.

AP 32: Identification of Substrates of Cyclin Dependant Kinases from Leishmania donovani.

Alakananda Goswami (Nag), Anup Kumar Maity, Manoshi Gayen*, Samiran Saha and Partha Saha

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata

Haldia Institute of Technology, Haldia, West Bengal

E-mail: alakananda.goswami@saha.ac.in

The cyclin-dependant protein kinases (Cdk) are the key regulators of eucaryotic cell cycle progression. Cdks control the cell cycle by phosphorylating many specific protein substrates involved in DNA replication, checkpoint pathways and mitosis. Like the higher eucaryotes, number of Cdk related kinases (CRKs) have their role in regulating the cell cycle progression of the protozoa Leishmania. A cyclin-CRK complex, viz., LdCyc1-CRK3 from L. donovani, the causative agent of Kala-azar, has been implicated in regulation of S-phase related activities. Since the conserved substrate-docking motif is present in LdCyc1, it is expected that the protein will interact with target molecules containing RRLFG-like Cy motif. So the protein molecules containing Cy motif and Cdk phosphorylation site may interact with LdCyc1-CRK3 complex and act as substrates. Based on such hypothesis, we have identified the putative open reading frames (ORFs) in the genomic sequence of Leishmania major that contain the Cy-motif and the Cdk phosphorylation site (S/T)PX(K/R) in order to identify the potential substrates of the kinase complex. We have selected 28 putative ORFs from L. major genomic database containing both Cy motif and Cdk phosphorylation site and successfully amplified six of them for protein expression in vitro. Finally, three proteins could be expressed in vitro by wheat germ based coupled transcription and translation reaction and all of them could be successfully phosphorylated in vitro by active LdCyc1-CRK3 complex. Further characterization of the identified substrates will be discussed during presentation.

Biochemistry

Department of Biochemistry, VRIPS, Nellore, AP.

BC Invited 1

Nitrosative stress response in yeasts.

Dr. Sanjay Ghosh

Department of Biochemistry

Calcutta University

Kolkata

Increasing number of evidences suggest that reactive nitrogen species (RNSs) and nitric oxide (NO) itself affect the cellular redox status like oxidative stress and modify cellular proteins reversibly or irreversibly. Thus a hostile environment is created which is termed as “nitrosative stress”. Yeasts are very good model for studying the effect of nitrosative stress on eukaryotic cells. In the present study, effects of different RNS donors at sub toxic doses on the cellular growth, glutathione status and enzymes like Glutathione Reductase (GR), Glutathione Peroxidase (GPx) related to glutathione redox cycle were examined. Cellular glutathione status showed differential sensitivity towards different RNSs in yeasts. The inhibition of GR activity by peroxynitrite both in vivo and under in vitro conditions was found due to the nitro tyrosine formation. GPx activity was inhibited only by NO in vivo but not in presence of peroxynitrite. Cellular protective measures against RNS have also been investigated. It reveals that Pap1 and Yap1, two known oxidative stress responsive transcription factors, may play some role in nitrosative stress response, too, in S. pombe and S. cerevisiae respectively. Cytosolic catalase gene expression was found to be induced following peroxynitrite challenge only in flavohemoglobin and GSNO reductase deleted strains of S. cerevisiae. Catalase also exhibited peroxynitrite reductase activity. Yeast glyoxalase-I and Glyceraldehyde-3-Phosphate Dehydrogenase were inhibited by GSNO. Interestingly, glyoxalase-I inhibition by GSNO was competitive in nature.

BC Invited 2

Heat Shock Protein 90 as Potential Drug Target Against Malaria

Utpal Tatu, Department of Biochemistry, Indian Institute of Science, Bangalore

My laboratory is interested in understanding roles of molecular chaperones in acclimatization of malarial parasite in human host. Over the years my laboratory has carried our extensive characterization of parasite coded heat shock proteins of the class PfHsp100, PfHsp90, PfHsp70, PfHsp60 as well as PfHsp40. In addition to cloning their genes we have studied their expression, localization, complexes and also functions in some cases. Using pharmacological inhibitors of Hsp90 we have shown a critical role played by PfHsp90 in growth of malarial parasite in human erythrocytes. Our results suggest PfHsp90 to be a potential drug target against malaria and inhibitors specific to Hsp90 as candidate anti-malarial drugs. To better appreciate the functions of PfHsp90 and other chaperones classes we have recently initiated a systematic analysis of chaperone complexes and their networks in parasite infected erythrocytes. The approach incorporates cell biological, bioinformatics as well as proteomic analysis of molecular chaperones in parasite infected erythocytes. The study will reveal critical processes regulated by parasite heat shock proteins, particularly PfHsp90 and uncover cellular events orchestrated by this important chaperone.

Relevant publications:

Systems analysis of Chaperone network in Plasmodium falciparum.

Pavithra, S, R., Kumar, R., and Tatu, U. PLoS Comp. Biol. Vol 3 (9) Sept (2007)

Chaperoning a cellular upheaval in malaria: Heat shock proteins in

Plasmodium falciparum. Acharya, P., Kumar, R., and Tatu, U.

Mol. Biochem. Parasitol. (2007) Jun; 153(2):85-94.

“Plasmo2D”: An ancillary proteomic tool to aid identification of proteins from Plasmodium falciparum. Khachane, A., Kumar, R.,, Jain, S., Banumathy, G., Singh, V., Nagpal, S., and Tatu, U. J. Proteome Res. (2005) 4, 2369-74.

ProteoMod: A new tool to quantitate protein post-translational modifications. Kumar, Y., Khachane, A., Belwal, M., Das, S., Somsundaram, K., and Tatu U.Proteomics, (2004) 4, (6): 1672-1683

Recurrent Fever Promotes Plasmodium falciparum Development in Human Erythrocytes. Pavithra, S R., Banumathy, G., Joy, O., Singh, V., and Tatu, U. J. Biol. Chem. (2004) 279 (45), 46692–46699.

Heat Shock Protein 90 Function Is Essential for Plasmodium falciparum Growth in Human Erythrocytes. Banumathy, G., Singh, V., Pavithra, S R., and Tatu, U. J. Biol. Chem. (2003) 278 (20), 18336–18345.

Invited BC 3

Plasmodium falciparum helicase 45 expressed during intraerythrocytic developmental cycle is essential for parasite growth

Renu Tuteja, Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067 email: renu@icgeb.res.in

Helicases are ubiquitous molecular motor proteins, which play important role in the metabolism of nucleic acids. Human malaria parasite Plasmodium falciparum contains a number of DEAD-box helicases. The gene encoding one of this helicase was cloned from Plasmodium falciparum. The polypeptide of 398 amino acids has a molecular mass of 45 kDa, contains striking homology to eukaryotic translation initiation factor 4A (eIF4A) and all the conserved domains of the DEAD-box family. The recombinantly expressed and homogeneously pure P. falciparum protein (PfH45) contains ATP-dependent DNA and RNA helicase, ATPase and ATP-binding activities. The deletion mutant studies show that the ssDNA-dependent ATPase activity resides in the N-terminal one third of the protein. PfH45 is phosphorylated by protein kinase C at Ser and Thr residues and this phosphorylation stimulated its DNA helicase and ssDNA-dependent ATPase activities. PfH45 is a unique bipolar helicase that contains both the 3’ to 5’ and 5’ to 3’ directional helicase activities and anti-PfH45 antibodies immunodeplete all its activities. PfH45 is expressed in all the intraerythrocytic developmental stages of the parasite and has a role in translation. The studies further indicate that the parasite cultures treated with PfH45 dsRNA or purified immunoglobulins against PfH45 exhibited ~60% and ~55% growth inhibition, respectively. This inhibitory effect was due to interference with expression of the cognate messenger and down-regulation of synthesis of PfH45 protein in the parasite culture and was associated with morphologic deformation of the parasite. These studies indicate that PfH45 is indispensable enzyme that is essential for growth and probably survival of P. falciparum.

References:

1.Tuteja, R. Helicases: feasible anti-malarial drug target for Plasmodium falciparum (2007) FEBS J. (in press)

2. Tuteja, R. Malaria-An overview (2007) FEBS J. (in press)

3. Tuteja, R. Malaria- The global disease (2007) FEBS J. (in press)

4. Pradhan, A and Tuteja, R. Bipolar, dual Plasmodium falciparum helicase 45 expressed during intraerythrocytic developmental cycle is required for parasite growth. (2007) J. Molecular Biology (in press)

5. Tuteja, R. and Pradhan, A. Unraveling the 'DEAD-box' helicases of Plasmodium falciparum. Gene, (2006), 376, 1-12.

6. Pradhan, A. and Tuteja, R. Plasmodium falciparum DNA helicase 60: dsRNA- and antibody mediated inhibition of the malaria parasite growth and down regulation of its enzyme activities by DNA-interacting compounds. FEBS J., (2006), 273, 3545-3556.

7. Tuteja, N., Tuteja, R. Helicases as molecular motors: an insight. 2006 Physica A, 372, 70-83.

8. Pradhan, A., Chauhan, V. S. and Tuteja, R. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. (2005), 144, 133-141.

9. Pradhan, A., Chauhan, V. S. and Tuteja, R. A novel ‘DEAD-box’ DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. (2005), 140, 55-60.

10. Tuteja, N. & Tuteja, R. DNA helicases: the long unwinding road. Nature Genet. 13, 11-12, 1996.

IT BC

Flavin Biosynthesis: Modeling of Riboflavin Kinase and FAD-Synthetase for Site-directed Mutagenesis

Srinivas Jayanthi, Nirupama Puvvada and Sobhanaditya Jonnalagadda

Department of Biochemistry, Osmania Univeristy, Hyderabad-7

In cells riboflavin is converted to active cofactors (FMN and FAD) by riboflavin kinase (EC:2.7.1.26), which phosphorylates riboflavin to FMN, and by FAD synthetase (EC:2.7.7.2), which adenylylates FMN to FAD. While most prokaryotes have a single bifunctional protein that carries out both activities, eukaryotes have two separate enzymes. Eukaryotic monofunctional riboflavin kinase is orthologous to the bifunctional prokaryotic enzyme. The N-terminal region of the bacterial bifunctional enzyme is similar to nucleotidyl transferases, hence, may be involved in the adenylylation reaction. The C-terminal portion of the bacterial bifunctional enzyme resembles the eukaryotic riboflavin kinase and may catalyze the kinase reaction. However, the eukaryotic FAD synthetase differs from its prokaryotic counterpart, and resembles a PAPS-reductase.

Homology modeling, using human enzymes as templates, of both riboflavin kinase and FAD synthetase from Neurosporsa crassa highlighted conserved residues that may be involved in catalysis. Most residues in the Neurospora crassa riboflavin kinase can be folded to an ordered minimum structure except for the unique stretch of poly-Q rich segment. The stretch is not represented in a current hypothetical model. The invariant residues Asn36 and Glu86 of riboflavin kinase are implicated in catalysis, the kinase follows an ordered sequential mechanism. Replacement of E158A and N48S residues resulted in a less active enzyme however, replacements by charge conservation E158D and N48V resulted in the restoration of enzyme activity. Studies to ascertain the invariant residues for FAD Synthetase are required for substrate binding and for product catalysis are in progress. Presumably Neurosporsa crassa is very close to the starting point of functional divergence of riboflavin kinase and FAD synthetase activities into separate polypetides. The presence of a unique poly-Q stretch may be responsible for protein-protein interactions of riboflavin kinase and FAD synthetase that might bring the enzymes physically close to each other though they are functionally separate.

BC 1

Enzymatic syntheses of phenolic glycosides

R. Sivakumar^*, B. Manohar^†, S. Divakar^*

^*Fermentation Technology and Bioengineering,

^†Food Engineering,

Central Food Technological Research Institute, Mysore-570020.

Enzymatic syntheses of glycosides present a better option to chemical synthesis. The present work is focussed on the syntheses of dopa 1, dopamine 2 and curcuminyl 3 glycosides through enzymatic method using amyloglucosidase from Rhizopus mold and b-glucosidase isolated from sweet almond in organic media. Glycosylation of the above mentioned aglycon were attempted with D-glucose 4, D-galactose 5, D-mannose 6, D-fructose 7, D-ribose 8, D-arabinose 9, maltose 10, lactose 11, sucrose 12, D-sorbitol 13 and D-mannitol 14. The reactions were optimized in terms of incubation period, pH, buffer, enzyme and substrate concentrations. Among the glycosides prepared, dopa-D-glucoside 15 (62%), dopamine-D-glucoside 16 (65%) and curcuminyl-D-glucoside 17 (44%) gave the best yields compared to the other carbohydrates employed. The glycosides were analyzed by HPLC, separated through Sephadex G15 column chromatography and characterized by UV, IR, mass spectroscopy, optical rotation and 2D HSQCT NMR spectroscopy. Glycosylation occurred at the phenolic OH group with the anomeric C1 a-, b- and/or C6 OH positions of the carbohydrates employed. The results from these investigations will be presented in detail.

BC 2

Enzymatic syntheses of pyridoxine and ergocalciferol glycosides

R. Einstein Charles^*, B. Manohar^†, S. Divakar^*

^*Fermentation Technology and Bioengineering,

^†Food Engineering,

Central Food Technological Research Institute, Mysore-570020.

Glycosylation of vitamins improves the pharmacokinetic properties besides imparting water solubility, heat and light stability to vitamins. Enzymatic glycosylation of vitamin B₆ (Pyridoxine) and vitamin D₂ (Ergocalciferol) were carried out using amyloglucosidase from Rhizopus mold and β-glucosidase from sweet almond in diisopropyl ether non-polar medium. The carbohydrates employed for the glycosylation were D-glucose, D-galactose, D-mannose, D-fructose, D-arabinose, D-ribose, lactose, maltose, sucrose, D-mannitol and D-sorbitol. The reactions were optimized in terms of incubation period, pH, buffer, enzyme and substrate concentrations. The reactions were monitored by HPLC, the products separated by Sephadex G15 column chromatography and characterized by UV, IR, mass spectroscopy, optical rotation and 2D HSQCT NMR spectroscopy. Glycosylation occurred at the phenolic OH of ergocalciferol and primary OH of pyridoxine with C1 α and β of the carbohydrate. Yields in the range 5-42% were obtained. Glycosides of vitamin B₆ were found to be more stable and those of vitamin D₂ water-soluble. The results from these investigations will be presented in detail.

BC 3

Enzymatic syntheses of thiamin and α-tocopherol glycosides

T. Ponrasu^*, B. Manohar^†, S. Divakar^*

^*Fermentation Technology and Bioengineering,

^†Food Engineering,

Central Food Technological Research Institute, Mysore-570020.

Glycosylation of vitamins imparts water solubility, heat and light stability and improves their pharmacokinetic properties. Enzymatic glycosylation of vitamin B₁ (thiamin) and vitamin E (α-tocopherol) were prepared using amyloglucosidase from Rhizopus mold and β-glucosidase from sweet almond in diisopropyl ether nonpolar medium. The carbohydrates employed for the glycosylation were D-glucose, D-galactose, D-mannose, D-fructose, D-arabinose, D-ribose, lactose, maltose, sucrose, D-mannitol and D-sorbitol. The reactions were optimized in terms of incubation period, pH, buffer, enzyme and substrate concentrations. The reactions were monitored by HPLC, the products separated by Sephadex G15 column chromatography and characterized by UV, IR, mass spectroscopy, optical rotation and 2D HSQCT NMR spectroscopy. Glycosylation occurred at the phenolic OH position of α-tocopherol and the primary OH of thiamin with C1 α and β of the carbohydrate. Yields in the range 11-23% were obtained. Glycosides of vitamin E were found to be water soluble and those of thiamin glycosides were stable with an acceptable taste. The results from these investigations will be presented in detail.

BC 4

Microbial amino acid oxidases for production of optically pure amino acids.

Susmita Singh*, B.K.Gogoi, R.L.Bezbaruah.

Division of Biotechnology, North East Institute of Science & Technology (formerly Regional Research Laboratory), Jorhat- 785006, Assam, India.

*email- sushmitazone@yahoo.co.in

A large number of microbial strains available in the repository of North East Institute of Science & Technology, Jorhat were grown for amino acid oxidases. Among them, fungal strain P-13 showed the highest activity. The physico-chemical characteristics such as temperature, pH of the growth medium, various carbon and nitrogen sources, metal ions were optimized. Effect of inducer concentrations, cell permeabilization by various chemicals, effect of surfactants and their concentrations on enzyme release from cells and substrate specificity were studied. The preliminary study showed the formation of optically pure amino acids. This result will be further confirmed by HPLC, using chiral column.

BC 5

Characterization of a thrombin-like, pro-coagulant serine protease from venom of Indian Russell’s viper (Daboia russelli) and some medical application of the enzyme

ASHIS K. MUKHERJEE

Department of Molecular Biology and Biotechnology

Tezpur University, Tezpur-784 028, Assam, India

Email: akm@tezu.ernet.in

Russell’s viper envenomation is a serious problem in South East Asia particularly in the rural tropics. Russell’s viper venom contains a variety of proteolytic enzymes affecting the coagulation process of the victims. One of theses enzymes is a serine protease which resembles in part thrombin, a multifunctional protease that plays a key role in coagulation. These enzymes, known as snake venom thrombin-like enzymes (SVTLEs) are directly involved in the envenomation process with a range of life-threatening activities. Because of the potential therapeutic application of SVTLEs in myocardial infraction, ischaemic stroke and thrombic diseases; during the recent years, there is a tremendous interest for the isolation, purification and characterization of novel SVTLEs.

In the present study, a thrombin-like serine protease was isolated from the venom of Daboia russelli by a combination of ion-exchange, gel filtration and reverse-phase HPLC techniques. SDS-PAGE analysis shows subunit molecular mass of RVBCMP is about 28 kDa and it appears to consist of a single polypeptide chain. The enzyme displayed caseinolytic (protease) and fibrinogenolytic activities and specifically cleaves the Aα chain of fibrinogen releasing the fibrinopeptide A. In in vitro condition, the enzyme displayed dose-dependent coagulation activity and was able to convert soluble fibrinogen from bovine plasma to insoluble fibrin clot demonstrating the thrombin like nature of this enzyme. Serine-protease inhibitors such as PMSF and DIFP at a concentration of 2.0 mM completely abolished the coagulant as well as protease activities of this enzyme which shows these two activities are correlated and this enzyme is a serine protease. On the other hand, metal ion chelator EDTA did not inhibit the protease activity reinforcing the serine protease nature of this enzyme. Inhibition of this enzyme activity by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure.

The enzyme showed stability at 4 ºC and post storage of enzyme at this temperature for 14 days, only 14 % activity was lost. Stabilization of enzyme activity can be enhanced if 0.5 mM concentration of Ca²⁺ ion is added in the enzyme preparation, which may presumably by preventing the autolysis of enzyme or by some other mechanism that enhances the stability. Since this pro-coagulant enzyme was not inhibited by heparin, the enzyme can be used as a research tool in quantitative determination of Fg, specially in the plasma of patients under heparin treatment as well as in the coagulation laboratory for routine assays of coagulation factors.

BC 6

Identification, purification and some properties of a protein inhibitor of glyceraldehyde-3-phosphate dehydrogenase

Amrita Roy, Manju Ray, Subhankar Ray

Department of Biological Chemistry, Indian Association for the Cultivation of Science,

Kolkata 700 032, INDIA; emailamrita_roy242000@yahoo.com

A 90,000 molecular weight protein, which is a strong inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been purified from mouse skeletal muscle. It consists of two uneven subunits of molecular weight 70,000 and 20,000. The presence of this protein has also been detected in Ehrlich ascites carcinoma cells (EAC) and in sarcoma tissue of mice, developed either by carcinogen (3-methylcholanthrene) or existing sarcoma cells (Sarcoma-180). The inhibitor is equally effective in inhibiting GAPDH of either normal or malignant cells. The inhibitor, a cytosolic protein does not irreversibly inactivate GAPDH. The inhibitory activity increases with increase in pH and maximum inhibitory activity is at pH 9.0. Increasing the concentration of glyceraldehyde-3-phosphate (GAP), a substrate of GAPDH significantly diminishes the inhibitory potency of the protein. However the other substrates NAD and phosphate have no such effect. A protein inhibiting the catalytic activity of GAPDH is hitherto unknown. So if this inhibitor is ubiquitously present then it will generate new insight in the control of glycolysis i

BC 7

Role of the aromatic interactions in mediating the activity of a viral serine protease domain

Smita Nair*, P. Gayathri^# M.R. N Murthy^# and H.S. Savithri*.

Dept of Biochemistry*, Molecular Biophysics Unit^#, Indian Institute of Science, Bangalore, India. nairsmita@biochem.iisc.ernet.in

Sesbania mosaic virus is a positive sense RNA virus that infects Sesbania grandiflora. The central part of the genome codes for two polyproteins of which one is expressed by ribosomal frame shifting. The N-terminal of both the polyproteins has the serine protease domain followed by VPg. The C-terminus differs by the presence of a RNA dependent RNA polymerase domain in polyprotein2ab and two domains, of sizes 10 and 8 kDa respectively of unknown functions in polyprotein 2a. It has already been shown that the serine protease catalyses the polyprotein processing and that the protease is inactive by itself in trans but active when expressed as a fusion protein with the VPg domain. The residue W43 of VPg was shown to mediate aromatic stacking interaction with the protease domain thereby making it active. These aromatic interactions were revealed by the presence of a positive circular dichroism peak at 230nm. In the current study, the role of the residues present in the protease domain that are involved in the aromatic interaction were identified and mutational analysis was carried to understand the importance of such interactions in the activation of the protease domain.

The crystal structure of the protease domain reveals a stretch of stacked aromatic residues F269, W271, Y315 and Y319 that are exposed to the solvent. To identify the residues involved in stacking interaction with VPg, site directed mutants of these residues were generated. Of these Y315A and Y319A mutants did not affect the cis or trans activity of the protease domain in protease-VPg fusion protein, suggesting that these were not involved in the aromatic interactions. The F269A mutant showed both, the loss in cis and trans activity as well as the loss in the positive CD peak at 230nm. The W271 was mutated to F and to A. Of these the F mutant was partially active and retained the 230nm positive peak whereas the A mutant showed complete loss of activity and the positive peak. The results suggest that both F269 and W271 are involved in stacking interactions with the W43 of VPg. In the vicinity of W271, is H275, the side chain of which faces the substrate binding pocket and H-bonds with H298. H298 is in turn H-bonded to the Glu of the substrate. Thus, it was proposed that the H275 could be essential in maintaining the negative charge of the substrate. The H275A mutant protease was completely inactive. But the CD spectra of this mutant surprisingly showed the absence of positive peak at 230nm also. This result suggests that unlike in the protease domain where H275 is not a part of the aromatic stack, in the protease-VPg fusion protein this histidine is a part of the aromatic stack along with F269, W271 and W43 and hence modulates the activity of protease.

BC 8

Phenanthrene Degradation: Metabolism of 1-Hydroxy-2-naphthoic acid by 1-Hydroxy-2-naphthoic acid hydroxylase

Jaigeeth Deveryshetty and P S Phale

Biotechnology Group

School of Biosciences and Bioengineering

IIT Bombay, Powai, Mumbai

jaigeethd@yahoo.co.in

In bacteria, phenanthrene is metabolized via 1-hydroxy-2-naphthoic acid, which is further degraded either via o-phthalate (Phthalate pathway) or via 1,2-dihydroxynaphthalene (naphthalene pathway). Oxidation of 1-hydroxy-2-naphthoic acid to 1,2-dihydroxynaphthalene is catalyzed by a 1-hydroxy-2-naphthoic acid monooxygenase. Balashova et. al. suggested that conversion of 1-hydroxy-2-naphthoic acid to 1,2-dihydroxynaphthalene is catalyzed by salicylate 1-monooxygenase. This study together with previous studies suggested that the enzymes of naphthalene pathway are sufficient to catalyze mineralization of phenanthrene. Alcaligenes strain PPH isolated from soil sample by enrichment technique utilizes phenanthrene as the sole source of carbon and energy via naphthalene pathway but failed to grow on naphthalene. Identification of metabolites from the spent media, biotransformation studies, whole-cell O₂ uptake and enzyme activity studies suggest that phenanthrene is degraded via 1,2-dihydroxynaphthalene, salicylate and catechol. 1-Hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase activities were observed in the cell-free extract. Whereas, the activity profile for both enzymes revealed induction of 1-hydroxy-2-naphthoic acid hydroxylase at 14 h and salicylate hydroxylase at 16 h of growth on phenanthrene. This observation suggests the presence of two different hydroxylases, one involved in the oxidation of 1-hydroxy-2-naphthoic acid to 1,2-dihydroxynaphthalene and other involved in the oxidation of salicylate to catechol.

BC 9

Corticosterone regulates adenosine deaminase of chicken in

an age- and tissue- specific manner

Piyali Bhattacharjee and R. Sharma

Department of Biochemistry, North Eastern Hill University, Shillong-793 022, India.

E-mail: piyali_bh@yahoo.com

Adenosine deaminase (ADA; EC 3.5.4.4), the purine salvage pathway enzyme, catalyses the irreversible hydrolytic deamination of adenosine and 2'- deoxyadenosine to inosine and 2'- deoxyinosine, respectively. In the study undertaken, the aim was to investigate the effect of corticosterone, a glucocorticoid, on the activity of ADA in various tissues of chicken at two different postnatal ages. Corticosterone was injected, intraperitoneally, in two different ages (day 10 and 60) of male chicken of strain BV-380 and its effect on ADA activity was studied in different tissues of GIT (crop, esophagus, proventriculus and small intestine) and spleen. Our studies showed that corticosterone significantly inhibited the ADA activity in all the tissues studied except proventriculus, in an age- and tissue- specific manner. In the crop, corticosterone inhibited the activity at both ages, but the magnitude of inhibition was more pronounced in day 60 (-79%) than day 10 (-56%). The activity of ADA was significantly inhibited by corticosterone in the esophagus, but more at day 60 (-41%) in comparison with day 10 (-23%). In small intestine, the activity was seen to be more reduced at day 60 (-41%) than at day 10 (-36%). In the spleen too, the effect of repression was more at day 60 (-40%) than day 10 (-32%). These results were confirmed by Slot blot analyses using anti-ADA antibody, indicating that the inhibition of activity was indeed at the ADA protein expression level. The studies indicated that the magnitude of ADA activity inhibition was more pronounced in the later stage of development in the GIT and spleen of chicken. The age- and tissue-specific inhibition may be correlated to the differential adaptive role and maturation of corticosterone action mechanism, its receptor and post-receptor events.

BC 10

Ficin from the latex of Ficus carica: purification and thermal stability in presence of cosolvents

K.B. DEVARAJ and V. PRAKASH

Department of Protein Chemistry and Technology

Central Food Technological Research Institute Mysore-570020

E-mail: devkadur@rediffmail.com

Ficin is a proteolytic enzyme isolated from the latex of Ficus trees and this enzyme is known to occur naturally in multiple forms distinguishable by ion-exchange chromatography. A major ficin (EC 3.4.22.3) from the crude preparation of Ficus carica latex is purified by cation-exchange chromatography and characterized. The purified enzyme was homogeneous both in Sodium dodecyle sulphate-PAGE and gelfitration chromatography on FPLC. The enzyme was active at neutral pH showed optimum activity between pH 6.5-7.0. The enzyme is a single polypeptide chain of molecular weight of 23100 ± 500 Da as determined by Matrix Assisted Laser Desorption Ionization Mass Spectroscopy –Time of Flight. The N-terminal sequence determined was L P E V D W A X F G A V N…. aligned with N-terminal sequences of other cysteine proteases. The many residues are conserved at amino-terminal sequences of cysteine proteases. The purified enzyme was completely inhibited by iodoacetamide and sodium tetrathionate confirming cysteine being the anchor amino acid in its active site. The thermal stability of the ficin was determined in presence of cosolvents namely sorbitol, trehalose, sucrose and xylitol. The effectiveness of these cosolvents against thermal denaturation of ficin was found to follow the order of trehalose>sorbitol>sucrose>xylitol as determined by residual activity measurements at 70 °C. In presence of 40% sorbitol and 40% trehalose apparent Tm increases from control value of 72 °C to 83 °C and 85 °C respectively. The presence of cosolvents has no effect on the fluorescence spectra and far-UV CD spectra of ficin. These above results indicate that the cosolvents stabilize the ficin without altering its structure.

BC 11

Polyol induced stabilization of endoglucanase from Aspergillus aculeatus

GAJENDRA.S, PURNIMA KAUL and V.PRAKASH

Department of Protein Chemistry and Technology,

Central Food Technological Research Institute, Mysore-570 020, India.

Email: rohangj@yahoo.com

Fungal cellulases are found in multiple forms varying in sizes and substrate specificity. Aspergillus aculeatus is known to produce complete set of cellulolytic enzymes including endoglucanase as a major component. Endoglucanase is a 45-kDa protein showing higher carboxymethyl cellulose (CMC) activity. Co solvents are known to stabilize the protein structures against thermal denaturation. The effect of various co solvents like glycerol, sorbitol and sucrose on the activity and stability of the endoglucanase have been studied. All the three cosolvents are found to increase the activity of the endoglucanase by about four folds in comparison with the control. Apparent thermal transition temperature studies show increase in the T_m value ranging from 57°C for control to 64.5°C in presence of different cosolvents. Native endoglucanase loses 50 % residual activity at higher temperature (90°C), but in presence of cosolvents the enzyme was able to retain complete activity. The enzyme after removal of the different cosolvents by gel filtration was able to retain only 60 – 70 % of its residual activity. This shows that the effect of cosolvents is reversible to some extent. Far UV-CD studies of the endoglucanase in presence and absence of cosolvents were similar at 25°C. Far UV-CD studies of the native enzyme at 70°C show changes in the structure where b-sheets are reduced and concomitant increase in the random structure. In the presence of cosolvents at 70°C, there was no change in the enzyme structure. This shows that the cosolvents protect the endoglucanase without altering its structural conformation and also has positive influence on the enzyme activity.

BC 12

The significance of amino acid utilisation in acute organophophate poisoining.

Mahalakshmi C, Landge Anil V and Oommen A,

Neurochemistry Laboratory, Christian Medical College, Vellore.

E-mail: mahachandran@yahoo.com

Organophosphate pesticide poisoning is a common method of suicide in south India and a lead cause of death. Organophosphates inhibit acetylcholinesterase leading to an accumulation of acetylcholine at cholinergic synapses in brain and at the neuromuscular junction. The resulting cholinergic stimulation and muscle weakness that occurs soon after poisoning rapidly utilize significant levels of energy. This negative energy balance if not corrected can lead to muscle wasting. Muscle wasting in relation to over use of energy implicates a role for amino acids in gluconeogenesis. It is not known if this occurs in organophosphate poisoning but would be useful to clarify as the morbidity and mortality of hospitalized organophosphate poisoned patients are associated with neuromuscular weakness and muscle wasting.

The aim of this study was to determine amino acid utilization in organophosphate poisoning in relation to muscle degradation.

Rats were subjected to severe monocrotophos poisoning (0.8 LD₅₀) and skeletal muscles isolated from animals sacrificed at 2.5 hours, 24 hours, 7 days, 15 days and 1 month after poisoning. The animals were completely paralyzed 2.5 hours after poisoning but recovered from paralysis within 12 hours. The muscles were assayed for acetylcholinesterase and subjected to SDS-PAGE to determine protein profiles. Plasma amino acids were estimated by cation exchange HPLC. The study was approved by the Institutional Animal Ethics Committee.

Skeletal muscle acetylcholinesterase was significantly inhibited (>80%) when animals were paralyzed. Muscle protein degradation occurred for one week after poisoning. Early in poisoning (2.5 hours) plasma levels of essential and non-essential amino acids (688 ± 113 µM and 576 ± 40 µM respectively) were significantly decreased compared to non poisoned animals (977 ± 189 µM and 985 ± 385 µM. P<>

The significance of amino acid utilization following energy depletion in organophosphate poisoning will be discussed in relation to understanding muscle wasting that occurs in the poisoning.

BC 13

Effect of temperature and pH on stability of porcine pancreatic lipase in presence of polyols and sugars

Gangadhara, and V. Prakash

Department of Protein Chemistry and Technology

Central Food Technological Research Institute

Mysore – 570 020, India

E-Mail: gangu_bn@yahoo.co.in

Porcine pancreatic lipase (triacylglycerol acylhydrolases - E.C. 3.1.1.3) is a key enzyme for the intraluminar digestion of fats and is one of the most widely used enzymes for biotechnological aspects. Pancreatic lipase is a single polypeptide chain of 50 kDa. The effect of polyols and sugars on lipase from porcine pancreas activity, kinetic measurements and stability were studied by comparing the enzymatic activity and conformation of the enzyme at optimum, lower pH and high temperature conditions. The polyols and sugars used (glycerol, xylitol, sorbitol, trehalose and sucrose). At optimum condition these additives didn’t show any change in the activity. But at lower pH and higher temperature these additives were protecting the enzyme in terms of both activity and conformational stability. The fluorescence spectra shows blue shift with increase in relative fluorescence intensity and far UV-CD studies shows these additives protecting the structure of the enzyme with increase in α-helical content. At lower pH (pH 4.0) native enzyme loses 90% of the enzyme activity but it retains 24, 44, 61, 35 & 48% of the activity in 40% glycerol, xylitol, sorbitol, sucrose & trehalose with increase in k_cat values minimizing K_m values. The data support that these additives were very effective in providing protection against loss of activity at higher temperature and at low pH by shifting the denaturation equilibrium towards the native state.

BC 14

Title: Effect of fish oil administration on isolated rat colonocytes

Agnihotri N.*, Sarotra P.*, Singh P.K.*, Aggarwal R.**, Vidhya K.*

*Department of Biochemistry, Panjab University, Chandigarh.

** Department of Pathology, PGIMER, Chandigarh, India.

email:navneetneeraj@hotmail.com

Background –Dietary n-3 polyunsaturated fatty acids acts on diverse physiological processes influencing normal health and chronic disease and are found to be very sensitive to peroxidation. Because the colonic mucosa may be subjected to significant changes after fish oil administration, knowledge of the oxidant defense mechanisms in the colon is of biologic and potential clinical importance.

Aim – The purpose of the present study was to determine the effects of dietary fish oil on the balance between generation of reactive species and the antioxidants in isolated colonocytes.

Method –The animals were divided into two groups. Control group was fed standard pellet diet while the test group was given 1 ml fish oil orally for 21 days in addition to standard pellet diet. Indexes of oxidative stress (lipid peroxidation, nitrite levels) and protective antioxidants (superoxide dismutase, catalase, glutathione-S-transferase and reduced glutathione) were estimated in the isolated colonocytes.

Result – As compared to the control group, the test group showed-

• A significant increase in lipid peroxidation, nitrite levels and in the superoxide dismutase and glutathione-S-transferase activity,
• A significant decrease in catalase activity and
• No significant change in reduced glutathione levels

Conclusion – The results of the present study suggested that the consumption of fish oil altered the balance between prooxidant and antioxidant levels leading to oxidative stress in colonic mucosa.

BC 15

A screen for inhibitors of M tuberculosis Dxs (1-deoxy-D-xylulose-5-phosphate synthase) and Dxr (1-deoxy-D-xylulose-5-phosphate reductoisomerase)

Vaishali Humnabadkar, Ramesh K. Jha, Nuzhat Ghatnekar and Sunita M de Sousa

AstraZeneca India Pvt Ltd, Hebbal, Bellary Rd, Bangalore 560024

email - Vaishali.Humnabadkar@astrazeneca.com

Deoxy-D-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (Dxr), the first two enzymes in the non-mevalonate pathway for isoprenoid synthesis, are good targets for anti-infective drug discovery. The aim was to develop an assay to screen for inhibitors of these enzymes from Mycobacterium tuberculosis (Mtu).

Dxs converts glyceraldehyde-3 phosphate (G-3-P) and pyruvate to 1-deoxy-D-xylulose-5-phosphate (DXP). Dxr reduces and isomerizes DXP to methylerythritol phosphate (MEP) with concomitant oxidation of NADPH. The products of Dxs cannot be monitored directly by absorbance or fluorescence measurements. DXP, the substrate of the Dxr reaction, is both expensive and difficult to synthesize whereas NADPH, the other substrate of the reaction can be monitored spectrophotometrically. Hence, a coupled Dxs-Dxr assay was designed in which the oxidation of NADPH was followed. This coupled assay system allowed us to overcome the disadvantages of each of the individual Dxs/Dxr assays.

Mtu Dxs and Dxr were cloned and expressed in Escherichia coli. Dxs, Dxr, pyruvate, G-3-P and NADPH were incubated in a microtitre plate to allow formation of MEP; the reaction was monitored by the oxidation of NADPH. The quantity of Dxs substrates was chosen carefully, to have a good signal and to select uncompetitive and/or potent competitive inhibitors. Two different assay formats were investigated- a discontinuous and a single step assay.

The biggest challenge was to establish the quantity of Dxr and Dxs in the coupled assay such that the assay remained sensitive to inhibitors of both enzymes. Additional assays were designed to distinguish between Dxs or Dxr inhibitors. The assay was validated by fluoropyruvate and fosmidomycin, known inhibitors of Dxs and Dxr, respectively. These inhibitors showed similar EC₅₀ values in both the coupled assay and the assays for individual enzymes. The interaction of fosmidomycin with Mtu Dxr was further characterised by isothermal calorimetry.

The Dxs-Dxr coupled assay is amenable to a high throughput format and can be used to select inhibitors of two essential enzymes. Inhibitors can be de-convoluted by testing them in the assays for individual enzymes.

BC 17

Prenatal exposure to lindane and adult responsiveness of cerebral and hepatic cytochrome P450s

Johri, A., Dhawan, A., Singh, R.L.¹, Parmar, D.

Developmental Toxicology Division, Industrial Toxicology Research Centre, Lucknow, India; ¹Dr. RML Avadh University, Faizabad, India

Email: ashu_itrc@rediffmail.com

The consequences of prenatal exposure to environmental chemicals could be enormous owing to the limited xenobiotic metabolizing capabilities of the developing fetus and neonate. Studies were initiated to ascertain the effects of prenatal exposure to low-levels of pesticides, such as lindane, an organochlorine insecticide on the responsiveness of xenobiotic metabolizing cytochrome P450s (CYPs) in rat offspring to a later exposure to the CYP inducers, namely phenobarbital (PB) and 3-methylcholanthrene (MC). Prenatal exposure to 0.0625-, or 0.125- or 0.25 mg/kg (1/1400^th or 1/700^th or 1/350^th of LD₅₀) of lindane was found to produce dose dependent alterations in the ontogeny of CYP1A1, 1A2, 2B1, 2B2 and 2E1 in brain and liver of the offspring, as determined by RT-PCR, western blotting and enzymatic studies. Interestingly, the effects were found to persist upto pnd 60. That the imprinted overexpression of cerebral and hepatic CYPs in the prenatally exposed offspring may modulate the inductive responsiveness of these CYPs to a challenge by the CYP inducers of the offspring when young at age was demonstrated by an enhanced responsiveness of cerebral and hepatic CYPs in offspring prenatally exposed to lindane. As demonstrated by RT-PCR, western immunoblot studies and enzymatic assays, a much higher mRNA and protein expression of CYP1A and 2B isoenzymes was observed in animals pretreated with lindane (0.25 mg/kg b. wt., p. o. to mother) during gestation and challenged with MC (30 mg/kg b. wt., i. p. x 5 days) or PB (80 mg/kg b. wt., i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC (30 mg/kg b. wt., i. p. x 5 days) or PB (80 mg/kg b. wt., i. p. x 5 days) alone. Furthermore, when the offspring prenatally exposed to lindane were subsequently challenged with a single dose (30 mg/kg b. wt.) of lindane, it lead to the worsening of the toxicity symptoms. While the onset of convulsions was decreased, the incidence was increased indicating higher vulnerability of the offspring in the prenatally lindane exposed group. Our data indicating the enhanced responsiveness of CYPs during adulthood may assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring could modify the adult response to a later exposure to xenobiotics including drugs many of which are metabolized by this enzyme system.

BC 17

Molecular cloning and in silico analysis of teleostean carbonyl reductase-like 20-hydroxysteroid dehydrogenase promoter motifs

G. Sreenivasulu and B. Senthilkumaran*

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad – 500 046. Presenting author: sreenivasgunti@gmail.com

*author: bsksl@uohyd.ernet.in

Meiotic maturation of prophase-I arrested oocytes is induced by the maturation inducing hormone (MIH). 17-20-dihydroxy-4-pregnen-3-one has been identified as MIH in several teleost fish species. Under the influence of gonadotropins, MIH is synthesized in the ovarian follicle layers from the precursor steroid 17-hydroxy progesterone by the enzyme 20-hydroxysteroid dehydrogenase (20-HSD). Cloning and characterization of 20-HSD cDNAs (E.C 1.1.1.53) demonstrated striking similarity to carbonyl group reducing enzymes that belong to short chain alcohol dehydrogenase/reductase superfamily. Besides their fundamental role in reproduction, 20-HSDs have wide range of substrate specificities towards xenobiotic and bioactive carbonyl compounds. Expression of 20-HSD is primarily regulated by gonadotropins via cAMP pathway. Recently, induction of carbonyl reductase-like 20-HSD by endocrine disrupting chemicals has also been identified. However, factors that regulate the transcription of 20-HSD are largely unknown. Here we report, for the first time, the isolation of upstream elements of carbonyl reductase-like 20-HSD in a siluriform teleost fish and identified the transcription factor binding sites by in silico analysis. A 2.0 kb fragment upstream of 20-HSD was isolated by genome walking approach from the air-breathing catfish, Clarias gariepinus. Transcriptional start site (TSS) was found to be at 59 nt upstream of the translational start point determined by 5’ RACE. In contrast to 5’RACE determined TSS, neural network promoter prediction software indicated TSS around the same position in reverse orientation. Analysis of promoter motifs by core promoter prediction software identified neither the TATA box nor DPE/inr elements. However, a TATA box-like element TAATAAA was observed at -44 position. Analysis of putative transcription factor binding sites by MatInspector and Transcription element search softwares demonstrated motifs for several transcription factors including cAMP and xenobiotic response elements that are present at the proximal and distal ends of promoter. Motifs for androgen/progesterone responsive elements, thyrotropic embryonic factor, CAAT enhancer binding protein, AP-1, SP-1, OCT1, GATA and few other factors involved in cancer were evident. Functional relevance of these elements by deletion mutants/site directed mutagenesis approach will provide insights into the transcriptional regulation of carbonyl reductase-like 20-HSD and helps in delineating its role in reproduction and house keeping function.

BC 19

Over expression of NSs, a suppressor of gene silencing from PBNV(To) and its functional characterization

Lokesh Bhushan, Srisathiyanarayanan. D, and Savithri. H. S

Department of Biochemistry, Indian Institute of Science, Bangalore, India. lokesh@biochem.iisc.ernet.in

Peanut bud necrosis virus (PBNV) belongs to the genus Tospovirus, a unique member of Bunyaviridae, which infects several economically important crops. The virus has three genomic ssRNA segments namely S (ambisense), M (ambisense) and L (negative sense). The S RNA codes for Nucleoprotein (NP) and Non-Structural protein (NSs) from viral complimentary and viral strand respectively. Many viral nonstructural proteins such as NS3 of Hepatitis C virus, Yellow fever virus, Dengue virus, cytoplasmic inclusion protein of Tamarillo mosaic potyvirus and SV40 large antigen of Simian virus are known to exhibit RNA/DNA (nucleic acid) stimulated NTPase, dNTPase and helicase activity. NSs of PBNV does not have any sequence similarity with any of the above mentioned viral RNA/DNA helicases but has a NTP binding domain. However, it has been implicated as suppressor of gene silencing in vivo. With a view to elucidate the mechanism by which NSs could act as a suppressor of gene silencing, the PBNV (To) NSs gene was amplified by RT-PCR, cloned into pRSET C vector and overexpressed in E.coli C43 (DE3) plysS cells. The recombinant NSs protein was purified by Ni-NTA chromatography.

In vitro studies with the purified NSs suggest that it exhibits the RNA stimulated NTPase activity with ATP as a preferred substrate. Optimum temperature and pH for this activity are 25^oC and 7.0 respectively. NTPase activity of rNSs is metal dependent and is inhibited by 5 mM of EDTA as well as an ATP analog. The rNSs could also hydrolyze dNTPs. In addition to NTPase and dNTPase activities, rNSs exhibited the 5’ RNA/DNA phosphatase activity also. We propose that NSs removes the 5’ phosphate from dsRNA, as a result of which Dicer is not able to recognize the dsRNA as a substrate. Hence, PTGS pathway is not triggered upon infection of PBNV.

BC 20

A comparative evaluation of different assay for the diagnosis of Tuberculous meningitis.

KHUSHBOO J. NAGDEV¹, P. S. DESHPANDE¹, R. S. Kashyap¹, H. j. purohit², g.m. taori¹ and H. F. daginawala¹

¹Biochemistry Research Laboratory, Central India Institute of Medical Sciences, 88/2 Bajaj Nagar, Nagpur-440010, India.

²Environmental Genomics Unit, National Environmental Engineering Research Institute, Nehru Marg, Nagpur-440020, India.

khushbu.nagdev@gmail.com

Tuberculous meningitis (TBM) is one of the most common forms of chronic infection of central nervous system. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods which are easy and reliable are required to diagnose TBM at an early stage. In our laboratory for the rapid detection of Mycobacterium tuberculosis in the CSF samples, we are routinely doing various assays viz. indirect Enzyme-Linked Immunosorbent Assay(ELISA) to detect antigen 85 (Ag85) complex, Adenosine deaminase (ADA) Spectrophotometric assay and Polymerase chain reaction (PCR) to detect a repeated insertion sequence IS6110 (Genei M.tuberculosis amplification kit). CSF samples from patients with clinical diagnosis of TBM were taken. All the above three tests were conducted according to standard procedures. A comparative evaluation was performed to find the sensitivity and specificity of the above mentioned three tests. The sensitivity of the individual test i.e for PCR, ELISA, ADA was 70.58%, 82.35%, 64.70% and specificity 75%, 87.5%, 79% respectively .Thus ELISA, PCR and ADA assays in CSF appear to be reliable and rapid diagnostic tools for the diagnosis of TBM. However, when all the three tests are used, they show higher sensitivity and specificity as compared to any single test for the diagnosis of TBM.

BC 21

Diagnostic application of CIIMS In house developed ELISA kit for pulmonary and extrapulmonary tuberculosis.

Sonali s. Ramteke¹, R. S. Kashyap¹, H. j. purohit², g.m. taori¹ and H. F. daginawala¹

¹Biochemistry Research Laboratory, Central India Institute of Medical Sciences, 88/2 Bajaj Nagar, Nagpur-440010, India.

²Environmental Genomics Unit, National Environmental Engineering Research Institute, Nehru Marg, Nagpur-440020, India.

sonaliramteke8@yahoo.co.in

In countries like India, most of the diagnostic tests available for tuberculosis (TB) are costly, time consuming and laborious. In our laboratory, we have developed simple and rapid in-house ELISA method for the demonstration of antigen 85 (Ag 85) complex for the diagnosis of TBM, which was identified in cerebrospinal fluid of TBM patients and characterized previously in our laboratory. Body fluids (serum, CSF, urine, ascitic, pleural and synovial fluids) were collected from confirmed, /suspected TB patients with age and sex matched control groups and subjected to indirect in-house ELISA based method using monoclonal antibodies (mAb) against the purified Ag 85 complex. Overall the ELISA method yielded 85% sensitivity and 90% specificity for the diagnosis of TB using mAb to Ag 85 complex antigen. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option which is rapid, sensitive, cost effective and simple to perform as compared to other costly methods.

BC 22

BENEFICIAL EFFECTS OF MATURE COCONUT WATER ON ALLOXAN INDUCED DIABETES RATS

Preetha P.P. Girija Devi V & T. Rajamohan

Department of Biochemistry, University of Kerala; Kariavatom, Thiruvananthapuram-695 581

e-mail: trmohan@sancharnet.in

The present study was conducted to evaluate the beneficial effect of mature coconut water on serum glucose lipids and lipid peroxides in serum, pancreas and liver and hepatic antioxidant enzymes in alloxan induced diabetes rats. In addition the histopathology of pancreatic β-cells was studied. Male albino rats weighing 100-120 gm were divided into three groups (8 rats each) and fed as follows, group I-control, group II-diabetes and group III-diabetes + mature coconut water treated rats. Diabetes was induced by a single intraperitonial injection of alloxan at a dose of 150 mg/Kg body weight. Mature coconut water was fed by gavage tube to rats (4 ml/100 gm body weight) for 45 days. Body weight and glucose levels were recorded weekly. At the end of the experiment the levels of serum glucose, lipids, lipid peroxides and activities of antioxidant enzymes were investigated.

Histopathology method

In alloxan induced diabetes rats, increased levels of total cholesterol, LDL cholesterol, triglycerides, TBARS and serum glucose were observed. Activities of reduced glutathione and antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase were decreased significantly. HDL cholesterol was significantly decreased in diabetes rats. On the other hand rats treated with mature coconut water showed significant decrease in serum glucose, TBARS, total cholesterol, LDL cholesterol, triglycerides and increase in HDL cholesterol. Significant increase in the level of reduced glutathione and increased activities of antioxidant enzymes viz: catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase were observed in this group. Histpathological studies showed that the β-cells were destroyed and shrunkenness of islets in diabetes rats, whereas large islets and regeneration of islets of ducts were seen in the mature coconut treated rats. The results suggest that mature coconut water has significant beneficial effect in alloxan induced diabetes rats.

BC 23

Identification of an essential Histidine Residue for EcoP15I DNA Methyltransferase activity as probed by chemical modification and site-directed mutagenesis

N. Madhu, Arathi S and Prashanth S Jois

Department of Biochemistry, Indian institute of science, Bangalore, 

madhu_n@biochem.iisc.ernet.in

DNA methylation is catalysed by a diverse group of enzymes that are uniformly dependent on S-adenosyl-L-methionine (AdoMet) as a methyl group donor. EcoP15I DNA methyltransferase (M.EcoP15I) transfers methyl group from AdoMet to the second adenine in the recognition sequence 5'-CAGCAG-3'. To achieve catalysis, the enzyme requires magnesium ion. Fenton chemistry affinity cleavage assay revealed that magnesium ion binding like motif to amino acids 355-377 is important for the critical for positioning of magnesium ions.

We have investigated the role of histidines in catalysis of EcoP15I DNA methyltransferase. EcoP15I DNA MTase, when incubated with diethylpyrocarbonate (DEPC), a histidine-specific reagent, shows a time and concentration -dependent inactivation to methylate DNA containing its recognition sequence. UV absorption spectrometer showed the loss of enzyme activity was accompanied with an increase in absorbance at 240 nm. A difference in spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that the enzyme preincubation with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the fifteen histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of histidine 335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases as evident from CD spectra, native-polyacrylamide gel electrophoresis and gel filtration chromatography. Substitution of His with Ala at position 335 results in a mutant enzyme which is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus, we have shown in this study through a combination of chemical modification and site-directed mutagenesis experiments that histidine 335 plays an essential role in DNA methylation reaction catalysed by EcoP15I DNA methyltransferase.

BC 24

Studies on Proteins & Protease Inhibitors in the Seeds of Mucuna

Siddalinga Murthy K R *, Ramachandra Swamy N *, Chandrashekharaiah K S **

* DOS in Chemistry, Central College Campus, Bangalore University, Bangalore, India,

e–mail krsmurthy2001@yahoo.com, **Dept. of Biotechnology (PG),PES Institute of Technology, Bangalore University, Bangalore, India

Mucuna is one of the lesser known legumes & under utilized as a plant protein source. To meet the protein demands in developing countries like India, there is a need to source plant proteins from lesser known non–traditional crops. Mucuna species is well recognized as a cover crop for high biomass production, weed suppression & beneficial impacts on main crop yields. Most of the species have been used as animal feeds. Some of the species have been used for human consumption, especially by tribal people. Mucuna seeds are rich in protein content (23-35%), but also contain anti–nutritional factors like protease inhibitors, phenolics, flatulence factors, tannins, phytic acid & saponins. Hence the objective of the study is to analyze the protein & protease inhibitor content in the seeds of different varieties of Mucuna & the effect of germination on protein & protease inhibitors in the seeds of Mucuna pruriens.

The proteins were extracted from the defatted seeds of Mucuna utilis using acetate buffer pH 4.5, citrate buffer pH 5.5, distilled water, phosphate buffer pH 7.0, tris-HCl buffer pH 8.5, tris-borate buffer pH 9.5 & 0.5 M sodium hydroxide. The quantitative analysis of proteins & protease inhibitors (trypsin & chymotrypsin inhibitors) showed that high amount of proteins (317mg/gm of defatted seeds) & low protease inhibitor activity (4.4 CIU & 5.0 TIU) were extracted with 0.5 M NaOH. Extraction with phosphate buffer pH 7.0 resulted in a high protease inhibitory activity. The protein & protease inhibitor extraction was carried out in defatted soaked seeds of 8 different varieties using phosphate buffer pH 7.0 & the analysis showed that both proteins & protease inhibitory activity were more in Mucuna pruriens (253.44mg/gm of defatted soaked seeds, 7.3 CIU & 13.6 TIU). The seeds of Mucuna pruriens were germinated for 6 days & the quantitative analysis showed that protein content increased up to 3 days & then decreased. Similarly there is only a slight variation in the protease inhibitory activity during germination. The qualitative studies were carried out by PAGE in 10 % gel. The electrophoretic patterns showed 5 major & 4 minor bands of proteins, and 4 major & 2 minor bands of trypsin & chymotrypsin inhibitor.

BC 25

Isolation of Tamarind Seed Protein, Polysaccharide and Cellulase from the Seeds of Tamarindus indica.

Shlini Purushothaman*, Siddalinga Murthy K R**

* Dept. of Biochemistry (PG), T. John College, Bangalore University, Bangalore, India,

e–mail, shlini_p@yahoo.co.in, ** DOS in Chemistry, Bangalore University, Bangalore, India,

Tamarind (Tamarindus indica L) is an economically important tree of India which grows abundantly in the dry tracts of Central and South Indian States. The life span of the tree is long (80 – 120 years) and yields about 150,000 tons of tamarind seeds per year. The seeds form 30 % of the whole fruit and 30 % of the seed is testa (seed coat) and 70 % is endosperm or seed kernel. The seed kernel is rich in proteins (15–21%), polysaccharide (65 – 72 %) and oil (4 – 8 %). The major industrial use for the seeds is in the manufacture of Tamarind Kernel Powder, which is an important sizing material for jute & textile industries. Tamarind Seed Polysaccharide is used as gelling agent, thickener, stabilizer and in the preparation of derivatives. The seeds are gaining importance as an alternative rich source of proteins containing high levels of methionine and cysteine. Also the seeds have been used as food in times of scarcity either alone or mixed with cereal flours. The seed oil is finding use in the manufacture of edible refined oil and soaps.

The proteins were extracted from the defatted tamarind kernel powder using distilled water, sodium chloride solution, 70 % alcohol and sodium hydroxide. The results indicated that glutelins constitute the bulk of the seed proteins. Majority of the proteins were extracted by sodium hydroxide. The fractions were also analyzed by carrying out PAGE and SDS–PAGE. The insoluble residue was the pure tamarind seed polysaccharide. Viscosity measurement was carried out using Brookefield Viscometer. The tamarind seeds were germinated for four days after treating the seeds with 50 % sulphuric acid. The endosperms were then separated, and acetone powder was prepared. The acetone powder was extracted with phosphate buffer, pH 7 and analyzed for cellulolytic activity using purified tamarind seed polysaccharide. The extract exhibited high cellulolytic activity and viscosity reduction assays suggested the enzyme to be an endocellullase.

BC 26

Isolation and Biochemical Characterization of Fungal Cellulases

Rashmi Raghunath*, Varsha Parekh*, Deepti*, Siddalinga Murthy K R^#

*Dept. of Biochemistry, Center for PG Studies, Sri Bhagawan Mahaveer Jain College,

Bangalore, India, e–mail rashmikrishna1@yahoo.co.in, ^# DOS in Chemistry,

Central College Campus, Bangalore University, Bangalore, India,

There is a growing demand for specific, efficient and cheap cellulase for use in formulation of washing powders and in textile industries. The cellulases produced using purified tamarind polysaccharide as substrate can be used to modify the characteristics of tamarind polysaccharide for use in various food formulations, textile industries for the manufacture of derivatives and for drug delivery. It is therefore of great interest to gain more information about the production of these enzymes by the microorganisms.

Altogether, 150 fungal isolates were isolated from various sources such as soil, leaf litter, wood, compost and saw dust from different locations of Bangalore. The potential producers were screened on the basis of area of zone of clearance on solid minimal medium containing de-defatted and deproteinzed tamarind seed polysaccharide as the sole source of carbon. The results of the initial screening were further confirmed by biochemical assay using DNS method. The higher activities were found to be produced by species of Cladosporium, Cephalosporium, Fusarium and Aspergillus. The organism with the highest production under conditions of submerged fermentation was identified to be species of Aspergillus and the corresponding enzyme was used for biochemical characterization. The activity was highest on five and seven days after inoculation and the enzyme was found to be membrane bound and diffusible. It showed four major protein bands on PAGE and a single cellulolytic enzyme. Viscosity reduction assays and thin layer chromatography suggested the enzyme to be an endocellullase.

BC 27

Altered LDH activity observed in the diseased human heart tissue.

V.Sasi Swetha, P. Anitha, A. Baby Shalini, P Hyma, U. Kanchana Ganga, D. Krishnaveni, P. Priyadarshni, K.V.N. Ratnakar reddi, B. Usha, G.P.Vishnu Vardhan, V.G. Yugandhar, O. Hari Prasad, and P.V.G.K.Sarma.

Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati. 517507.

Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme. Fluctuation in LDH levels in serum are used to diagnose myocardial infarction, haemolytic anaemia, hepatocellular damage, carcinoma and necrosis condition. The conversion of lactate to pyruvate with concomitant conversion of NAD+ to NADH is catalysed by LDH. LDH is a tetramer and it exists in five different isoenzyme forms. LDH was purified from human liver, kidney and heart tissues and the LDH activity was highest in kidney tissue followed by liver and least in heart tissue, however, the K_M for liver was found to be 20.8mM, heart 40mM and for kidney 80.1mM indicating importance of this enzyme in liver and heart. K_M of LDH observed in the heart tissue was same observed in normal individuals, however, altered LDH activity was observed in the heart tissue obtained from the surgery department indicating that the diseased condition of the heart.

Presenting Author:

V.Sasi Swetha

Phone: 91-877-2287777 ext.2394, 2395.

E.mail: sasi_pdl@yahoo.co.in

BC 28

P-glycoprotein ATPase from the resistant pest Helicoverpa armigera; Purification, characterization and reconstitution

Ravindra.M.A, Senigala K. Jayalakshmi, and K.Sreeramulu

Department of Biochemistry Gulbarga University, Gulbarga-585106, India

Plasma membrane P-glycoprotein (P-gpATPase) is known as an ATP dependent drug efflux pump that confers multidrug resistance. The P-gp was detected in a membrane preparation from the insecticide resistant pest Helicoverpa armigera using C219 antibodies. This protein was purified by 60% ammonium sulphate precipitation followed by conA affinity chromatography and Gelpermition chromatography by using (Sephadex G-50) the glycoprotein shows ATPase activity.SDA PAGE confirmed that a high molecular mass P-glycoprotein (150 KDa) was over expressed in resistant pest, this protein was characterized, by kinetic parameters such as effect of temperature, pH, metal ions organic solvent, inhibitors, and pesticides on Pgp ATPase. the PgpATPase was reconstituted into proteoliposomes by using phospholipids ( phosphatidyl choline, phosphatidyl serine) it was found that some pesticides such as ethylparaoxon, parathion, monocrotophos, cypermethrin, and fenvalarate stimulate the PgpATPase.Orthovanadate is a potent inhibitors of its ATPase activity, inhibiting it by 90% at concentration of (1.5μm) and verapamil (5μm) induce the Pgp ATPase.Other inhibitors, such as EDTA, sodium azide, ouabain, EGTA and sodium molybdate resulted in only 20% decrease in activity. Details of the structure and function of Ha-Pgp will be important in the development of strategies to overcome insecticide resistance in this pest.

Corresponding author: Dr. K. Sreeramulu

Fax: +91 8472 245927

Tel. No: +91 8472 263290

E-mail: ksramu@rediffmail.com

BC 29

Effect of Maternal Diabetes On Postnatal Development Of Intestine In Rats

*Ruchi Sharma, Jyotdeep Kaur and Akhtar Mahmood

Department of Biochemistry, Panjab University, Chandigarh (INDIA)

Gestational diabetes mellitus is one of the few metabolic disorders, which changes the intrauterine environment in which the development of fetus occurs. It affects nearly 3% -5% of pregnant women all over the world. Organs such as kidneys, lungs and heart are affected depending upon the severity of diabetic condition. In the present study, the effect of maternal diabetes was investigated on the postnatal development of intestinal functions in rats. Alloxan, at a dose level of 120 mg/kg body weight was injected to pregnant female wistar rats on the day 7^th of pregnancy. Postnatal studies were conducted in pups sacrificed at 5, 10, 14, 21, 30 and 45 days after birth. Body weight, intestinal length and weight of pups born to diabetic mothers were significantly low as compared to their respective controls. Weight gain during gestation was low in diabetic mothers as compared to controls though no change was observed in gestation period and litter size. A significant increase in the brush border alkaline phosphatase (p< style=""> aminopeptidase (p<>

*E-mail : ruchi _208@yahoo.com

BC 30

Characterization and viscosity parameters of seed oils from wild and cultivated plants

R.D. Gaikwad., G.S. Shamsunder., S. Paramjyothi*

Department of Biochemistry, Gulbarga University, Gulbarga. 585106, Karnataka, India.

e-mail: gaikwadrd@rediffmail.com

*Corresponding author: Department of Biochemistry, Gulbarga University, Gulbarga. 585106, Karnataka, India.

Kinematic viscosity and physico-chemical properties of Madhuca longifolia L seed oil and viscosity profiles of Sterculia foetida L, which grow in wild along with two cultivated varieties of Hibiscus cannabinus L namely Black and Red, were investigated.

M. longifolia had a significantly high content of oil 58%(w/w). The oil appeared to be stable and nondrying one based on the low peroxide and iodine value of 0.24 mEq kg^-1 and 70.2 g/100g respectively.

M. longifolia had the lowest density 0.9111 g cm^-3 at 30 ºC while S. foetida was denser than the other oils, with value of 0.9281 g cm^-3 at 30 ºC. The viscosity values were much higher in the range of 148.120 -361.687 cSt with the highest for S. foetida at 30 ºC. The oils showed a negative dependence on temperature in the range 50 –70 ºC i.e., reduction in viscosity was more or less 30%, which is generally desirable in lubricating oils so as to attain better performance over a range of temperature. Reduction in viscosity at 70 ºC by over 60% of their values at 30 ºC was observed for all oils with the highest by 77% for S. foetida.

BC 31

Correlation of Plasma nitrite and nitrate levels with plasma lipoprotein patterns as well oxidative stress and antioxidant status in human alcoholics with an impact of gender

G.Kavitha*, K. Rameshwar Reddy and N.Ch. Varadacharyulu

Department of Biochemistry, Sri Krishnadevaraya University

Anantapur - 515 003, India

Presenting Author E-mail: kavi13g@gmail.com

Levels of nitrite and nitrate, total cholesterol, triglycerides, lipoprotein cholesterol patterns ( HDL,LDL,VLDL-C) in plasma and activities of red cell antioxidant enzymes viz catalase, superoxide dismutase, gamma-glutamyl transferase, glutathione reductase were determined in male and female chronic alcoholics to compare them with respective age matched controls (each group consisted of 26 volunteers). Results of the present study suggested that chronic alcohol consumption leads to cardiac risk in both sexes, and, alcoholic females are being more sensitive to alcohol and are prone for cardio vascular risk when compared to alcoholic males. It is also evident from the study that lipoprotein patterns were found to be in correlation with plasma nitrite and nitrate concentrations in both sexes suggesting the role of NO in these changes. Increase in lipid peroxidation and other changes revealed that alcohol consumption enhances free radical induced oxidative stress leading to damage. Besides decreased antioxidant status in alcoholic groups of both sexes is evident from the study. These latter effects are more prominent in females. Further, evidences clearly suggested the involvement of NO in all the above changes and also for the observed gender dependent responses.

BC 32

Title: Serine/threonine protein kinases F and regulation of its substrate.

Azeet Narayan^1,2, Preeti Sachdeva¹, Anil K Tyagi², Yogendra Singh¹

1. Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi, 110007, India.

2. Department of Biochemistry, University of Delhi South Campus, New Delhi, 110021, India.

azeet.narayan@igib.res.in

Several pathogenic microorganisms utilize their eukaryotic type serine/threonine protein kinases (STPKs) and phosphatases as virulence factors to subvert the host cell's normal processes while aiding their survival inside the host. Reversible protein phosphorylation by these STPKs plays a key role in regulating plethora of cellular processes including stress response, regulation of cell cycle, and development. M. tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A number of published reports suggested that many of these kinases are absent in other fast growing mycobacterial genomes and their absence was directly correlated with the virulence of the slow growing pathogen. We have identified STPKs of genus Mycobacterium, made ortholog assertions and prediction of novel functions using a combination of contextual information. We extensively used genome context to infer phylogenetic relationship among the mycobacterial kinases and also predicted the novel functions and regulatory network of these kinases. We identified that PknA and PknB along with PknL are the key regulators of cell division and cell wall synthesis, a major virulence factor of M. tuberculosis. Using computational tools, we have identified pathogen specific protein kinases in M. tuberculosis. pknF, a pathogen specific kinase is known to regulate the cell division and septum formation moreover it is also implicated in the regulation of glucose metabolism. But the mechanism still remains to be elucidated. In this study we also endeavored to elucidate the mechanism of action of PknF. Our results suggest that pknF is required for the in vivo survival of pathogens and regulates its substrate, an FHA domain containing protein in a phosphorylation dependent manner. The involvement of PknF in mycobacterial physiology will be discussed.

BC33

Using protein stability information to optimise conditions for protein purification and ligand binding

Debasmita Sarkar, Suresh Solapure & Janani Venkatraman

AstraZeneca India Pvt Ltd, Bellary Road, Hebbal, Bangalore 560024

The thermal stability, Tm, of a protein can be measured using fluorophores that monitor protein unfolding. Changing solvent composition results in shifts in the Tm value, as does the presence of other interacting molecules such as ligands. These thermal shifts are convenient tools to query protein-ligand binding and to optimise buffer conditions for maximal protein recovery during purification.

We present here the test case of a bacterial kinase that is difficult to purify in large quantities, as it is unstable under standard experimental conditions. We have used thermal shift information to triage buffer conditions under which the protein is most stable and hence has maximum yields. We discovered during the course of the study that this kinase requires high ionic strengths for stability. This often means using high concentrations of salts such as sodium or potassium chloride.

However, high salt concentrations are frequently incompatible with ligand binding characterized by electrostatic interactions, as is the case between kinases and ATP. We discuss our efforts to identify the optimum salt ion and the salt ion concentration that reconcile solvent requirements for stability with those for detection of ligand binding.

BC 34

Purification and characterization of thermostable

α-galactosidase from Aspergillus tereus_GR

*Shankar S. K. and Veerappa Mulimani

Department of Biochemistry, Gulbarga University, Gulbarga, 585106, Karnataka, India.

e-mail:shankar_sk@rediffmail.com

An Extracellular thermostable α-galactosidase producing Aspergillus terreus_GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by (NH₄)₂SO₄, gel filtration and DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after SDS-PAGE. The molecular weight of the purified enzyme after SDS-PAGE was 108 KDa. The purified enzyme showed optimum pH and temperature of 5.0 and 65⁰C respectively. Enzyme is found to be thermostable as it was not inactivated after heating at 65⁰C for 40 minutes. The Km for pNPαGal, oNPαGal, raffinose and stachyose are 0.1, 0.28, 0.42, 0.33 mM respectively. Inhibitors such as 1, 10, Phenanthroline, PMSF, EDTA, mercaptoethanol and urea have no effect on enzyme activity whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg²⁺, Ni²⁺, Ca²⁺, Co²⁺ and Mn²⁺ had no effect on enzyme activity, but metal such as Ag²⁺, Hg²⁺ and Cu²⁺ have inhibited complete activity.

B C 35

Three phase partitioning and concentration of alpha-galactosidase

Dhananjay.K* and Mulimnai V.H

Department of Biochemistry,

Gulbarga University Gulbarga, Karnataka,

India

Four hour simple, attractive and versatile technique, three phase

partitioning (TPP) was used to upstream to isolate/concentrate

α-galactosidase from fermented media of Aspergillus oryzae.

t is said in this technique, that it is not necessary, one to use t-butanol

and ammonium sulphate to facilitate TPP. Therefore as ‘proof’ of

concept various organic solvents like C₄, C₅ and C₆ solvents

and different salts like kosmotropic, chemotropic and neutral salts

required for attracting efficient purification of α--galactosidase

factions were optimized. n-Butanol, t-butanol, isopraponol and

tetrahydrofuron able to form solvent protein complex and able

to push the protein at the interphase in presence of 30% ammonium

sulphate. The ratio of t-butanol to crude extract of 1:1 and 30%

ammonium sulphate gave best result at second phase of TPP.

Alpha-galactosidase under optimal conditions gave recovery of 92%

enzyme activity with 23 fold purification. The final purified enzyme

from Aspergillus oryzae showed single band on SDS-PAGE with

a mol.wt of 64 kDa. The processes were also optimized for

simultaneous purification of invertase and α-galactosidase

from fermentation media.

BC 36

Immobilized Aspergillus oryzae for the production of

alpha-galactosidase and its application

Praveen Kumar.S.K.* and Veerappa Mulimani

Department of Studies in Biochemistry, Gulbarga

University, Gulbarga-585 106,

Karnataka-585 106, India

The Aspergillus oryzae has been immobilized in sodium alginate, for the production of α-galactosidase. The various parameters for production of enzyme was optimized, such as concentration of sodium alginate, calcium chloride, carbon source, nitrogen source & other parameters were optimized. The rate of enzyme produced by both immobilized and fee Asp.oryzae cells were almost comparable. The immobilized cells were used for the hydrolysis of Raffinose family oligosaccharides in Soymilk in continuous mode. 93.66% hydrolysis of these RO’s oligosaccharides was observed in continuous mode in fluidized bed reactor. The hydrolyzate was conformed by HPLC and TLC. The immobilized cells were used up to 5 cycles without loss in the enzyme production. This technique is proved to be very effective for both production of α-galactosidase as well as for hydrolysis of oligosaccharides (Raffinose family oligosaccharides) in soymilk.

BC 37

CHANGES IN POLY ADP- RIBOSYLATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND ITS IMPLICATION IN ANGIOGENESIS

M.S. Kiran, V.B. Sameer Kumar, R.I. Viji, P.R. Sudhakaran

Department of Biochemistry, University of Kerala

Thiruvananthapuram, Kerala, India – 695 581.

kiranmsk112@rediffmail.com

Vascular endothelial growth factor is a key angiogenic growth factor which promotes cell survival, migration, proliferation and differentiation of endothelial cells and plays important role in angiogenesis. To date, five different VEGF have been described, namely VEGF-A, VEGF- B, VEGF-C, VEGF-D and VEGF-E or OV-VEGF. Among them VEGF-A is mainly involved in angiogenesis and atleast five different isoforms of VEGF-A have been reported of which VEGF165 is the predominant form. VEGF acts through two tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The biological effect of VEGF varies with cells to cells due to (a) the variation in the expression of receptors for VEGF (b) the ability to bind to the extracellular matrix whereby their availability to the cells vary (c) the post translational modification of VEGF. One of the major post translational modifications of VEGF is the poly ADP-ribosylation of VEGF. In order to examine the relation between PAR modification of VEGF and the angiogenic potency of VEGF, human umbilical vein endothelial cells were maintained in culture under different conditions and analysed the PAR modification of VEGF. Use of VEGF with varying PAR modification in CAM assay for angiogenesis showed that greater the PAR modification less the biological activity of VEGF to induce angiogenesis. This was further confirmed by the use of inhibitors of Poly ADP-ribose polymerase (the enzyme that catalyses PAR modification), where increased biological activity of VEGF was observed, suggesting the importance of this modification of VEGF in regulating angiogenesis.

BC 38

Role of nitric oxide in alprazolam induced biochemical changes in plasma lipid profile as well oxidative stress

A. Hela kiranmai, R. Purushotham, B. Saradamma, N.Ch. Varadacharyulu.

Department of Biochemistry, Sri Krishnadevaraya University

Anantapur - 515 003, India

Presenting author E-mail: kiran_hiay@yahoo.co.in

Alprazolam, a benzodiazepine compound is commonly used drug throughout the world for its anxiolytic, sedative and hypnotic anti convalescent properties as it is believed to be fairly safe and rapidly reduces the symptoms of anxiety. However, reports reveal that certain side effects result in by the acute administration of the drug. It is surprising to note that no chronic data is available on alprazolam use in humans related to biochemical changes in Nitric oxide (NO) generation as well plasma biochemical profile. Therefore this study is designed to understand the biochemical changes in plasma profile and NO generation in male volunteers receiving alprazolam 0.5 mg/day for the past five years to compare them with controls as preliminary attempt. Results of the present study, chiefly the increased levels of plasma nitrite and nitrate, increased plasma lipid peroxidation (increased oxidative stress) followed by elevated HDL-C, VLDL-C and triglyceride levels plus a decrease in LDL-C with no change in total cholesterol in plasma of volunteers using alprazolam suggested a mixed response (a beneficial as well an adverse changes) in alprazolam users when compared to age and sex matched controls who do not use this drug. Evidences suggested the involvement of the modestly increased NO.

Key words: Alprazolam, nitric oxide, lipid peroxidation, HDL, LDL

BC 39

Determination Ascorbic acid in tear samples of control subjects
and contact lens wearers by HPLC – A preliminary study.

Saijyothi AV, Vidhya S, Angayarkanni N, Ramakrishnan S

Vision Research Foundation, Sankara Nethralaya,
18, College Road, Chennai – 600 006. India,
Ph: 28271616, Email: drak@snmail.org

Purpose: The tear fluid protects the external surface of the eye from photo- induced oxidative damage due to reactive oxygen species (ROS). Compounds in tears such as ascorbic acid, glutathione, uric acid, tyrosine etc., oppose such damage by acting as antioxidants. Ascorbic acid apart from acting as an in situ antioxidant also acts as an anti-inflammatory and a wound-healing component in tear. The aim of this study to determine the ascorbate levels in tear specimen of the contact lens wearers wherein local hypoxia induced oxidative damage has been reported.

Methods: Reflex tears were collected by schirmer strip (sterile and graduated cellulose acetate strip) from 3 contact lens wearers (soft daily wear, for less than one year) along with 10 age and sex matched apparently healthy controls with a mean age of 24 ± 3), by placing the Schirmer strip in the cul de sac of the lower eye lid. The subject keeps the eye open, with no direct light or air falling on the eye to enable reflex tear collection. Reflex tears were also collected using capillary tubes for comparison with the schirmers value in the control subjects. Tear ascorbic acid (AA) was measured by High performance liquid chromatography using C18 column, with isocratic elution in 0.1% orthophosphoric acid: methanol (1:1) mobile phase with pH: 2.8 and detection of Ascorbic acid at 245nm using Diode Array detector with in 30 min of collection of the tear. A questionnaire was used to rule out ocular complications and vitamin supplementation for the past one month in all the subjects.

Results: Ascorbic acid (AA) determined in the Schirmer collected tear specimen of the healthy subjects, was found to be significantly higher (p = 0.03) than the capillary AA level. The mean AA in reflex tears collected by schirmers showed. 0.14 ± 0.03 mmoles/L and that in the contact lens wearers the AA levels increased to a value of 0.246 ± 0.118 mmoles/L that was significant (p value: 0.017). The mean Schirmer strip value, a measure of the tear production was not significantly different in the contact lens wearers from that of the controls.

Conclusions: The ascorbate levels in tears showed variation in collection procedure. This is reported to be owing to the corneal/conjunctival cells that adhere to the Schirmer strips. Still the tear collection by Schirmer was done, as this was not possible in all cases when the tear was insufficient to collect by the capillary. Ascorbate levels of contact lens wearers was found to be significantly higher which could be due to the protective effect of the ocular surface by the hypoxia induced oxidative stress, as it has been reported that there is a constant supply from the main lacrimal gland secretion and any marked increase in the level is an indication of corneal damage. Therefore the preliminary results of this study are indicative of a possible corneal damage in the contact lens wearers.

BC 40

INDUCTION OF GLUTATHIONE S-TRANSFERASES IN RAT BRAIN

Uma A, Ruxana Begum Sk^#, Sandhya D, Sreenivasulu D, Lakshminarasaiah,U and Thyagaraju K

Department of Biochemistry, Sri Venkateswara University, Tirupati 517502

Andhra Pradesh, India

#shaikruxana@yahoo.co.in

Abstract

The glutathione S transferases (GSTs, EC 2.5.1.18) of rat brain were purified to electrophoretic homogeneity and determined their anionic and cationic proteins using isoelectric focusing as eight and five, respectively, ranging the pI values from 5.1 to 9.8. The SDS-PAGE and substrate specificity analysis of rat brain GSTs on treatment with Phenobarbital (PB) ranging from 12 to 48mg the expression of GST subunits on long term and short-term treatment was specific in the order of Yb, Yδ and Yc. To observe the cell modification histology and immunohistochemistry studies were conducted on the sections of the rat brain. At the level of cell, the PB and other molecules caused damage to brain. Gliosis was predominant in the rat brain. The GST proteins can be used as markers at first stage by observing Yb subunit, later at second stage by observing Yδ subunit and finally at third stage by observing the prolonged production of Yc subunit which is more useful to remove the hydroperoxides. Therefore the results related to GSTs as marker proteins at various levels of the presence of inducers in rat brain shall be discussed during the conference.

Supported by: DST, GOI, New Delhi.

BC 41

BIOCHEMISTRY OF BRAIN AMMONIA DURING MALARIA INFECTION

Anju Agrawal

Reader Department of Zoology, S.N.SenBVPGCollege, Kanpur

Anjuaa@rediffmail.com

Cerebral malaria is one of the major manifestations of severe malaria and is defined as an acute encephalopathy, arising from Plasmodium falciparum infection, accompanied by variable neurological signs in an individual in whom other causes of coma can be excluded. The central importance of ammonia in cerebral nitrogen metabolism and on the pathological consequences of ammonia metabolism during malaria infection is discussed. The increased concentration of ammonia during malaria infection alters the physiological properties of blood-brain-barrier thereby increasing the permeability of large biomolecules besides secondary symptoms like reduced arousal, development of seizures, lethargy and reduced mental activity.

BC 42

Increased plasma nitrite and nitrate levels with enhanced oxidative stress as well increased antioxidant status in human male volunteers exposed to long term allethrin inhalation

M. Narendra, A. Hela kiranmai, P. Padmavathi and N.Ch. Varadacharyulu

Department of Biochemistry, Sri Krishnadevaraya University

Anantapur - 515 003, India

Presenting Author E-mail: lipidnaren143@gmail.com

Allethrin, a pyrethroid, is commonly used for domestic and agricultural purposes to get protection from mosquitoes and other insects. Use of this type I pyrethroid for longer durations (inhalation 8 hrs per day) and prolonged periods in the form of coils and liquid vaporizers result in biochemical and biophysical changes in erythrocyte membrane and cell. Plasma nitrite and nitrate concentrations, activities of red blood cell enzymes such as acetyl cholinesterase (Ach) and antioxidant enzymes (SOD, catalase, GPx) and the content of GSH and erythrocyte membrane lipid peroxidation were measured in human male volunteers exposed to allethrin inhalation for the past 5-7 years to compare them with controls. Increased plasma nitrite and nitrate, activities of defense enzymes as well LPO with an inhibitory effect on Ach were observed in chronic Allethrin users when compared with normal healthy humans who do not use Allethrin at all as mosquito repellent. This study revealed a modest increase in nitric oxide production, increased antioxidant status followed by enhanced oxidative stress. A possible role of nitric oxide in the above changes cannot be ruled out. Further this study confirmed the earlier finding the marked inhibition of acetyl cholinesterase by the pyrethroid allethrin.

BC 43

Isoleucine and Leucine uptake by HPLC analysis in CHO-K₁ cells exposed to amino acids mixture in high glucose environment

N. Angayarkanni, R. Selvi and S. Ramakrishnan

Biochemistry Research Department, Vision Research Foundation, Sankara Nethralaya, Chennai-600 006, email: drak@snmail.org

Purpose: Isoleucine, a branched chain amino acid, has been shown to play an important role in the improvement of glucose metabolism. Oral administration of Isoleucine has been demonstrated to decrease the plasma glucose level by increased glucose uptake and decreased hepatic gluconeogenesis. The aim of this study is to see the uptake of the branched chain amino acid namely, Isoleucine and Leucine when added along with other amino acids as a mixture, to the CHO-K₁ cells, and also see the effect of the amino acid mixture on the glucose uptake, in high glucose environment

Method: The Amino acids uptake with respect to the namely Isoleucine, Leucine, and Alanine in CHO-K₁ cells exposed to amino acids mixture - AAM (Ile, Leu, Ala, Arg, Asp, Glu, Lys) of varying concentrations (1, 2.5, 5 mM over and above the amino acid level in the media), was seen in terms of the decrease in the levels of these specific amino acids in the medium as determined by HPLC. The AAM was added along with glucose in increasing concentration of glucose (5, 10, 20 mM). These specific amino acids were determined in the medium before and after (24 hours) addition to the medium. The experiments were done in 24 well plates with a CHO-K₁ cell density of 3 X 10⁴ cells per well in F₁₂-K₁ serum free medium. Glucose uptake in terms of labeled U¹⁴C glucose in the presence of 100nM of insulin. The cell viability was measured by MTT assay.

Result: In the presence of 5mM glucose, the uptake of Ile and Leu was increased by 30- 40% depending on the concentration of the amino acid mixture, as seen at the end of 24 hours of addition of glucose and amino acid mixture. But there was no such increase in the uptake of Alanine, a neutral non-branched amino acid. In the presence of 10mM glucose, the entry of the Ile and Leu showed a similar increase but only at 5mM concentration of the amino acid mixture. The cells were found to be viable(100%) in the conditions of added amino acids in the presence of glucose. The glucose uptake was significantly increased in the presence of 5mM amino acids mixture and 5mM Glucose(p< style=""> compared to the absence of amino acid mixture. But at 10mM glucose concentration, only 10mM AAM showed significant glucose uptake (p=0.01).

Conclusion: There is increased glucose uptake in the presence of AAM in CHO-K1 cells exposed to high glucose environment. The present study reveals that the uptake of Ile and Leu is increased in the presence of high glucose concentrations. The study shows, by and large, that maximum effects are obtained if glucose and amino acids are in stochiometric concentration.

BC 44

Identification and characterization of a super-stable, anti-inflammatory, anti-fungal, anti-bacterial SOD enzyme from leaves of turmeric (Curcuma longa L)

Sunita Kochhar *, V.K. Kochhar

National Botanical Research Institute, Lucknow-226001, India

Reactive oxygen species (ROS) and free radicals have been involved in many degenerative processes. Superoxide dismutases (SODs; EC1.15.1.1) have been known to play a significant role in defense against oxidative stress caused by ROS. Infact, they constitute the first line of defense against many stress responses and are present in all organisms

We report a novel super stable superoxide dismutase (SOD) extracted from the leaves of Curcuma longa - a post-harvest waste. The scavenging activity of this SOD remains intact both in crude and purified forms before and after heating at boiling temperatures (80-100°C) up to 20 minutes, autoclaving (6-20 bars up to 10 minutes) and micro waving (frequency of 2450 megahertz -MHz or million cycles per second for 1-3 minutes). This SOD has significant shelf life at room temperature (25-35°C) and is stable for at least 18 months at 4°C and with the retained activity of 82% at –10°C and 88% at –20°C without any infection or contamination. The enzyme is stable under wide range of pH, alcohol and SDS concentrations as compared to commercial SOD from Horse radish available from Sigma Chemicals and SOD extracted from mungbean and ginger leaves.

The activity staining through native PAGE, purification of the enzyme protein and inhibitor studies have shown that this form of enzyme has a native molecular weight of 32 kDa and requires Cu/Zn as co factor. The crude and partially purified enzyme preparations exhibit anti-fungal, anti-inflammatory and anti-bacterial properties. This is a unique example of the presence of a super stable superoxide dismutase from a medicinal plant.

BC 45

ANTIHYPERTENSIVE EFFECTS OF TENDER COCONUT WATER ON FRUCTOSE INDUCED HYPERTENSION IN RATS

Bhagya.D , Dr.T Rajamohan

Department of Biochemistry, University of Kerala , Trivandrum e-mail trmohan@sancharnet.in

In the last few decades there has been a shift in the types of nutrients included in the diet. The consumption of fructose has increased, largely because of an increased consumption of soft drinks and many other beverages that are high in fructose and also because of the consumption of foods such as breakfast cereals, baked goods, condiments and prepared desserts sweetened with sucrose and high fructose corn syrup. Fructose consumption induces insulin resistance, hypertriglyceridemia and hypertension in animal models. This cluster of changes appears to be major risk factor in the pathogenesis of coronary artery disease.

Tender coconut water (TCW) is a rich source of active biological components, vitamins, minerals and aminoacids.It is reported to possess antihepatotoxic, anti-inflammatory, antioxidant, antiplatelet and hypolipidemic activities.

The biochemical effects of tender coconut water on fructose induced hypertension was studied in male Sprague – Dawley rats (n=24). Rats consumed a fructose enriched diet (containing 71% of total calories as fructose) for 30 days. After 2 weeks the rats developed hypertension which were then infused continuously during the last two weeks with tender coconut water (5ml /kg body weight/day). At the end of this period, significant decreases in the mean systolic and diastolic blood pressure were observed for groups receiving TCW. Increased lipid levels and electrolyte imbalance were attenuated in rats infused with tender coconut water. These data support that tender coconut water rich in active biological components possess antihypertensive effects.

BC 46

Synthesis and Characterization of Phthalamide-O-aminophenylenediamine, its complexes and biological activity.

Dr. Y.Lingappa, S.Sreenivasa Rao, Suresh, M.Raghu and Vedavathi.

Department of Chemistry, S V University, Tirupati – 517 502.

In continuation of our studies and lacuna identified on metal complexes of phthalamide derivatives considerable interest has been shown in the preparation of sulphur and nitrogen containing ligands and their metal complexes due to their physiological activity. Cu (II), Co (II), Ni (II), Hg (II) and Mo (IV) complexes are synthesized with phthalamide-o-aminophenylenediamine (PTAPDA) and characterized by Electronic, IR, Mass and Elemental analysis.

The ligational behavior of phthalamide-o-aminophenylenediamine (PTAPDA), towards Cu (II), Co (II), Ni (II), Hg (II) and Mo (IV) have been described. It should be interest to study ligands containing both amino and thiol moieties, as ligands containing mixed functions are expected to show interesting structural and functional properties. For this reason we have studied complexes of phthalamide-o- aminophenylenediamine. All synthesized compounds were screened for their antibacterial activity against various bacteria obtained from the NCIM (National Collection on Industrial Microorganisms, NCL, Pune, India). The bacteria included Gram positive bacterial isolates –Staphylococcus aureus (NCIM no. 5021, ATCC no. 25923) – Bacillus faecalis (NCIM no. 2063, ATCC no. 6633) and four Gram negative bacteria – Escherichia coil (NCIM no. 2931, ATCC no. 25922), Pseudomonas aeruginosa (NCIM no. 5029,ATCC no. 27853), Salmonella typhimurium (NCIM no. 2501, ATCC No. 23564) and Klebsiella pneumoniae (NCIM no.2957). The bacteria are rejuvenated on Hi-media nutrient agar and sub-cultured as needed. The inhibitory power of the complexes is greater than that of the control sample (DMSO). The activity ranges of these complexes are determined by following Disc and Minimum Inhibitory Concentrations (MIC) methods.

BC 47

Comparative studies on the evaluation of Antioxidant properties and Antioxidant capacity of Clitoria ternata and Eclipta prostrata

D. Bhaskar Rao, M. Rama Rao#and T. Raghava Rao

Department of Biochemistry, College of Science & Techhology, Andhra University, Visakhapatnam, Andhra Pradesh, India.

#Department of Biochemistry, College of Science, GITAM, Visakhapatnam, Andhra Pradesh, India.

Email: bhasidadi@qmail.com

Free radical-mediated oxidative stress is believed to be the primary cause of many age related disorders such as cardiovascular diseases, brain dysfunction, cataracts, diabetes mellitus, arthritis, cancer, ageing etc. In treatment of these diseases, antioxidant therapy has gained an utmost importance in the recent days. Current research is now directed towards finding naturally occurring antioxidants of plant origin. In indian system of medicine Clitoria ternata and Eclipta. prostrata are the important medicinal plants, which have a wide range of applications in traditional medicine. To understand the mechanisms of pharmacological actions, evaluation of the enzymatic antioxidants such as superoxide dismutase, catalase, peroxidase, glutathione peroxidase and ascorbate oxidase, non-enzymatic antioxidants include reduced glutathione, vitamin C and total phenolics and the in vitro antioxidant capacity of aqueous extracts of Clitoria ternata and Eclipta prostrata are investigated. In both the plant extracts of present study, significant levels of enzymatic, non-enzymatic antioxidants are observed. In addition both the extracts also showed potent total antioxidant power, reducing activity and radical scavenging activities.

BC 48

Production, purification and characterization of an oxidant, SDS, and organic solvent-stable protease from a newly isolated Bacillus sp. RKY3

L.V.A. Reddy ^{a, b}, Young-Jung Wee ^b, Jong-Sun Yun ^c, and Hwa-Won Ryu ^b

^{a, b} Yogi Vemana University, Vemanapuram, Kadapa-516003 India

^b School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea,^c Institute of Biotechnology, BioHelix, Naju 520-811, Korea

A protease enzyme was produced from a newly isolated Bacillus sp. RKY3 by using economic medium components such as corn starch and corn steep liquor. The enzyme, protease was purified in two steps through anion exchange chromatography after concentrated the crude enzyme by ammonium sulfate precipitation. The purified protease had a molecular mass of around 38 kDa. It was much active over a broad range of pH between 7.0 and 9.0 and also stable in a wide pH range from 5.0 to 11.0. Though the optimum temperature for enzyme activity was 60^oC, it was rapidly deactivated above 60^oC. It has good stability at 50^oC with a 70 min half-life. Ca²⁺ ions did not enhance much both the activity and stability of the enzyme. PMSF (1 mM) completely inhibited the protease activity so the purified protease is considered to be serine protease. The purified protease was stable with oxidants (H₂O₂, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. With these properties the purified protease seems to be useful in the protease-based detergents production.

Bioinformatics

Department of Biochemistry, Raos Degree College, Nellore, AP

BI 1

Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major

Rajesh K. Yadav, Subhankar Dolai, Swati Pal, and Subrata Adak

Division of Structural Biology and Bioinformatics Indian Institute of Chemical Biology

4, Raja S.C. Mullick Road Kolkata – 700 032.

E.mail: biochemrajesh@yahoo.co.in

Unlike most eukaryotes, Leishmania lacks catalase and selenium-containing glutathione peroxidases, enzymes capable of rapidly metabolizing high levels of H2O2. Our lab has cloned, expressed and purified an unusual plant like ascorbate peroxidase from L. major (LmAPX) and characterized its catalytic parameters under steady state condition. Primary structural analysis has shown LmAPX is similar with cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) placing it in class I peroxidase. In present work, the role of conserved proximal tryptophan (Trp-208) residue has been addressed by point mutagenesis in controlling catalysis. Interestingly it has been found that Trp208Phe mutant is 1000 times less active than the wild type for cytochrome c oxidation although apparent dissociation constant of enzyme-cytochrome c complex , k_m value for cytochrome c and second order rate constant for compound I formation remain unaltered indicating no perturbation in other structural features. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H₂O₂ to form compound-I (compound-II type spectrum), which was quite different from that of compound-I in W208F mutant as well as horseradish peroxidase (HRP). Since kinetic for the formation and decay of compound-I & -II of mutant enzyme was almost identical with wild type enzyme in enzyme-H₂O₂-ascorbate system, it is distinct that there is two unique pathway of electron transfer for cytochrome c (through tryptophan cationic radical) and for ascorbate or guaiacol oxidation (using porphyrin radical). Thus LmAPX is a functional hybrid between CCP and APX.

BI 2

Molecular Characterization of Mycobacterium tuberculosis PII protein

Anannya Bandyopadhyay *, Amit Arora, Chhabinath Mandal, Vladimir A. Ivanisenko,

Souvik Maiti, Srinivasan Ramachandran

Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, *anannya@igib.res.in

Tuberculosis is a major killer disease worldwide. In efforts to identify new drug targets, it is important to characterize proteins of biochemical pathways critical for cell survival. The Nitrogen assimilation and regulation is one such step. The signaling protein PII senses the status of intracellular carbon and nitrogen and is involved in regulating the expression and activity of Glutamine synthetase, which synthesizes glutamine. In order to characterize the PII protein from M. tuberculosis (Mtb), we have cloned and expressed it in Escherichia coli. The His-tagged recombinant PII had mol. wt. of 15.9 kDa. Peptide mass fingerprinting using MALDI-TOF MS reconfirmed the identity of purified protein as Mtb PII.

Unlike E. coli PII, the Mtb PII did not display any post-translational modification in nitrogen optimum, excess and starvation conditions. These results either indicate the inability of orthologous enzyme of E. coli to modify Mtb PII or a novel intrinsic character of Mtb PII. These results are strikingly new considering that PII is a universally conserved protein. The E. coli PII has 69% identity with Mtb PII. Unlike E. coli, Mtb has only one copy of PII gene a feature similar to other actinobacteria. Phylogenetic analysis revealed that Mtb PII and other mycobacterial PII proteins form a distinct lineage distant from the proteobacterial PII lineage. Multiple alignment using ClustalW showed that residues forming the T-loop including Tyr 51 (site for post-translational modification) and the ATP binding site are conserved, whereas the differences are scattered throught the protein sequence.

In order to gain insights into its structural and functional aspects, a homology model was generated using SWISS-MODEL, Insight II using high resolution X-ray crystal structures of orthologs 1QY7 (Synechococcus sp. PCC 7942) and 2PII (E. coli). The Mtb PII modeled structure had rmsd values of 0.312 Å and 0.442 Å respectively, with these proteins. The structure revealed that it may bind to ATP. Also, 2-ketoglutarate (intracellular carbon status indicator) binding site has been predicted using AUTODOCK and MOLKERN programs. It binds in the lateral cleft region adjacent to the ATP binding site. Residues from adjacent monomers of PII are suggested to interact with the 2-keto glutarate molecule. The estimated free energy of binding of 2-ketoglutarate to PII is not affected significantly by the presence or absence of ATP, although van der Waals and electrostatic interactions have been predicted between ATP and 2-keto glutarate.

Presence of ordered secondary structure in the protein was confirmed through CD spectroscopy. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirm the binding of ATP (K_d = 6.44 ± 1.35 µM). Interaction of Mtb PII with ATP remains unaffected by the presence of α-ketoglutarate (K_d = 8.87 ± 2.82 µM). These findings appear to be supported by the modeling studies wherein it was observed that the binding site of 2-keto glutarate is adjacent to ATP binding site and the binding of these two molecules are not affected by the presence of each other. However, van der Waals and electrostatic interactions may be possible. The study also revealed that PII binds to GTP (K_d = 8.09 ± 5.04 µM) and CTP (K_d = 8.44 ± 2.21 µM) with almost equal affinity as ATP. UTP (K_d = 31.5 ± 0.72 µM) and ADP (K_d = 29.3 ± 7.82 µM) however show lower affinity. This had so far remained unexplored. Interaction energetics revealed the exothermic binding nature of the nucleotides to PII.

BI 3

Homology modeling of 5-Lipoxygenase, docking studies and hints towards

selective inhibitor design

P. Aparoy¹, Lalitha Guruprasad², Nageswar Reddy³, M. Rami Reddy⁴, P. Reddanna^1*

¹Department of Animal Sciences, School of Life Sciences, University of Hyderabad.

Hyderabad – 500 046. e-mail: aparoy@gmail.com, preddanna@yahoo.com*

²School of Chemistry, University of Hyderabad, Hyderabad – 500 046.

³Rational Labs Pvt. Ltd., Plot No: 177, Phase II, IDA Mallapur, Hyderabad, India

⁴ Metabasis Therapeutics, 11119 North Torrey Pines Road, La Jolla, CA 92037, USA

Lipoxygenases (LOXs) represent a heterogenous family of non heme, non sulphur, iron containing dioxygenases that catalyze the regioselective and stereo selective dioxygenation of fatty acid substrates containing one or more (Z,Z)-1,4-pentadiene moieties. The primary reaction products are hydroperoxides of conjugated (E, Z)-dienes that are further metabolized to various bioactive lipid mediators such as leukotrienes, lipoxins, hydroxyeicosatetraenoic acid (HETEs) and hepoxilins. Lipoxygenases (LOXs) are classified according to their positional specificity of arachidonate oxygenation into 5-, 8-, 9-, 11-, 12- and 15-LOXs. The prominent animal LOXs are 5-LOX, 8- LOX, 12-LOX and 15-LOX, while the plant LOXs are mostly 5-LOX and 15-LOX. The 5-LOX pathway is the source of potent pro-inflammatory mediators. LOX metabolites are potent physiological effectors in a variety of cellular responses, associated with normal host defense and inflammation. Inhibitors of the 5-LOX pathway, therefore, have a therapeutic potential in a variety of inflammatory and allergic diseases. As the crystal structure of the protein is yet to be elucidated, modeling studies will be helpful to obtain a consistent representation of the enzyme-inhibitor recognition step at the molecular level and furthermore, provide new insights that can be used to design novel therapeutic agents. In the present study, the homology modeling technique has been used to construct the structure of potato 5-Lipoxygenase. The conserved structural pattern of all lipoxygenases mainly at the active site residues formed an advantage for building up an accurate homology model. The protein consists of 864 amino acid residues and treated as the target. The amino acid sequence identity between the target protein and sequence of template protein 1NO3 (Soybean LOX-3) searched from NCBI protein BLAST was 63%. Based on the template structure, the protein model was constructed by using the InsightII/Homology program. The protein model was briefly refined by energy minimization steps and validated using Profile-3D in InsightII and by web available servers such as ERRAT and PROCHECK. The results showed that 99.3% of the amino acids were in allowed regions of Ramachandran plot, showing that the model is accurate and its stereochemical quality good. Like all LOXs, 5-LOX also has a two-domain structure. The small N-terminal β-barrel domain and a larger catalytic domain containing a single atom of non-heme iron coordinating with His525, His530, His716 and Ile864. Asn720 is present in the fifth coordination position of iron. The sixth coordination position faces the open cavity occupied here by the ligands which are docked. Our model of the enzyme is validated by examining the interactions of ligands whose inhibition has been reported earlier. Five ligands were selected for our current docking studies, Nordihydroguaiaretic Acid (NDGA) having IC50 of 1.5 mM and analogs of benzyl propargyl ethers having an IC50 of 45mM, 60mM, 760 mM and no inhibition respectively to study the interactions with our model, thus validating it further. Our results correlated well with the experimental data reported earlier which proved the accuracy of the model. This model generated can be further used for the design and development of more effective 5-LOX inhibitors.

BI 4

A High Resolution Crystal Structure of Cyclophilin from Leishmania Donovani

Elucidation of Its interactions with Drugs and Proteins

V. Venugopal^*, Banibrata Sen^#, Alok Datta^#, Rahul Banerjee^*

^* Saha Institute of Nuclear Physics, Kolkata-64

^# Indian Institute of Chemical Biology, Jadavpur, Kolkata-32

The crystal structure of cyclophilin from Leishmania donovani (LdCyp) has been determined and refined at 1.97 Å resolution to a crystallographic R factor of 0.178 (R_free = 0.197)¹. The structure was solved by molecular replacement using cyclophilin from Trypanosoma cruzi as the search model. LdCyp exhibits complete structural conservation of the cyclosporin-binding site with respect to the homologous human protein, as anticipated from LdCyp-cyclosporin binding studies. Comparisons with other cyclophilins show deviations primarily in the loop regions. The solvent structure encompassing the molecule has also been analyzed in some detail. The coordinates of this high resolution structure were used to build homology models of different mutants to elucidate its interaction with L.donovani adenosine kinase (LdAdK) and Arg147 in LdCyp was found to play a key role in reactivating LdAdk².

¹ V. Venugopal, Banibrata Sen, Alok K Datta, Rahul Banerjee, Structure of cyclophilin from Leishmania donovani at 1.97 Å resolution. Acta Cryst F., 2007, F63; 60-64.

² Banibrata Sen, V. Venugopal, Anutosh Chakraborty, Rupak Datta, Subhankar Dolai, Rahul Banerjee, Alok K Datta, Amino Acid Residues of Leishmania Donovani Cyclophilin Key to Interaction with Its Adenosine Kinase: Biological Implications, BioChem., 2007, 46(26); 7832-7843.

BI 5

Homology modelling of Human 12R-Lipoxygenase and

Hints towards selective drug design

Aparoy P¹, Leela T¹, Lalitha Guruprasad², M. Rami Reddy³, Reddanna P¹

¹School of Life Sciences, University of Hyderabad, Hyderabad – 500 046.

e-mail: aparoy@gmail.com, preddanna@yahoo.com*

²School of Chemistry, University of Hyderabad, Hyderabad – 500 046.

³Metabasis Therapeutics, 11119 North Torrey Pines Road, La Jolla, CA 92037, USA

Lipoxygenases (LOXs) represent a heterogenous family of non-heme, non-sulphur, iron containing dioxygenases that catalyze the regioselective and stereo selective dioxygenation of fatty acid substrates containing one or more (Z,Z)-1,4-pentadiene moieties. The primary reaction products are hydroperoxides of conjugated (E, Z)-dienes that are further metabolized to various bioactive lipid mediators such as hydroxyeicosatetraenoic acid (HETEs), leukotrienes, lipoxins, and hepoxilins. The uncontrolled production of theses lipoxygenase products is associated with many inflammatory disorders, including psoriasis and disrupts the epidermal barrier functions. A recognized feature of psoriasis and other proliferative dermatosis is accumulation in the skin of the unusual arachidonic acid metabolite, 12R- hydroxyeicosatetraenoic acid (12R-HETE), a product of 12R Lipoxygenase. 12R-LOX is a potential drug target for psoriasis and skin cancers. Little structural information is available regarding 12R-LOX.

The present study is aimed to understand the structural details of the enzyme in silico by Homology modelling of 12R-LOX (target) by taking Rabbit reticulocyte 15-LOX (1LOX) as a template. Even though the sequence similarity between the target and template sequence is low (~36%), by taking advantage of the conserved structural pattern mainly at active site residues such as metal binding residues, steriospecific residues and positional specific residues which are highly conserved in all LOXs an accurate model can be built by employing homology modelling. Based on the template structure, the protein model was constructed by using the InsightII/Homology program. Energy minimization steps briefly refined the protein model. The protein model was validated using Profile-3D in InsightII and PROCHECK. The superimposition of predicted structure with that of template structure, 1LOX reveals an RMSD of 1.2 Ǻ. Like all LOXs, 12R-LOX also has a two-domain structure, the small N-terminal β-barrel domain and a larger catalytic domain containing a single atom of non-heme iron coordinating with His398, His403, His578 and Ile701. Asn582 is present in the fifth coordination position of iron. The sixth coordination position faces the open cavity occupied by the substrate/ligand.

The present study should help us to understand the structural importance of 12R-LOX in catalytic activity and providing a clear vision of the active site residues and their involvement in positional and steriospecificity. The present study assumes importance as no structure of enzymes that catalyse with R-stereochemistry has been reported. This model can be considered as a working tool for generating hypotheses which can be used in drug design studies leading to drug discovery.

BI 6

Structure based drug-designing studies of gymnemic acid derivatives based on binding studies of sweeteners and inhibitors on the proteins involved in hyperglycemia

Mrs.G.V.Padmavathi¹, Prof.D.Saralakumari¹, Mr.P.Nataraj Sekhar².

1. Dept.of Biochemistry, Srikrishna Devaraya University, Anantapur

2. Dept. of Genetics, Osmania University, Hyderabad.

padmavathi1515@yahoo.com

Computer-aided drug designing has come into focus with the discovery of new software’s that is made available to us today in the market. This has prompted us to take up the present research work with the following objectives. The first one is to dock the sweeteners and known inhibitors against crystal structures of receptors and enzymes involved hyperglycemia. The next is to design new inhibitors using gymnemic acid as a core molecule with different side chains. To dock the molecules against homology models of some human proteins involved in hyperglycemia using OPENEYE, commercial software. Finally to predict drug likeliness of inhibitors and derivatives. These studies revealed that gymnemic acid and its derivatives are binding with high affinity against T1R2 an important receptor in taste perception and not with any other enzyme and receptor. The studies also showed that gymnemic acid derivatives are binding with affinity compared to sweeteners and known inhibitors against GLUT proteins and crystal structures of liver, muscle and with the homology model of brain glycogen phosphorylaseA. it is hoped that these newly designed molecules if synthesized and tested in animal models hold promise for anti - hyperglycemic activities.

BI 7

Homology modeling of Plasmodium Vivax Protein phosphatase 5 and validation of the structure

K. Venkateswara swamy¹, Anita Suri², Babajaan¹, Chaitanya¹, Chitta Suresh Kumar¹,

V. Kothekar²

¹ Bioinforamtics Centre, Department of Biochemistry,Sri Krishnadevaraya University,Anantapur, Andhra Pradesh, India.

² Biotechnology and Bioinforamtics Institute, D.Y.Patil Univeristy, Akurdi,Pune, India

Malaria is a major international public health problem, and the disease affects more than 500 million people and causes 2-3 million deaths each year. Among the most interesting antimalarial target proteins currently studied are proteases, protein kinases, glycolytic enzymes and enzymes involved in lipid metabolism and DNA replication Due to the rapid development of resistance to clinically used drugs like chloroquine and mefloquine and the increasing risk of resistance to artemisinins, novel effective and affordable antimalarial agents are urgently required. PP5 is likely player in parasitic signal transduction and hence a potential target for antimalarial drug designs. PP5 is destined to have a definitive role in parasitic growth and signaling pathways.

Protein sequence of Plasmodium vivax PP5 was subjected to BLAST against Protein data bank (PDB) and results has shown 45% homology with human PP5 protein. Homology model of Plasmodium vivax Protein Phosphatase 5 (PP5) was modeled by selecting serine/threonine protein phosphatase 5 (PP5) (1WAO: PDBID) as best template with Modeller 9v1 and energy minimized under the GROMOS96 force field. Modelled PP5 protein structure was evaluated with PROCHECK and Verify_3D has given satisfactory results for further analysis of docking studies with suitable lead molecules to innovate new drugs for Malarial disease.

Biotechnology

SDHR PG and UG Institutions, Rayachoti and Tirupati

Invited Talk

EXPERIMENTAL DENGUE VACCINE

Navin Khanna

International Centre for Genetic Engineering & Biotechnology, New Delhi, India

There is currently no vaccine to prevent dengue (DEN) virus infection, caused by any one of four closely related serotypes, DEN-1, DEN-2, DEN-3, or DEN-4. A DEN vaccine must be tetravalent, as immunity to a single serotype, does not offer cross protection against the other serotypes. We have developed a novel tetravalent chimeric protein by fusing the receptor-binding envelope domain III (EDIII) of the four DEN virus serotypes. This protein was expressed in the yeast, Pichia pastoris, and purified to near homogeneity in high yields. Antibodies induced in mice by the tetravalent protein, formulated either in alum or Montanide ISA 720, neutralized the infectivity of all four serotypes with titers of >1:80. This, coupled with the high expression capacity of the P. pastoris system and easy one-step purification, makes the EDIII-based recombinant protein a potentially promising candidate for the development of a safe, efficacious, and inexpensive, tetravalent DEN vaccine.

BT 1

Bioconversion of D-xylose to xylitol by yeast strain KB-41.

Jyoti Rekha Saikia, B.K. Gogoi and R.L. Bezbaruah

Biotechnology Division, NEIST (formerly RRL) Jorhat-785006, Assam

e-mail: jyt_saikia@yahoo.com

Xylitol is a five carbon sugar alcohol that has attracted the attention of food, odontological and pharmaceutical industries. It is non-cariogenic and can be used by diabetics. Xylitol is industrially obtained by a chemical process from wood hydrolyzates. In the present study, various microbial strains were isolated from soil samples collected from sugarcane growing areas. The microbes which can utilize xylose as the sole source of carbon were tested for xylitol production. Out of the strains KB-41 showed highest production of xylitol. The physico-chemical parameters for optimum production of xylitol were studied. A temperature of 30⁰C was found to be suitable. pH plays an important role in bioconversion and pH 6.5 was optimum for this strain. A substrate concentration of 5% or more gave a good conversion to xylitol. Aeration also plays an important role on production of xylitol and lower rate of aeration favors xylitol production. Fermentor will be used to optimize the bioconversion process.

BT2

Studies on the microbial population diversity with respect to changes in enzyme activity in a composting process

Sutripta Sarkar¹, Madhura Ghosh², Sunanda Chanda¹, Subrata Pal²

Indian Statistical Institute, 203 B.T. Road, Kolkata-108 ¹, Department of Life Science and Biotechnology, Jadavpur University, Kolkata-32 ²

Email; sutripta_99@yahoo.com

Composting is a method of transforming bioorganic wastes into products that can be safely used as soil conditioner or biofertilizer. The process involves degradation of complex macromolecules by microbial communities. High microbial activity results in evolution of heat that causes increase in temperature The objective of this work was to determine the changes in microbial population diversity with respect to the changes in enzyme profile during the different stages of composting, with special emphasis on the heating or thermophilic phase.

The work involved monitoring the changes in microbial community structure, dynamics and physiological activity with changes in physical parameters viz. temperature, pH, moisture content etc. during composting. Mesophilic and thermophilic culturable bacterial population were enumerated in different growth medium viz. Luria broth agar, Casein agar, Starch agar and carboxymethyl cellulose agar. Dehydrogenase, β- glucosidase, protease and amylase activity present during the different phases of composting were measured.

The initial phase of composting showed a high count of mesophilic population. With the onset of the thermophilic phase there was a sharp decline in the total mesophilic bacterial count and an increase in the thermophilic bacterial count. However both mesophilic and thermophilic populations show an increase post thermophilic phase of composting.Then a decline was observed in the maturation phase. The cellulolytic, proteolytic, amylolytic activities and total microbial activity as measured by dehydrogenase assay, increased at the end of the thermophilic phase. The enzyme activity indicates that in the thermophilic phase the degradation is essentially done by thermophiles.

From the above study it can be concluded that that extracellular enzyme activities serve as a good indicator of the physiological and microbial changes occurring in the process of composting.

BT 3

Isolation and screening of microorganisms for laccase production for potential application in remediation of effluent water from the pulp and paper industry

SHIKHA AND SHIV SHANKAR

Department of Environmental Science, Babasaheb Bhimrao Ambedkar University,

Vidya-Vihar, Rae bareli Road, Lucknow (U.P.) Pin-226 025 India

E mail: dr_shikha2003@yahoo.co.in

The pulp and paper industry generate effluent which is heavily polluted with lignin, inorganic salts and hydrogen sulphide that arise from the pulping process that uses sodium sulphite for the extraction of lignin from the pulp. Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp, effluent decolourization and detoxification. It is essential to find novel, efficient enzymes to further develop these applications. Fungi and bacteria from diverse sources (soil, Kraft mill effluent, decaying wood, etc.) were cultivated on solid media containing indicator compound that enabled the detection of laccases as specific colour reactions. The screening work resulted in isolation of 06 positive fungal and 02 positive bacterial strains. However, a bacterium isolated from decaying wood sample by differential screening of a number of strains was found to be the most potential candidate giving a unit activity of 1.66 Units/ml in submerged culture. The taxonomic characterization of this strain indicates that it belongs to the genus Bacillus, and has the ability to produce laccase (EC 1.10.3.2). We studied the laccase activity with specific substrates such as syringaldazine and guaiacol. The optimum pH and temperature, obtained for the polyphenol oxidases, was at about pH 5.5 and 55 °C, respectively. Plate-test screening based on polymeric dye compounds, and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be efficiently performed with guaiacol.

BT 4

Alkaline amylases from an obligate alkalophilic actinomycete sp A 01.07. 716

Geetha S., Saravanan A., Lalitha Kumar, Srinivasan M C^▲ and M V Rele*

Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India

E-mail: g.subramanian@ncl.res.in

Most of the alkaline amylases reported in the literature were from the genus Bacillus; however, we isolated an alkalophilic actinomycete capable of secreting alkaline amylases from the Lonar lake region, Buldhana District, Maharashtra. Preliminary morphological studies indicated the organism to be a Streptomyces species. To date, there are only two reports of alkaline amylases from Streptomyces sp, both of which have been patented by the Japanese (Moriyami et al., 2000; Nakai et al., 1986). Besides this, there is only one published report of an alkaline amylase from a Nocardiopsis sp (El-Meleigy et al., 1996). The Streptomyces strain 716 secretes saccharifying alpha-amylases that are active at alkaline pH. This strain grew optimally at 28⁰C in alkaline medium (pH 10) and there was no growth in neutral medium. The optimal growth was obtained at 28⁰C at pH 8.0 – 9.0. Raw starch was found to be a superior inducer than soluble starch. Approximately fifty percent of the control activity was induced by maltose. Glucose is known to repress amylase activity However, in the present studies, it was observed that a minimal level of enzyme was produced in the presence of 3% glucose alone. In a glucose- malt extract medium (MGYP), where the glucose concentration was 5% substantial production of the enzyme was observed. Eighty percent of the control value was obtained although maltose induces the enzyme, enhanced production cannot be explained by this alone .The role of malt extract in enzyme production is being investigated.

The alpha-amylase from this strain has maximal activity at pH 9 and at 45⁰C. The enzyme is also active over the temperature range of 30⁰C to 50⁰C at pH 9 and pH 10. The alpha-amylase from this strain has maximal activity at pH 9 and at 45⁰C. The enzyme is also active over the temperature range of 30⁰C to 50⁰C at pH 9 and pH 10. These characteristics would make this enzyme suitable for detergent, leather and textile applications. The enzyme retained 40% of its activity in the presence of 0.2% Surf Excel detergent at 40-50⁰C. The removal of starchy stains using formulations containing 0.2 mg/ml and 7 mg/ml of Surf supplemented with 5 IU/ml of enzyme was superior as compared to the effect by detergent alone. All these observations suggest a potential application of this enzyme in the detergent industry.

^▲ Retired scientist

BT 5

Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants

Saurabh Agarwal, Shweta Jha, Indraneel Sanyal and D.V. Amla

Plant Transgenic Lab, National Botanical Research Institute, Lucknow, India

saurabhagarwal2@gmail.com

Transgenic plants hold promising alternative as bioreactor for large-scale sustainable production of safe and functionally valuable recombinant proteins of biopharmaceutical, industrial and therapeutic applications. Engineering plants as biological factories is often limited, due to poor expression levels of heterologous genes that requires extensive modifications in the coding region and flanking regulatory sequences for high-level expression, proper folding, stability and sub-cellular localization of the expressed foreign proteins. We have analyzed, designed and synthesized modified gene coding human α-1-antitrypsin (AAT) protein for high-level expression and localization in dicot plants. In modified AAT gene, about 205 codons, out of total 394 were replaced with dicot-preferred codons and 281 nucleotides were altered for 45.5% G+C content. Six putative polyadenylation signals (AATAAA and variants), five mRNA instability sequences (ATTTA and variants) and potential mRNA splicing sites were eliminated and long hairpin loops were avoided. The native signal peptide sequence of AAT gene was substituted with either modified signal peptide sequence of tobacco pathogenesis related protein PR1a, sweet potato sporamineA (SPS) or with dicot preferred native signal peptide sequences of the gene at the N-terminal end while a ER retention signal KDEL was incorporated at C-terminal end for anchoring and accumulation of mature protein in endoplasmic reticulum of transgenic plant cells. A dicot plant preferred translation initiation context sequence (TAAACAATGG) was also introduced at 5’ end for efficient initiation of translation. The full-length modified AAT gene was synthesized by PCR-based strategy using overlapping oligos of 50-55 mer length. Tomato plants were genetically engineered by nuclear transformation with the constructs pPAK, pSAK and pNAK having different signal peptide sequences to express the modified synthetic AAT gene under the control of DECaMV35S promoter along with AMV 5’ UTR sequence. The incorporation, expression and stability of recombinant AAT gene and protein was examined by PCR, RT-PCR, Southern, DAC-ELISA, SDS-PAGE, Western immunoblotting and residual porcine pancreatic elastase (PPE) activity assays. The T₀ plants raised with pPAK, pSAK and pNAK constructs accumulated recombinant AAT protein at an average of 1.0 ± 0.28, 0.67 ± 0.15 and 0.66 ± 0.26% of total soluble protein respectively with biological activity at an average of 0.39 ± 0.17, 0.40 ± 0.21 and 0.34 ± 0.25% of total soluble protein respectively. The glycosylation of plant expressed recombinant AAT protein was established in comparison to unglycosylated E.coli expressed protein by SDS-PAGE and immunoblotting analyses. Our results demonstrate the high-level expression of biologically active, glycosylated human AAT protein in transgenic tomato plants.

BT 6

Bio-diesel-A New Approach for Fuel Crises

Srikanth S

anits_srikanth@yahoo.co.in

T Satyanarayana Reddy

Bio-diesel is produced from vegetable oil through a refining process called "trasnestrification",a chemical process that mixes vegetable oil with methyl or ethyl alchol in presence of base catylst ( NaOH or KOH).The chemical reaction produces biodiesel and glycerine.The process is adapted for the development of the various biodiesel technologies at an industrial scale from a variety of vegetable oil feed stocks .In this paper ,some of the industrial processes in action across the globe are revived giving flow diagrams and details . A few expriments were carried out to produce Biodiesel based pongamia oil .The samples were test for density ,viscosity,flash point distillation chrecteristics and cetane no. .They were also tested for performance in a diesel engine .The exprimental results are presented in this paper.

BT 7

Standardization of efficient regeneration protocol in cotton and transformation with Brassica juncea annexin and npr1 genes for conferring abiotic and biotic stress tolerance respectively

K. Divya¹, P. B. Kirti^1*

¹Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, India

*Corresponding Author: pbksl@uohyd.ernet.in

High frequency in vitro recurrent shoot bud differentiation and multiple shoot production from hypocotyl segments of 8 to 10-d-old seedlings of cotton has been achieved. Out of the growth regulators tried, a combination of 2 mgl^-1 dm^-3 thidiazuron (TDZ) and 0.05 mgl^-1 naphthaleneacetic acid (NAA) was most effective with (76%) shoot regeneration and a maximum of 10.6 shoots per responding explant has been attained. Combination of the cytokinins Benzyl aminopurine(BAP) and Kinetin induced better regeneration response than their individual treatments. Culture medium was also augmented with ethylene inhibitor silver nitrate (AgN0₃) and efficient phenol adsorbent activated charcoal. Optimal rooting was obtained on half-strength MS medium with 1 mgl^-1 indole butyricacid(IBA) and activated charcoal. Scanning electron micrographs of cultured explants revealed de novo formation of shoot primordia. The transformation procedure was optimized using Agrobacterium strain GV2260 harboring pCAMBIA2301 GUS-INT.

Annexins in mammals and plants had been proved to play a role in the oxidative stress response. These belong to a family of structurally related proteins containing approx. 70-75aa conserved repeating unit with endonexin fold, which bind to the phospholipids in the membranes in a Ca²⁺ dependent manner. Annexins are divergent in function and are activated by ABA, osmotic stress, salinity, oxidative stress and heavy metal stress etc. thus proposed to confer multiple stress tolerances. We are aiming at the generation of transgenic cotton plants expressing Brassica juncea annexin gene with multiple stress tolerance. Southern and northern analyses of the putative transgenic plants show the integration of the annexin gene. Various stress treatments using Mannitol, NaCl, Polyethylene glycol, H₂O₂ and CdCl₂ in T₁ plants using leaf disk assay shows clear discrimination in the chlorophyll content of the control and transgenic samples after 72hrs incubation.

The Brassica juncea NPR1 protein is characterized by having two protein-protein interaction domains, an ankyrin repeat, BTB/POZ (broad complex, tramtrack, Bric-a-Brac, Zinc finger) domain, a putative nuclear localization signal and phosphorylation sites. Upon pathogen attack or Salicylic acid treatment NPR1 oligomer in split into monomer followed by nuclear localization, binding to TGA factors, and subsequent activation of PR genes which confer resistance to various pathogens. Genetic transformation of cotton using NPR1 gene was confirmed by southern, northern and western analyses.

References:

Plant Physiology 126, pp.1072-1084, 2001.

Physiological and Molecular Plant Pathology 68, pp. 128-137, 2006.

Cells Tissues Organs; 185:51–60, 2007

Int. J. Cancer (Pred. Oncol.): 84, 174–178 (1999)

BT 8

Patents & its impact on Biotechnology

Sowbhagya Rani.V. and Uma Devi.K.

Department of Law, Sri Padmavathi Mahila Visvavidyalayam, Tirupati. A P. India.

The term biotechnology means “the technical applications of biological processes in microorganisms, plants or animals”. We gain benefits from biotechnology in the food industry, agriculture and medicine. India is innovating its way out of poverty using biotechnology tools. It could revolutionize biotechnology on the basis of its large and increasingly well-educated work force, just as it did in Information technology. The biotech industry is globalizing rapidly. The world market for genetics was expected to increase significantly in the next few years as several major drugs lost patent protection. Advancements in biotechnology have a drastic impact on drug development in general, the health care policies of developing countries in particular.

With the advent of new biotechnology methods, for developing “New Chemical Entities” (NCE’s) has changed rapidly in the last 30 years. From the 1980’s onwards biotechnology also allowed new approaches in the production of drugs, for example genetically modified micro-organism were employed to produce desired proteins with complex molecular structure, such as human Insulin, which could not be synthesized through classical chemical methods. The results related to the Biotechnological inventions that can be patented and biotechnological inventions that cannot be patented will be the subject of the paper.

BT 9

Effect of theromotolerant Bacillus sp. on decolorization of distillery effluent.

Rajeeva Gaur and N.S. Darmwal

Department of Microbiology

Dr. Ram Manohar Lohia Avadh University, Faizabad (U.P.), India

E-mail: rajeevagaur@rediffmail.com

A strain of Bacillus sp, isolated from effluent of distillery showed ability to remove melanoidin by 50% at pH 5.5 and temperature 45°C, further addition of yeast sludge of fermentor, 3g/l and cellulose, 0.5g/l increase the colour removal by 15%. A strain of Pseudomonas sp. having ability to decolorize melanoidin by 40% level was also used in mix culture with the Bacillus sp. No further, decolorization was reported in the same fermentation condition, therefore, the Bacillus sp. can alone be used for decolorization of distillery effluent at industrial level.

Cancer Biology

Nagarajuna Degree colleges, Kadapa

IT CAN

Autoantibodies in Oral Cancer

Surekha M Zingde

Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, India. szingde@actrec.gov.in

The development of cancer is strongly influenced by the host immune system. It is now demonstrated that cancer may elicit autologous immune response involving cytotoxic T-lymphocytes, T-helper cells as well as antibodies. There are reports of circulating antibodies to tumor antigens. It is becoming apparent that immune system responds to self-antigens, which are increased in expression, mutated, post translationally modified, or present in an altered cellular location. Though the role of autoantibodies in cancer biology is not very clear but evaluation of tumor antigens by antibody response could be used to identify biomarkers for early detection, prognosis and recurrence.

There are several studies in literature which evaluate the utility of single biomarkers for their clinical relevance for head and neck cancers. The capability of these markers for diagnosis, prognosis etc is controversial for several different reasons. The process of transformation is complex, multistep and multifactorial and it is apparent that multiple markers are required to define the transformed state. This has necessitated global profiling of proteins from tumor tissues/fluids etc. Recent studies use 2D/LC based separation of tumor tissue/cell line proteins and immunoblotting followed by mass-spectrometric for the identification of tumor antigens.

This presentation will summarize the information on auto antibodies in cancer and the identification of tumor antigens, which elicit an antibody response in cancer of buccal mucosa using immunoproteomics. Sera from patients with buccal mucosa cancer and healthy controls have been analysed for antibody-based reactivity against proteins from the KB cell line. Proteins were separated by 2D-PAGE and immunoblotted with sera IgGs of individual patients and healthy controls. Several antigens were detected by sera of cancer patients. Antigens detected by the patient’s sera were different among different individuals with presence of any single antigen ranging from 7-79%. Several of these antigens have been identified by mass-spectrometry and confirmed by immunostaining. These antigens are three forms of Enolase-I, Peroxiredoxin-VI, HSP-70, Pyruvate kinase, Annexin-II, ATP synthase, -tubulin, -tubulin, Phospho glycerate mutase, Triose phosphate isomerase, Aldose reductase and cyclophilin-A. Except HSP 70, these antigens are being reported in cancer of buccal mucosa for the first time. Initial results also show that autoantibody response against Enolase-I, HSP 70, Annexin II, Peroxiredoxin-VI, and Aldose reductase are also seen in patients with leukoplakia of buccal mucosa, which suggests early occurrence of these autoantibodies during the process of carcinogenesis. Work is ongoing towards generating a multiplex array of antigens to be used for validation and to assess their utility for early detection.

IT CAN

Junctional adhesion molecule A and its role in cancer cell metastasis

Meghna U. Naik, Tejal U. Naik and Ulhas P. Naik

Department of Biological Sciences, University of Delaware, Newark, DE 19716 U.S.A

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanism of regulation of this cellular invasiveness is poorly understood, however, it is believed that increased migratory behavior is associated with the highly invasive metastatic phenotype. Here we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is found to be expressed in breast cancer tissues. In breast cancer cell lines, its expression is differentially regulated and correlates inversely to the known ability of these cells to metastasize. A similar inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed. T47D cells, which migrate least, are found to express high levels of JAM-A followed by MCF-7 and MDA-MB-468 cells. MDA-MB-231, which is highly migratory, is found to express the least amount of JAM-A. Overexpression of JAM-A inhibited MDA-MB-231 cell migration as well as invasion through collagen gel. Furthermore, knockdown of JAM-A using siRNA enhanced invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion was found to be due to formation of functional tight junctions. These results identify, for the first time, an Ig-superfamily cell adhesion protein expressed at tight junctions as a key negative regulator of breast cancer cell invasion and possibly metastasis. These results also suggest that JAM-A may serve as a useful marker for invasive breast cancer.

IT CAN:

Aberrations in Transforming Growth Factor Beta Signaling in Human Cancers

Devarajan Karunagaran

Department of Biotechnology, Indian Institute of Technology Madras, Chennai – 600 036

Loss of sensitivity to Transforming Growth Factor Beta (TGF-β) is known to occur in human carcinomas due to inactivating mutations in genes encoding TGF-beta signaling intermediates. TGF-β functions through its transmembrane receptors, TGF-β Receptor I and TGF-β Receptor II and intermediate signaling molecules, the Smad proteins that act as transcriptional regulators and tumor suppressors. Activation of the signaling pathway results in phosphorylation of Smads and their translocation to the nucleus to bring about transcriptional regulation of target genes. We probed, for the first time, for alterations in Smad 2 and Smad 4 genes using human cervical cancer cell lines and human cervical tissue samples. Using PCR/RT-PCR, single stranded conformation polymorphism analysis and DNA sequencing, we observed a deletion of ‘G’ in the L3 loop (crucial in Smad-receptor interaction) in C-33A cells, and an insertion of ‘A’ in codon 122 (loss of MH2 domain) from a cervical tumor sample, both of which caused frame shift and pretermination in Smad 2. In addition, a G/A transition at 31 bp upstream-nontranslated regions of exon 8 of Smad 4 was found in Bu 25TK cells. Smad 2 expression was less in some of the cervical tumor samples than that of nonmalignant samples and 6 cancer samples showed C-terminal deletions that abolish Smad 2 phosphorylation sites. The loss of expression of Smad 4 found in some cervical tumor samples was due to transcription loss rather than deletion of the gene. Our results highlight an important role for Smad 2 and Smad 4 in human cervical tumors.

We also focused on the mechanisms of TGF-β/Smad signaling using human ovarian cancer and normal tissues. Mutational screening showed 25% of the tumor samples harboring the low penetrant tumor marker TGF-βRI 6A*allele. Two samples were found to have a deletion of forty-five nucleotides in exon I of TGF-β RI. 20% of the samples analyzed had mutations in the Big Adenine repeat of TGF-b RII; this was accompanied by a loss of the protein in many of the samples analyzed. Another marked feature was the loss of Smad-4 protein expression in some of the tumor samples. TGF-β signaling pathway is emerging as an attractive target in cancer research. Future studies correlating TGF-βRI 6A*allele incidence to the occurrence of cancers and identification of the key regulators of the TGF-β signaling will be of immense help to clinical research aimed at better prognosis and improved survival of ovarian cancer patients.

IT CAN

POLY N-ACETYLLACTOSAMINE SUBSTITUTED β 1,6 BRANCHED N-OLIGOSACCHARIDES FACILITATE LUNG-SPECIFIC METASTASIS OF MELANOMA CELLS.

Rajiv D. Kalraiya, Nithya Srinivasan

Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)Tata Memorial centre, Kharghar, Navi Mumbai.

Cancer cells show several metastases associated surface modifications. One such consistent modifications is the expression of β,6-branched N-oligosaccharides. The cell lines bearing these oligosaccharides metastasize either to the lungs or the liver. To understand their role in, and determinants of, lung or liver specific metastasis, the lung colonizing low (B16F1) and high (B16F10) metastatic variants of B16 melanoma were employed.

We earlier demonstrated that poly N-acetyl lactosamine (polylacNAc) substitutions on β1, 6 branched N-oligosaccharides facilitate adhesion of B16F10 cells to lung vascular endothelium via galectin-3- the high affinity lectin receptor for polylacNAc. However, for successful organ colonization, these cells need to displace endothelium, degrade vascular basement membrane, invade and interact with the parenchyma. These studies investigate if Galectin-3 - a multi-functional protein expressed at the highest levels in lungs and polylacNAc expressed on melanoma cells play a role in these later events of organ colonization.

These studies demonstrate that the melanoma cells show metastasis dependent adhesion and haptotactic motility towards galectin-3, which was inhibited by specific disaccharide lactose but not sucrose. Galectin-3 appeared to influence matrix degradation by inducing secretion of MMP-9 in a metastasis and dose dependent manner. Galectin-3 was found to be expressed in all the major tissue compartments of the lungs, thus facilitating these processes.

PolylacNAc is expressed on both N- and O- linked oligosaccharides; however, inhibition of N-but not O-linked oligosaccharides inhibited adhesion and metastasis. Inhibition of O-linked oligosaccharides in fact augmented metastases, apparently due to increased β1,6 branching on these cells. Antibodies to LAMP1, the major carrier of polylacNAc on N-oligosaccharides, significantly inhibited adhesion to galectin-3 and metastasis.

The results thus demonstrate that polylacNAc specifically on N-oligosaccharides on cancer cells and galectin-3 on the lungs facilitate all major steps of lung colonization.

IT CAN:

Identification of glioblastoma biomarkers with prognostic and therapeutic value using microarray

Kumaravel Somasundaram

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. Diffuse astrocytoma may be classified (as per WHO classification) as low-grade diffuse (DA; Grade II), anaplastic (AA; Grade III) and glioblastoma multiforme (Grade IV; GBM), in the order of increasing malignancy. Currently, these classifications are based on the observed histopathological characteristics of the tumor, which are sometimes subjective and inconsistent. GBM constitutes more than 80% of malignant gliomas and patients with GBM have a median survival of less than one year. Current treatments, including surgery, radiation therapy, and chemotherapy, unfortunately have not changed the natural history of these incurable neoplasms; and the prognosis of patients with GBMs has not improved significantly in the past 30 years. To find new diagnostic and therapeutic strategies, a better understanding of the biological pathway(s) leading to glial tumorigenesis is warranted.

With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray. We identified several differentially regulated genes between normal brain tissue and astrocytoma- DA and AA; progressive astrocytoma (DA, AA, secondary GBM) and primary GBM as well as between secondary and primary GBM. In addition, the expression pattern of a few novel markers characteristic to GBMs, in particular primary GBMs was confirmed by quantitative real time RT-PCR analysis on an independent set of 100 samples. The grade specific expression of some of the markers was also confirmed by immunohistochemical staining. Of the identified markers, we have demonstrated the utility of some of them as prognostic markers and therapeutic targets. The details will be presented. Thus we were able to identify potential astrocytoma biomarkers of diagnostic, prognostic and therapeutic value.

Can 1 Protein kinase C in Prostate Cancer: Bioinformatic and Bioenergetic Implications

Shailza Singh^*1 and D.K. Sharma¹

¹Center for Energy Studies, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016,India

E-mail: shailza_iitd@yahoo.com

The concept of cell signaling mechanism integrates bioenergetics and bioinformatics to account for living processes on the molecular level. To ensure signaling fidelity, kinases must be sufficiently specific and act only on a defined subset of cellular targets; this precision of specificity is essential for the integrity of signal transduction. Defects in protein kinase function result in a variety of diseases and kinases are major targets for drug design. The recent progress made in the crystallization of protein kinases ( > 50 crystal structures of protein kinases, in most cases complexed with ATP site-directed inhibitors, are available) has confirmed that the ATP-binding domain of protein kinases is indeed an attractive target for drug design. The structural information from the theoretically modeled complex may help us to clarify the catalytic mechanism of enzyme. Protein kinase C (PKC) involved in Prostate Cancer is an adapter protein implicated in the regulation of a large spectrum of signaling pathway. PKC is regulated by two distinct mechanisms: by binding 3 molecules of ATP which regulates the active site and subcellular localization of the enzyme, and by second messengers (Ca2+, diacylglycerol, phorbol esters) which promote PKC's membrane association and result to pseudosubstrate exposure. Here, in order to understand the mechanisms of ligand binding and the interaction between the ligand and the protein kinase C (PKC), a three-dimensional (3D) model of PKC is generated. The investigation is made to gain further insight into the structural basis for complex of PKC with ATP. In order to design novel and higher affinity ligands, an understanding of the interaction between ATP and PKC at the molecular level would be valuable. For the reason of taking the interacting mode of PKC with ATP, AUTODOCK was used to perform the automated molecular docking. The best binding structures of the ligand to the receptor based on the energy of the ligand/receptor complex were automatically found and in this procedure a combination of Monte Carlo type and stimulated annealing procedure to dock a guest molecule with a host was employed.

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Can3

Effect of experimental diabetes on DNA degradation, GLUT4 translocation and Membrane linked function in rat tissues and their reversal by insulin and antidiabetic compounds.

Pardeep Kumar ^{1, 2}, Asia Taha^1, M.R. Siddiqui,¹ R.K.Kale² , D. Sharma¹, and Najma Z. Baquer¹

¹ Neurobiology Laboratory, ² Cancer and Radiation Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

E-mail: epardeep@gmail.com

Metabolic disturbances and their consequences in diabetes mellitus one well knows. Our laboratory has been carrying out extensive work on the regulation and control of metabolic pathways in experimental diabetes and the reversal of the diabetic complication using insulin, vanadate and plant products at physiological, biochemical and molecular levels. In this report we present the changes in four very important metabolic parameters in diabetes and their removal by insulin, Trigonella seed powder (TSP) and a trace metal vanadium. The four parameters studied include DNA degradation. Glut-4 translocation, Na⁺/K⁺ ATPase alpha isoform levels and monoamine oxidase activity.

Methods: Animals were made diabetes by injecting 15mg/100g body weight alloxan to each rat. Diabetes animals were given Trigonella seed powder (5% w/w) mixed with their standard food together with lower doses of vanadate in the drinking water (0.2mg/ml) for 21 days. DNA degradation in liver and whole brain was determined by using DNA laddering method. The expression and distribution of glucose transporter-4 (GLUT4) was measured by Western blotting and Immunohistochemical techniques in the membrane fractions of cardiac and skeletal muscle. Relative abundances of Na⁺/K⁺ ATPase subunit isoform in heart and kidney was determined by Western blot analysis. Monoamine oxidase (MAO) activity was measured in synaptosomes fractions of whole brain.

Results: The effect of parameters like body weight and plasma glucose levels of alloxan diabetic rats showed a near complete reversal expects in dose of Trigonella treatment where the glucose homeostasis was restored partially. The efficacy of the treatments was further examined by measuring the levels of glucose transporter (GLUT4) in both skeletal and cardiac muscle. Protein expression of alpha-1 isoform of Na⁺/K⁺ ATPase enzyme also showed a significant decrease in the heart and increase Kidney of diabetic animals. Activity of catecholamine degrading enzyme, MAO showed a significant increase in the synaptosomes membrane fractions of whole brain in diabetic animals. There was a marked decrease in the GLUT4 protein levels in the membrane fractions of the heart and skeletal muscle of diabetic animals. Immunohistochemical data of the diabetic skeletal muscle also showed the decreased distribution of GLUT4 protein. Conclusion: It was observed that Trigonella in combination with reduced doses of vanadate was most effective in correcting the variations in all the parameters chosen and can indeed be considered as an alternative to be explored further for clinical trials as a means of amelioration of diabetes in human subjects.

Can 4

Down-Regulation of Creatine/Creatine Kinase System in Sarcoma Tissue:

A Potential Diagnostic Marker for Tumor Progression

Soumen Bera, Alok Ghosh, Subhankar Ray and Manju Ray

Department of Biological Chemistry, Indian Association for the Cultivation of Science,

Kolkata 700 032, India; email: soumen_bera@yahoo.co.in

In muscle cellular energy metabolism, the phosphocreatine/creatine/creatine kinase system plays a crucial role in maintaining the energy homeostasis by buffering the level of cellular ATP during muscle contraction and relaxation. Phosphocreatine is the main stored form of energy in muscle tissues, which can be readily utilized by the cells to regenerate ATP during need through creatine kinase catalyzed reaction. The demand for ATP is also very high in rapidly growing tumor cells. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with progression of malignancy. In the present study, sarcoma was developed in one hind leg muscle of mice by injecting either the carcinogen 3-methylcholanthrene or existing sarcoma 180 cells. Creatine, phospho-creatine and creatine kinase decreased with progression of malignancy and reached very low level in the final stage of sarcoma development. Whereas, all these parameters remained unaltered in the unaffected contra lateral hind leg muscle of the same animal. Immunoblot with antibodies against cytosolic muscle type creatine kinase (MCK) and sarcomeric mitochondrial creatine kinase (sMitCK) showed that both these isoforms decreased with progression of malignancy in mice. mRNA of MCK was apparently no longer expressed, and expression of sMitCK was severely down regulated. Significantly decreased levels of creatine and creatine kinase isoforms suggest (i) that the genuine muscle phenotype is lost during sarcoma progression, and (ii) these parameters can be used as diagnostic marker and prognostic indicator of malignancy in this tissue.

Can 5

Baicalein, the 12-Lipoxygenase inhibitor, and Celecoxib, the selective COX-2 inhibitor induce apoptosis in Skin cancer cell line (A431)

Smita Agarwal,Karnati R Roy,Chandrani Acharia, GV reddy and P.Reddanna

Department of Animal Sciences, School of Life Sciences, University of Hyderabad. Hyderabad – 500 046.

E-mail: smita_agarwal1978@yahoo.com

Poly unsaturated fatty acids (PUFAs), including Arachidonic acid (AA) and linoleic acid have been shown to induce tumorigenesis. Further studies on relationship between PUFA and carcinogenesis have led to the identification of new molecular targets in cancer chemoprevention and treatment .These targets include Arachidonic acid metabolizing enzymes namely the Cyclooxygenase(COX) and the Lipoxygenase(LOX), which lead to the formation of various eicosanoids involved in a variety of human diseases, such as inflammation ,fever, arthritis and cancer. Recent studies reveal mammalian skin as a tissue with an extensive and most diverse lipoxygenase catalyzed fatty acid metabolism.

In the present study the relative contribution of the various family members of AA metabolizing enzymes in A431 Human Skin Cancer cell line was determined. To study this question, the expression of five AA metabolizing enzymes (5-LOX,15-LOX,12-LOX,COX-1 and COX-2)as well as 5-lipoxygenase activating protein (FLAP) were analyzed in A431 Human Skin cancer cell lines using reverse transcription-PCR.These studies revealed the expression of COX-1, COX-2, FLAP and 12-LOX in the skin cancer cell line. Using selective biochemical AA metabolizing enzyme inhibitors, we then evaluated their effect on the proliferation of the cell line. These studies revealed potent inhibition in the growth of the skin cancer cell line by Baicalein,the 12-LOX inhibitor and Celecoxib,the selective COX-2 inhibitor.The combination of baicalein and Celecoxib,however,showed more potent inhibition in the growth of these cells. The molecular mechanisms involved in the induction of apoptosis in skin cancer cells by COX and LOX inhibitions will be presented These studies open up a new avenue for Skin cancer therapy involving inhibition of eicosanoids biosynthesis.

CAN 6

Imatinib-resistant K562 cells are more sensitive to celecoxib, a selective

COX-2 inhibitor: Role of COX-2 and MDR-1

Kalle M. Arunasree, D. Praveen, Karnati R. Roy, Kotha Anilkumar, A. Aparna,

Gorla Venkateswara Reddy, Pallu Reddanna

Department of Animal Sciences, University of Hyderabad, A.P., India – 500 046

Email: mkarunasree@gmail.com

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. More than 90% of CML cases and 30-40% of acute lymphoblastic leukemia (ALL) cases are associated with the presence of the Philadelphia chromosome (Ph⁺). The Philadelphia chromosome is the result of a reciprocal translocation between 9 and 22 chromosomes that fuses Bcr-encoded sequences to a truncated c-Abl. The fusion protein produced has increased protein tyrosine kinase (TK) activity of Abl that is responsible for the malignancy. The BCR/ABL tyrosine kinase in the cytosol activates various intracellular signaling pathways, those involving Ras, Rap1, B-Raf, Raf-1, Erk, PI-3K, STAT5 and NF-kB, which normally play role in the regulation of hematopoiesis by hematopoietic cytokines and other extracellular stimuli. Imatinib mesylate (Gleevec - 2-phenyl amino pyrimidine compound) a specific inhibitor of several TKs, ABL, ABL-related gene product (ARG), c-KIT and PDGFR, induce complete hematologic and cytogenetic remissions in most patients with chronic phase CML. Despite significant hematologic and cytogenetic responses with imatinib, resistance occurs, particularly in patients with advanced disease. In the present study we demonstrate that COX-2 and MDR-1 are over expressed in imatinib-resistant K562 (IR-K562) cells compared to imatinib-sensitive K562 cells. So, we sought to examine the effect of celecoxib, a selective COX-2 inhibitor, on imatinib-resistant K562 (IR-K562) cells. The results clearly indicate that celecoxib is more effective in inhibiting the growth of IR-K562 cells with a lower IC₅₀ value of 10 µM compared to an IC₅₀ value of 40 µM in K562 cells. In the IR K562cells, celecoxib effectively decreased PGE₂ production but showed no effect on BCR/ABL kinase expression and activity. The effects of celecoxib in IR-K562 appear to be mediated by a COX-2 dependent mechanism. Further studies revealed that celecoxib induces apoptosis in IR-K562 cells by decreasing the Bcl₂/Bax ratio, alteration in mitochondrial membrane permeability, cytochrome c leakage into the cytosol, PARP cleavage and nuclear disintegration. In the light of more potent effects of celecoxib in IR K562 cells than in K562 cells, further studies were undertaken to evaluate the synergistic effects of celecoxib and imatinib. These studies revealed that celecoxib at 1 µM concentration enhances the effects of imatinib by reducing the IC₅₀ value of imatinib from 10 µM to 6 µM.

In conclusion the present study demonstrates that the development of resistance in K562 cells is mediated through the over-expression of COX-2 and MDR-1 and hence IR-K562 cells respond positively to COX-2 inhibitors. The studies also point the possible utility of COX-2 inhibitors in adjuvant therapy to overcome drug resistance in chronic myeloid leukemia patients.

CAN 7

Apoptosis in Sarcoidosis

Archana Bhatnagar ¹, Sandeep Singla² and D Behera²

¹ Department of Biochemistry, Panjab University, Chandigarh, India. Email : abhat19@yahoo.co.in

² Department of Pulmonary Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh

Introduction:

Sarcoidosis is a multisystemic granulomatous disease primarily affecting lung and lymphatic system. The understanding of the pathogenesis of the disease has rapidly progressed which has been contributed significantly by the study of bronchoalveolar lavage cells. Activated macrophages and T-cells have been identified in different compartments of the sarcoid lung and the characteristics of activation suggest that the cells become activated in the course of normal immune response. Apoptosis is being increasingly recognized in granulomatous lung diseases. This study was carried out to assess the presence of Fas, Fas Ligand, Bcl-2 and Bax proteins in Bronchoalveolar lavage fluid (BAL) and peripheral blood mononuclear cells (PBMC).

Material & Methods:

Samples from 15 biopsy proven cases of sarcoidosis and 13 age and sex matched controls were taken for the study after informed consent. None of them was under treatment with corticosteroids. Bronchoscopy was performed and BAL fluid was collected. Fluorochrome labeled monoclonal antibodies were used to detect the percent positive cells expressing the particular molecule. Pearson’s correlation coefficient was used to establish correlation between the estimations and samples. Mann Whitney U test was used to compare the estimations between the BAL samples and peripheral blood samples.

Results:

The levels of apoptotic (both pro and anti) markers are as follows:

Fas expression :

In BAL – (patient)= CD4 68.56+ 10.58 %, CD8 = 32.71+ 5.6%, CD14 = 40.35 + 7.6%

(control)= CD4 6.33+ 1.66% , CD8 = 3.80+0.66%, CD14 = 7.71+0.67%

In PBMC - (patient) = CD4 18.92+ 4.07 %, CD8 = 10.38+ 2.5%, CD14 = 18.33 + 2.9%

(control) = CD4 2.48+ 0.67 %, CD8= 2.83 + 0.45%, CD14 = 3.95+ 0.32%

Fas Ligand expression :

In BAL –(patient) = CD4 60.51+2.70%, CD8 = 29.77+ 6.5%, CD14 = 62.35 + 5.6%

(control)= CD4 5.46+1.46%, CD8 6.08+0.89%, CD14 7.42+0.97%

In PBMC - (patient)= CD4 20.39+ 3.07 %, CD8 = 10.43+ 4.5%, CD14 = 20.03 + 2.7%

(control) = CD4 2.57+ 0.8%, CD8 2.05+0.34%, CD14 2.33+ 0.3%

Bcl-2 expression:

In BAL – (patient) = CD4 47.94+ 6.58 %, CD8 = 40.97+ 8.2%, CD14 = 43.45 + 6.23%

(control) CD4 4.73 +1.74%, CD8 6.65+ 0.6%, CD14 6.23+0.6%

In PBMC - (patient)= CD4 14.09+ 2.39 %, CD8 = 17.45+3.99%, CD14 = 21.37 + 4.3%

(control) = CD4 2.57+ 0.8%, CD8 3.15+ 0.5%, CD14 2.33+ 0.3%

Bax expression:

In BAL – (patient) = CD4 37.51+ 3.15 %, CD8 = 37.58+ 5.55%, CD14 = 57.56 + 4.3%

(control) CD4 6.33+0.86%, CD8 6.24+0.79%, CD14 6.2+0.5%

In PBMC - (patient)= CD4 9.62+ 3.07 %, CD8 = 16. 58+ 4.23%, CD14 = 20.63 + 2.7%

(control) = CD4 2.06+ 0.6%, CD8 2.33+ 0.29%, CD14 2.41+ 0.28%

CONCLUSIONS: Our results suggest that the apoptotic proteins are overexpressed in alveolar lymphocytes and macrophages, which characterize the alveolitis in sarcoidosis and could provide insights into the pathogenesis of the disease and prove useful as a marker of disease activity or response to therapy.

Can 8

15-LOX metabolites induce apoptosis in Jurkat cells by NADPH Oxidase mediated ROS generation and subsequent activation of extrinsic

and intrinsic death pathways

Anilkumar Kotha, Aparna A, Nishant P. Reddy, Arunasree MK, Reddy GV, Reddanna P

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad- 500 046, India

(E-mail:anilkumarkotha1231@rediffmail.com)

Lipoxygenases (LOXs) are a group of closely related non-heme iron containing dioxygenases, which catalyze the addition of molecular oxygen into polyunsaturated fatty acids (PUFAs) containing cis, cis 1-4 pentadiene structures to yield their hydroperoxy derivatives. LOXs are classified depending on their site of oxygen insertion on arachidonic acid (AA) into 5-, 8-, 12- and 15-LOXs and according to the positional specificity of arachidonate oxygenation into S and R isoforms. 15-LOX has two isoforms, 15-LOX-1 and 15-LOX-2. Linoleate (LA) is the preferred substrate for 15-LOX-1 which is metabolized to 13-(S)-HPODE that eventually gets reduced to 13-(S)-HODE. 15-LOX-2, on the other hand, oxygenates mainly AA to 15-(S)-HPETE that is reduced to 15-(S)-HETE. The role of various LOXs in regulating carcinogenesis was very well documented. 5-LOX, 8-LOX and 12-LOX were shown to have a procarcinogenic role, whereas 15-LOX was shown to be anti-carcinogenic. This study is designed to understand the molecular mechanisms mediating 15-LOX metabolite-induced apoptosis in human T cell leukemia cell line (Jurkat). Apoptosis induced by 15-LOX-2 metabolites in Jurkat cells was found to be very rapid and highly effective with hydroperoxy metabolite [15-(S)-HPETE] compared to the corresponding hydroxy metabolite [15-(S)-HETE]. 15-(S)-HPETE, the hydroperoxy metabolite of 15-LOX, inhibited the growth of Jurkat cells with in 3 h after exposure with an GI₅₀ value of 10 µM. The corresponding hydroxy metabolite (15-(S)-HETE), on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC₅₀ of 40 µM. The cells exposed to both 15-(S)-HPETE and 15-(S)-HETE showed increased expression of Fas ligand and caspase-8. The cells also showed Bid cleavage, cytochrome c release, caspase-3 activation, PARP-1 (poly (ADP) ribose polymerase-1) cleavage and DNA fragmentation. ROS generation studies using DCFH-DA by flowcytometry, inhibitory studies with DPI, a pharmacological inhibitor of NADPH oxidase, and NAC (N-acetyl cysteine), the precursor of glutathione, revealed that NADPH oxidase-mediated generation of ROS is responsible for 15-LOX metabolites –induced caspase-3 activation and Fas mediated apoptosis.

Can 9

Multidrug Resistance in Cancer: Role of Cyclooxygenase-2 and Reactive Oxygen Species

Roy KR, Nishant, PR, Reddy GV, Reddanna P.

Department of Animal Sciences, University of Hyderabad, Hyderabad- 500046, India

roykarnati@gmail.com

Multidrug resistance is the major limitation in cancer chemotherapy. The most important form of drug resistance is the over expression of the membrane-associated, 170 kDa, P-glycoprotein (P-gp) MDR1. To date, efforts to combat MDR1 have involved the use of functional modulators or reversal agents that block the MDR1 mediated efflux of anti cancer drugs. Intervention to prevent the transcription of MDR1 is important rather than blocking their function subsequent to its expression. Among different regulators of MDR1 transcription reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) were shown to be key players but the mechanisms underlying are not well elucidated. Using experimental and in silico approaches, an attempt was made in the present study to elucidate the role of ROS and COX-2 in the regulation of MDR1 expression and study the effect of antioxidant (C-Phycocyanin) and COX-2 inhibitor (Celecoxib) treatment on the regulation of MDR1 expression.

C-Phycocyanin (C-PC), a biliprotein from Spirulina platensis inhibited 2-AAF-induced expression of MDR1 in mouse macrophage cell line (RAW 264.7) (in vitro) and mouse model (in vivo). MDR1 induction by 2-AAF was dependent on ROS (reactive oxygen species)-Akt (protein kinase B)-NF-kB (Nuclear factor kappa B) signaling pathway. Generation of ROS, phosphorylation of Akt and corresponding nuclear translocation of NF-kB, the events that play a major role in the induction of MDR1 expression, were decreased significantly in C-PC treated cells. To further understand the mechanism, we created a computational model of the detailed ROS-Akt-NF-kB pathway. In silico results correlated well with the experimental trends.

The role of COX-2 in the regulation of MDR1 expression in hepatocellular carcinoma cell line, HepG2, was studied. Celecoxib, a selective inhibitor of COX-2, increased the accumulation of doxorubicin in HepG2 cells leading to enhanced sensitivity of the cells to doxorubicin. This enhanced sensitivity of HepG2 cells to doxorubicin by celecoxib is mediated by the down regulation of MDR1 expression. PGE₂, the COX-2 product, increased the expression of MDR1 and it is mediated through the increase in nuclear levels of AP-1, which is activated by signal transduction pathways involving ERK, JNK and p38. This was demonstrated by experimental and in silico studies. These studies suggest that antioxidants and selective COX-2 inhibitors can be employed successfully to overcome drug resistance in patients undergoing cancer chemotherapy.

Can 10

Meningiomas and Gliomas types with their derived cell lines of Glycosidase activity exhibiting similarity in β-Galactosidase and Hexosaminidase.

Prabha.M*+#, .N.Ramachandra Swamy*,V. Ravi # and K.Taranath Shetty+** Biochemistry Section*, Chemistry Department, Central College, Bangalore-560001. Department of Neurovirology# and Neurochemistry+, NIMHANS, Bangalore-560029.

Glycosidases such as β-Galactosidase, β-Glucosidase and Hexosaminidase showed higher specific activity in case of Brain tumors as compared to normal brain. β-Galactosidase was higher in all meningiomas and in majority of gliomas, while passages expressed more activity in majority of meningiomas and in almost all gliomas. β-Glucosidase exhibited almost similar activity in tumors to that of normal brain. Cell culture showed lower activity in passages except in few meningiomas atypical meningioma and in anaplastic astrocytoma gemistocystic corpus collusum. Hexosaminidase exhibited higher activity in all meningiomas and in majority of glioma tumors and in passages of all cell culture except fibrous meningioma. Therefore β-Galactosidase and Hexosaminidase exhibited similar activity in tumors and in their cultures of meningiomas and gliomas and confirms they exhibited similar activity in metabolism (catabolism) of tumors.

Key words: Glycosidases, β-Galactosidase, β-Glucosidase, Hexosaminidases, meningiomas, gliomas.

References:

David J. Kurz, Stephanie Decary, Ying Hong and Jorge D. Erusalimsky‡ Senescence-associated -galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. Journal of Cell Science. 2000; 113: 3613-3622.

De Martinez N R, Neme N, Canto J A. Alterations of glycosidases in benign, premalignant and malignant human lesions. Cancer Detect Prev. 1984; 7(1): 37-43.

Newmark J, Brady RO, Grimley PM et al. Amygdalin (Laetrile) and prunasin beta-glucosidases: distribution in germ-free rat and in human tumor tissue. Proc Natl Acad Sci .U S A. 1981; 78 (10): 6513-6.

O’Brien, John S., Okada Shinlaeo. Chem., Agnes and Fellerup Dorolthy L Sach’s Disease. Detection of heterozygotes and homozygotes by serum hexosaminidase assay. The New England Journal of Medicine. 1970; 283/1: 15-20.

Ramsey R. B., K. R. Smith Jr, D. C. Crafts, H. D. Chung and M. Fredericks. Hydrolytic enzyme activities of the nervous system. 1980; Arch Neurol. Vol 37, June: 356-359.

CAN 11

Chemopreventive effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei on 1, 2-Dimethyl hydrazine induced genotoxicity and preneoplastic lesions during colon carcinogenesis in rats.

Nikhlesh Kumar Singh*, Arvind Kumar and P.R. Sinha

Division of Animal Biochemistry, National Dairy Research Institute, Karnal, India

E-mail- nikhil24216@yahoo.com

Colon cancer is a major cause of death from cancer in western world. Diet is reported to account for about one-third of all cancer deaths in developed nations. There is an interest in the potential protective role of fermented milk containing probiotic cultures against colon cancer. In our lab a probiotic dahi was prepared by inoculating Lactobacillus acidophilus NCDC 14, Lactobacillus casei NCDC 19 and dahi culture Lactococcus lactis biovar diacetylactis NCDC-60. The dahi was of good flavour, body and texture having pH 4.6, titrable acidity 1.08, viscosity 0.270 Pa.s, fat 3% and 10⁸ cfu of above mentioned probiotic microorganisms. The antihypertensive, antidiabetic, antiatherogenic and immunomodulatory effects of the probiotic dahi had already been tested in rats. The present study was designed to determine the effect of probiotic dahi on 1, 2 dimethyl dihydarzine (DMH) induced colon carcinogenesis in wistar rats. Four experimental groups were used: 1) non-treatment control; 2) DMH control; 3) dahi-DMH-dahi group: dahi administered before and after DMH; 4) dahi-DMH: dahi given only 4 weeks before DMH. At 10 weeks of age, all animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20mg/kg body weight, once weekly for 15 weeks. The animals were sacrificed one week after the last injection, and the aberrant crypt foci (ACF, preneoplastic lesion for colon cancer) formation were visualized under light microscopy. Probiotic dahi significantly prevented the development of ACF (69%), decreasing the total number of AC and inhibiting cyst formation. Comet assay in lymphocytes was done to asses the DNA damage. A significant decrease in DNA damage (48%) was observed in dahi fed group as compare to DMH control group (94%).The feeding of probiotic dahi to rats significantly enhanced the activity of glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in the liver and colon cells. We speculate that the acidophilus casei dahi exerts chemoprevention against cancer development at extra hepatic sites by modulating hepatic biotransformation enzymes and antioxidant status. Furthermore, the findings also suggest that acidophilus casei dahi inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.

Can 12

Molecular genetic analyses of tumours from colorectal cancer patients suggest existence of alternate tumourigenesis pathway(s) in young patients from India

R Ratheesh ^1*, G Swarnalata ², K Viswakalyan ¹, A K Chaudhary ³, E C Reddy ³,

M Srinivasulu ⁴, M Lavanya ⁴, S Patnaik ⁵, M Vamsy ⁵, R A Sastry ⁶, V Anjayneyulu ⁴,

M D Bashyam ^{1, 3}.

¹Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

²Apollo Hospitals, Hyderabad, India

³National Genomics and Transcriptomics Facility, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

⁴ MNJ institute of Oncology and Regional Cancer Centre, Hyderabad, India

⁵Indo-American Cancer Institute and Research Centre, Hyderabad, India

⁶Nizam’s Institute of Medical Sciences, Hyderabad, India

*ratheesh@cdfd.org.in

Similar to other solid tumors, colorectal cancer (CRC) is thought to be an age-related disease, and usually occurs in older patients (>60 years). In the western population, deregulated Wnt signaling has been reported in a majority (~80%) of CRC patients. Adenomatous Polyposis Coli (APC) is one of the key tumor suppressor genes in Wnt signaling; mutations in APC are known to result in chromosomal instability (CIN) leading to CRC. Another form of genetic instability, the microsatellite instability (MSI), has been shown to be responsible for approximately 15% of the CRC cases. Familial syndromes, which account for <10% st="on">India, a rise in incidence of CRC among the young has been recently reported. The young patients account for almost 1/4th of the total CRC incidence as per the local hospital registries, often succumb to aggressive metastatic tumors and interestingly, usually present with no family history. In order to understand the early occurrence and aggressive nature of CRC in India, we employed a multi-pronged strategy including a) identification of status of Wnt signaling through Immunohistochemistry-based determination of intra cellular localization of β-catenin, b) screening for mutations in the APC tumor suppressor gene and c) screening for MSI. Our results revealed that a majority (>75%) of tumors from young patients exhibited an inactive Wnt signaling pathway, as opposed to tumors from older patients, a majority of whom harbored an active wnt signaling pathway (>75%); a statistically significant difference (P<10^-5). In addition, we have identified several novel mutations in the APC gene in tumors with active Wnt signaling. We could not detect any other significant difference between the two classes of patients with respect to microsatellite instability, tumor grade and stage, tumor location, gender and life style, etc. Our results therefore suggest the existence of alternate tumorigenesis pathway(s) in young sporadic CRC patients in India. Array-based studies are currently underway to identify the pathways responsible for the early occurrence of CRC in India.

Can 13

Role of β-catenin/Lef/Tcf signaling in tumorigenesis and tumor progression in ENU Induced Transplacental Glioma Rat Model

Gangadhara Reddy Sareddy¹, Sundaram Challa², Anita Mahadevan², Prakash Babu Phanithi^1*

¹Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, India

²Department of Pathology, Nizam Institute of Medical Sciences, Hyderabad, India

*Corresponding Author: prakashbabuphanithi@gmail.com

Gliomas are the most common and deadliest form of central nervous system neoplasms. Better understanding of complex regulation and key molecules involved in glioma pathology are needed for designing new and effective treatment modalities. β-catenin has two distinct roles in E-cadherin mediated cell adhesion and carcinogenesis by activating the Wnt/ β -catenin signaling pathway. In the present study we hypothesize that Wnt/β-catenin/Lef/Tcf signaling may have a role in gliomagenesis and its progression.

In this study, N-ethyl N-nitrosourea induced glioma rat model and C6 rat glioma cell lines were used to understand the role of β-Catenin, Lef1 and Tcf4 (key components of Wnt signaling pathway) and their target genes like cyclinD1, c-myc, N-myc and c-jun in the progression of tumor malignancy. 12 glioma rat brains of early (P90) and progressive (P135 and P180) stages were used for histological, immunofluorescence and immunoblot analysis. Western blot analysis reveals increased levels of β-catenin, Lef1 and Tcf4 and their targets in tumor tissues in comparison to control tissues. Further immunohistochemical staining and immunofluorescence studies demonstrated that β-catenin was prominent in cytoplasm and nucleus (which is the hallmark of Wnt pathway activation) while other proteins were localized in the nucleus of tumor cells. This study may indicate that β-catenin/Lef/Tcf signaling plays crucial role in the early and progressive stages of glioma malignancy. These studies suggest that nuclear translocation of b-catenin and activation of Wingless/Wnt signalling may represent an early event in liver carcinogenesis, providing a growth advantage in a subset of hepatic tumors with a more differentiated

References:

Cancer Research 61, 2085–2091, March 1, 2001

Carcinogenesis vol.24 no.3 pp.435–442, 2003

Cells Tissues Organs; 185:51–60, 2007

Int. J. Cancer (Pred. Oncol.): 84, 174–178 (1999)

Can 14

Characterization of Cancer Stem Cells in Retinoblastoma

Balla Murali Mohan Sagar, Geeta K Vemuganti, Chitra Kannabiran, Santosh Honavar, Ramesh Murthy, Sudhakar & Sreekanth Ravi stem cell Lab,

L.V. Prasad Eye Institute, Hyderabad 500034

bsagar327@gmail.com

Purpose: Retinoblastoma is the most common childhood cancer, caused by inactivation of the both alleles of RB gene either as sporadic or inherited manner following the well understood Knudsons two hit hypothesis. Some of the cells in the tumors are in the proliferative compartment of the tumor, called the “S” phase fraction of the tumor cells, while others are either dead or differentiated. In recent years, evolved the concept of Cancer stem cells suggesting that each tumor may have a population of cells that possess the capacity to proliferate extensively, resist chemotherapy or radiotherapy and survive to form new tumors in the body. This study aims at evaluating the presence of stem cell and proliferative markers in the retinoblastoma tumors.

Methods: Expression of ABCG2, Ki67 and P53 was evaluated by Immunohistochemistry in n=8 samples of paraffin embedded sections of retinoblastoma tumors. Results were compared with differentiation of the tumor cells.

Results: The mean age of the affected patients was 4 years. Histologically, they were diagnosed as well differentiated (n=2), moderately differentiated (n=1) and poorly differentiated (n=5.). All cases showed large no of Ki67 and p53 positive cells ( range 50-70%) in the viable tumor region, independent of differentiation. ABCG2 expression was noted in 30-90% of cells and did not correlate with differentiation.

Conclusion: Evidence points towards existence of putative stem cells within the proliferating population of retinoblastoma tumor cells, thus warranting further studies for confirmation.

Can 15

HSP90 REGULATES THE ACTIVITY OF RAF PROTEIN KINASE AND P53 IN STRESSED FISH HEPATOCYTES

E.Padmini¹, M.Usha Rani²

¹ Reader in biochemistry, ²Research scholar.

*Corresponding author and Mailing address:

E. Padmini,

Reader in Biochemistry,

P.G. & Research Department of Biochemistry,

Bharathi Women’s College,

Affiliated to university of Madras,

Chennai-600 108.

Tel.: +91 44 25286411; Fax: +91 44 25280473.

E-mail addresses: dstpadmini@rediffmail.com, epi622001@yahoo.com

Organisms are challenged by a multitude of stresses throughtout their lifespan which cause irreversible damage to proteins that in turn could impair cellular processes. In a normal cell, there is always a balance between pro- and antioxidant pathways. Upon stress stimuli, an imbalance of the redox milieu develops, and leads to the accumulation of ROS and induction of stress situation. The stress response to any exogenous, or endogenous perturbation under such conditions is necessary for the protection of cells from damage, and for survival. The molecular chaperone or stress protein HSP90 in addition to playing an important role in response to proteotoxic heat shock and other stresses, is critical for maintaining normal cellular homeostasis. This study investigated the regulation of HSP90 protein by Raf, the upstream kinase of Raf-MEK-MAPK cascade and their role in cell survival during environmental pollutant induced stress situations in Mugil cephalus. Additionally, regulation of p53 protein by HSP90 was also studied. Our results have demonstrated that reactive oxygen species via Raf protein kinase, enhances HSP90 protein expression which in turn favours cell survival through its proliferative and anti-apoptotic role. Also, HSP90 downregulates the upregulation of p53 apoptotic events by binding to and stabilizing its mutated form (produced by stress) further promoting the rate of cell survival. Since HSP90 comprises the core of several multimolecular chaperone complexes which interact with signaling proteins at different stages of their maturation and with different functional consequences, our results suggests that probably under this situation p53 plays an important role as one of the client protein for HSP90 in facilitating cell survival.

*This is a part of the work funded by DST, Government of India.

Can 16

HSP70 OVEREXPRESSION DOWNREGULATES ASK-1 EXPRESSION IN POLLUTION STRESSED FISH LIVER

E.Padmini¹, B.Vijaya Geetha²

¹ Reader in biochemistry, ²Research scholar.

P.G. & Research Department of Biochemistry,

Bharathi Women’s College,

Chennai-600 108.

Living systems continuously interact with their environment, and many factors in the environment are not kind to the organisms and the reflection of this incessant antagonism between the organism and environment is perceived by the organisms or its cells as “Stress”. Oxidative stress is known to induce apoptosis, a molecularly regulated cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Fascinating biochemical and genetic parallels exist between the cell death pathways of different animal species. However, an even more highly conserved cellular response can be engaged as a consequence of stress, which functions to maintain cellular survival and this response is mediated by the heat shock or stress proteins . The 70 kDa stress protein HSP70 plays important roles in a variety of physiological processes, including protein chaperoning protection against apoptosis, steroidogenesis and general stress response in vertebrate organism and has also been proposed as a early and sensitive biomarker of environmental stress such as toxicant exposure. It functions as sensor in stress signaling and exhibits crucial function in the maintenance of cell homeostasis and considered to be an antiapoptotic molecule by acting as a negative regulator of ASK-1 (Apoptosis Signal –Regulating Kinase). ASK-1 , a serine threonine kinase seems to be a intracellular target of HSP70 and the inhibition of ASK-1 activation is an important component of the mechanism by which HSP70 modulates stress activated signaling .Our study focused on the pollution induced oxidative stress mediated liver HSP70 variation in the Grey mullets that are surviving in contaminated Ennore estuary .The survival of the fish under such environmentally stressed condition may be due to overexpression of HSP70 that is mediated by downregulation of ASK-1 expression which promotes cell resistance against contamination stress induced apoptosis .

*This is a part of the work funded by DST, Government of India

Can 17

Studies on chlorpyrifos induced DNA damage in rat tissues

Nalini Srivastava, Radhey Shyam Verma and Anugya Mehta

School of Studies in Biochemistry

Jiwaji University, Gwalior 474 011

Pesticides are the chemicals which are purposefully added in environment to control domestic and agricultural pests. Organophosphate (OP) pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests. Chlorpyrifos (O, O’- dithyl-O-3,5,6-trichloro-2-pyrydyl phosphorothionate, CPF) is among the leading OP pesticides used extensively throughout the world including India. The main target of OP pesticides is acetylcholinesterase (AChE), which hydrolyses acetylcholine (ACh) in cholinergic synapses and in neuromuscular junctions where this enzyme plays a key role in cell to cell communication. Pesticides are known to produce oxidative stress, extensive data suggest that oxygen free radical formation can be a major contributor to the toxicity of pesticides. Pesticides have been considered potential chemical mutagens. A number of pesticides have been tested in a wide variety of mutagenic assays testing for gene mutation, chromosomal aberrations and DNA damage. The present study was undertaken to test the in vivo genotoxic potential of CPF in rats, using the single cell gel electrophoresis (or comet) assay. The rats were given different high doses of CPF on one, two and three days as well as low doses for longer periods. The level of DNA damage was estimated in liver and brain of CPF exposed rats and compared with control. The results clearly indicate that exposure to CPF, acutely or chronically, caused a dose-dependent increase in DNA damage in the liver and brain of rats. From the present study, it can be concluded that CPF exhibits genotoxic potential in vivo.

Can 18

Monocrotophos induced DNA damage in rat tissues

Santosh Kumar Yaduvanshi and Nalini Srivastava

School of Studies in Biochemistry

Jiwaji University, Gwalior 474 011

Pesticides are the chemicals which are purposefully added in environment to control domestic and agricultural pests. Organophosphorus (OP) compounds represent an important class of pesticides widely used in agriculture and domestic purposes. Monocrotophos (dimethyl-E-1-methyl-2- [metgylcarbamoyl] vinyl phosphate, MCP), commonly known as azodrin or nuvacron, is one of the organophosphate (OP) pesticides extensively used in agricultural practices throughout the world including India. It is one of the most avian toxic OP pesticide. The present study was designed to determine whether MCP can damage DNA in tissues of pesticide exposed animals. Adult male albino rats of Wistar strain were used as the animal model in the present study. Different groups of rats were given ¼ LD₅₀ and ½ LD₅₀ equivalent of MCP once orally and tissues namely liver, kidney, spleen, brain and blood were collected and DNA damage was studied by comet assay. The rats were also given low doses of MCP for two months for study of chronic toxicity. Results showed that MCP exposure caused extensive DNA damage in all the rat tissues. The damage was repaired to some extent if the comet assay was performed 24 hr, 48 hr and 72 hr post MCP treatment suggesting a time dependent repair of the damage.

Cell Biology

SVDC and their Institutions, Kadapa and Tirupati

IT CB

Malarial infection induces hydroxyl radical -mediated mitochondrial-dependant hepatocyte apoptosis

Mithu Guha, Pallab Maity, Uday Bandyopadhyay

Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4, Raja, S. C. Mullick Road, Jadavpur, Kolkata 700032

Liver damage and hepatocyte dysfunction are common during malarial infection. But the mechanism of liver damage is largely unknown. We have shown that malarial infection induces apoptosis in liver through the augmentation of oxidative stress and activating the mitochondrial pathway. The induction of apoptosis has been confirmed by TUNEL assay, transmission electron microscopy and caspase-3 assay. The activation of mitochondrial pathway of apoptosis has been established by gene expression analysis using RT-PCR, which indicates the significant down-regulation of Bcl-2 and up-regulation of Bax expression in liver of malaria infected mice. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Interestingly, malarial infection significantly decreases cardiolipin content, which favors the release of cytochrome c in the cytosol to activate caspase-9. Malarial infection induces the generation of hydroxyl radical (^·OH) in liver and application of ^·OH scavengers protects liver in vivo from malaria-induced activation of the mitochondrial pathway as well as the development of oxidative stress. Thus, it can be suggested that the induction of oxidative stress and apoptosis by ^·OH may be the cause for liver damage and hepatocyte dysfunction during malaria.

IT CB:

Inhibition of RelA phosphorylation sensitizes apoptosis in constitutive NF-kappaB-expressing and chemoresistant cells

Sunil K Manna

Division of Immunology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nacharam, Hyderabad 500076, India

Nuclear transcription factor kappa B (NF-kB) is an important transcription factor and has a role in cell cycle and oncogenesis and its regulation is important for cancer therapy. Overexpression, amplification, and rearrangements of different genes related to NF-kB have been observed in tumors. Constitutive expression of NF-kB leads to activation of several factors involved in cell cycle progression and cell differentiation for cancer metastasis. Considering its role in normal cell division, subunit such as Rel A (p65) specific regulation is one of the important strategies. In spite of the growing evidence of the important role of NF-kB in tumorigenesis and resistance to chemotherapy, only few attempts have been made to understand the mechanisms of constitutive activity of NF-kB in tumor cells and its regulation for successful therapy.

The thiazolidones and thiadiazolie have drawn considerable attention for their anti-bacterial, anti-fungal, and anti-inflammatory activities. We studied the anti-tumorogenic activity of several thiazolidine derivatives and their role of nuclear transcription factor kappaB (NF-kB) in this process. In constitutive NF-kB-expressing cells, treatment with 5-(4-methoxyarylimino)-2-N-(3,4-dichlorophenyl)-3-oxo-1,2,4-thiadiazolidine (P₃-25), one of such derivatives inhibited expression of NF-kB-dependent reporter gene, adhesion molecules, and cyclooxygenase. It downregulated phosphorylation of p65 by inhibiting upstream kinases, such as protein kinase A and casein kinase II, but did not alter NF-kB DNA binding activity. Alone P₃-25 induced apoptosis in NF-kB-expressing and doxorubicin-resistant breast cancer cells and in presence of other chemotherapeutic agents it potentiated apoptosis. Overall, our results suggest that P₃-25 exerts anti-tumorogenic activity by inhibiting phosphorylation of p65, the transcriptional active subunit of NF-kB through inhibiting its upstream kinases, and potentiates apoptosis mediated by chemotherapeutic agents. These results suggest novel approaches for design of anti-cancer drugs for combination chemotherapy.

IT CB

Apoptosis in Spodoptera frugiperda (Sf9) Cells: Phosphorylation of eIF2α is a cause and consequence.

Kolluru V A Ramaiah, Department of Biochemistry, University of Hyderabad, Hyderabad- 500 046.

Translational eukaryotic initiation factor 2 (eIF2), a heterotrimeric protein with a, b and g- subunits, plays a key role in the initiation step of protein synthesis. Phosphorylation of the alpha-subunit by a family of eIF2a kinases inhibits protein synthesis in general or stimulates translation in a gene-specific manner. Phosphorylation of eIF2α is implicated in protecting and promoting apoptosis in mammalian cells. We have analyzed the phosphorylation status of eIF2α in the ovarian cells of Spodoptera frugiperda (Sf9), a lepidopteran insect and natural host of baculovirus, in response to wide variety of stress conditions that include DNA-damaging agents, virus infection and agents that promote stress in the endoplasmic reticulum (ER). Our observations suggest that eIF2α phosphorylation can occur in response to both ER and non-ER stress conditions and is correlated to the induction of an activated transcription factor-4 (ATF4) which in turn can induce genes involved in amino acid transport, glutathione biosynthesis and redox regulation. Phosphorylation of eIF2α is a cause and consequence of apoptosis in non-ER stress. Our observations also suggest that PKC activation is apparently involved in the UV-induced eIF2α phosphorylation and apoptosis. However, ER stress-induced eIF2α phosphrylation may be limited to inducing an unfolded protein response as seen by the induction an ER chaperone-like BiP but the incessant ER stress does not lead to apoptosis in Sf9 cells as has been observed in mammalian systems. (Support by DST to KVAR is acknowledged).

IT CB

Ocular Surface Reconstruction Using Autologous Oral Epithelial Cells

Yashoda Ghanekar¹, Geeta K. Vemuganti², Soundarya Laxmi Madhira¹, Anirban Bhaduri³, Virender Sangwan⁴ and Santosh Honavar³

¹Sudhakar and Sreekanth Ravi Stem Cell Laboratory, ²Opthalmic Pathology Services, ³Ocular Oncology and Oculoplastics, ⁴Cornea and Anterior Segment Services, L. V. Prasad Eye Institute, Kallam Anji Reddy Campus, Banjara Hills, Hyderabad 500034

Corneal epithelium, the outermost layer of cornea, is continuously regenerated by stem cells present in the adjacent limbal tissue. Limbal stem cell deficiency (LSCD), caused due to thermal/chemical injury or systemic inflammatory/autoimmune disorders, leads to loss of corneal epithelium and ocular surface integrity, finally leading to blindness. In such patients, ocular surface is reconstructed by culturing limbal stem cells from autologous or allogenous healthy eye and transplantation of sheet of cultured cells in the injured eye. Patients with bilateral LSCD, who need to undergo allogenous transplants, further need to take systemic immunosuppressants which compromise quality of life and are costly. To overcome these problems, sources of autologous stem cells that can functionally replace corneal epithelium are being explored. A possible alternative to limbal cells is oral mucosal epithelium, as it shares several morphological and phenotypic characteristics with corneal epithelial cells and is easily available. We have established and characterized cultures of oral mucosal epithelial cells using oral biopsies from healthy volunteers. Biopsies were cultivated as explant cultures on amniotic membrane. Cells from explant cultures formed healthy, stratified epithelial cultures. Electron microscopic studies showed that the epithelial cells formed gap junctions and tight junctions. Phenotypic characterization was also carried out to investigate expression of markers of corneal differentiation, stratified epithelia and epithelial stem cells by RT-PCR and immunohistochemistry. The results show that cultured oral epithelial cells express markers similar to cultured limbal cells and also harbor stem cells, making it a suitable substitute for limbal cells. Clinical trial to reconstruct ocular surface of patients with bilateral LSCD using these cultured cells is currently underway.

CB 1

Co-exposure of ethanol and rotenone on cultured rat hepatocytes leading to oxidative stress and cell death

S Mehrothra , A Gupta¹, S. Singh¹, and P Kakkar²

Department of Biochemistry, Lucknow University, Lucknow 226 001, UP.

Sudhirankush@yahoo.com

1 Department of Biochemistry, Dr RML Avadh University, Faziabad –1, UP

2 Herbal Research Lab, ITRC, MG Marg, PB 80, Lucknow, 226 001, UP.

The last two decades in particular have witnessed a growing scientific concern, public debate and media attention over the possible deleterious effects in human and wild life that may result from exposure to chemical insecticides that may have the potential to interfere with the biological systems. It is known that acute ethanol intoxication increases free radical production and oxidative stress related changes in hepatic tissues, and pesticidal activity to rotenone is attributed to irreversible binding and inactivation of complex I of mitochondrial electron transport chain, thereby inhibiting oxidative phosphorylation.

Our work with ethanol and rotenone induced decrease in cell viability and oxidative stress in fresh hepatocytes was studied in order to understand their role in cytotoxicity.Viability of hepatocytes by methyl thiazoletetrazolium (MTT) reduction method and release of lactate dehydrogenase along with melondialdehyde formation indicated hepatotoxic action of ethanol and rotenone. Results showed that both ethanol and rotenone are able to generate oxidative stress in hepatocytes individually and responsible for decreased cell viability, but in the co-exposure of ethanol and rotenone, it was found that large population of cell damage occurs even at low concentration of above.

CB 2

Oxidative stress and antioxidant status in the erythrocytes of amyotrophic lateral sclerosis patients

G. Nagesh Babu, Alok Kumar, Jayantee Kalita and U.K. Misra

Department of Neurology, SGPG Institute of Medical Sciences, Lucknow, UP, India

gnageshbabu@yahoo.com

Free radicals have been implicated in numerous disease processes including motor neuron degeneration observed in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects lower and upper motor neurons and causes significant mortality within five years of onset. Overproduction of hydrogen peroxide (H₂O₂), represented by increase in lipid peroxidation in erythrocytes compared to control, may be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). Owing to their ability to permeate through biological membranes, excess H₂O₂ may be present in the media surrounding motor neuron. Anti-oxidative defense enzymes (ADEs) [i.e., levels of Glutathione peroxidase (GSHPx), Superoxide dismutase (SOD) and Catalase (CAT) in erythrocytes are capable to detoxify the reactive oxygen species (produced endogenously or exogenously), but may also be structurally modified and inactivated by reactive oxygen species. Both balanced and coordinated ADEs activities are of utmost importance for their correct physiological function.

The extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 patients of SALS and 20 age and sex-matched controls. The result of the analysis of lipid peroxidation in MND patients confirmed that the levels of lipid peroxidation were much greater (2.581 ± 0.045) than in normal human (1.191 ± 0.036 nmol MDA/mg protein). Statistical evaluation revealed that the increase in the level of LPO in erythrocytes was (P<0.001)>2O₂ reduced/min./mg protein). Statistical evaluation revealed that the decrease in the Catalase activity in erythrocytes was significant (P<0.01).>

During ALS, it was found that, lipid peroxidation started to increase and catalase activity started to decrease as the disease progressed from 6 to 24 months. A second-degree polynomial regression was applied to the data to plot the correlation between the biochemical markers and the duration of the disease. On the other hand, erythrocytes of MND patients did not show any significant differences in GSHPx and SOD activities with respect to controls. This study confirms the involvement of oxidative stress in ALS and it appears that ALS progressed rapidly in the absence of a significant rise in the activities of GSHPx and SOD.

CB-3

Production of extra cellular neutral Protease by Immobilization ofAspergillus oryzae (NCIM 637)

K. Jaya Raju ¹ , E.Kanthi Sameera ²

Centre for Biotechnology,

Department of Chemical Engineering,

College of Engineering, Andhra University,

Visakhapatnam – 530 003, India.

¹ kj.raju.chemical@aucevizag.ac.in

^{2 kanthisameera@gmail.com}

The fungal spores of Aspergillus oryzae (NCIM 637) were immobilized in Calcium alginate beads and used for neutral protease production. The maximum protease activity (300μg/ml) was observed at 3% (w/v) Sodium alginate and 3% Cacl₂ concentration. These immobilized cells were grown in a medium containing fish meal (FM) of marine waste. Submerged fermentation was carried out in a basal medium containing 0.1% K₂HPO₄ and 0.05% MgSO₄.7H₂O, 1% of ammonium nitrate and 0.1% NaCl. The fermentation conditions like effect of incubation period (5 days), inoculum level (200 beads), temperature (30⁰C) and pH (7) on protease production were optimized. The effect of sodium alginate concentration, effect of CaCl₂ concentration, effect of number of beads and effect of varying bead size and reusability of gel matrix (repeated batch) were studied. The study indicates the suitability of fish meal as a substrate for the production of protease.

CB 4

Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species -mediated cardiolipin oxidation.

Subhankar Dolai, Rajesh Kumar Yadav, Swati Pal and Subrata Adak.

Division of Structural Biology and Bioinformatics , Indian Institute of Chemical Biology,

4, Raja S.C. Mullick Road, Kolkata – 700032, India

E.mail: subhankar_dolai@yahoo.co.in

Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions have revealed that LmAPX is localized within the mitochondria by N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western-blot analysis with anti-LmAPX antibody was further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that mature enzyme (~3.6 kDa shorter than theoretical value of whole gene) was present in intermembrane space side of inner membrane. Moreover, expression of LmAPX gene is increased by treatments with exogenous H₂O₂, indicating that LmAPX is induced by oxidative stress. To investigate the biological role of LmAPX we have generated Leishmania cells overexpressing LmAPX to mitochondria. Flowcytometric analysis and IC₅₀ measurement suggest that overexpression of LmAPX causes depletion of mitochondrial ROS burden, and confers a protection against mitochondrial cardiolipin oxidation and an increase in tolerance to H₂O₂. These results suggest that single copy LmAPX gene plays a protecting role against oxidative damage.

CB 5

Isolation of Stem Cells from Human Umbilical Cord Blood

Nishanth P. Reddy¹, Ramakrishna BS¹, Mohan C. Vemuri², Reddanna P¹

¹Department of Animal Sciences, University of Hyderabad, A.P., India – 500 046

²Senior scientist-Stem cells, Reprogenetics, West Orange New Jersy 07052, USA

email: nishant.reddyp@gmail.com

Umbilical cord blood (UCB) is gaining more prominence in recent times as a source of nonembryonic multipotent stem cells. Global annual human birth rate (100 million) presents UCB as the largest non-controversial stem cell source, with an added advantage of naive immune status. UCB is a rich source of hematopoietic stem and progenitor cells but contains fewer T cells than bone marrow, which may permit greater degree of mismatch without increased graft versus host defense (GvHD). Cord blood stem cells are routinely utilized in stem cell transplantation in leukemia patients and carry huge potential to treat other human malignant and non-malignant diseases with less concern of rejection. Studies have shown that the rate of neutrophil and platelet engraftment correlates with the number of CD34⁺ cells. Because UCB contains low number of stem cells, their use is associated with significant delays in engraftment of neutrophils and platelets. Development of reliable methods for isolation and expansion of cord blood stem cells is critical for consequent clinical application. In the present study methods were developed for isolation of maximum number of CD34⁺ cells from UCB, collected from local hospitals. We were successful in isolation of 0.44% CD34⁺ cells when compared to maximum of 0.37% cells, reported by other research groups till now. Several research groups have evaluated a number of formulations for their ability to support survival and expansion of hematopoietic stem cells (HSCs) from UCB. Key cytokines have been identified as extracellular regulators of hematopoiesis and have been routinely used in the isolation and expansion of human UCB stem cell population. Some cytokine combinations are G-CSF, SCF, Flt-3 ligand (FL), thrombopoieten (TPO), megakaryote growth and development factor, several interleukines (IL-1_, IL-2, IL-3, IL-6 and IL-7) and interferon-ﻻ. In particular, SCF, TPO and G-CSF were found to have beneficial effect during ex vivo expansion of human UCB stem cells grown in a serum/animal protein-free medium. As we have successfully developed a method for optimal isolation of CD34+ cells now the main focus of our lab is to evaluate existing approaches and to develop novel ex-vivo expansion methods for optimal growth of UCB stem cells.

CB6

Estimation of Magnesium uptake in Yeast by PIXE and its affect on invertase production

Mary Anupama,P¹*, Guru Mahesh G², Venkateswaulu.P³, Naveen Kumar.T⁴

1,2,4 Dept. of Biotechnology, ANITS, Sangivalasa, Visakhapatnam, A.P, India.

1,2,4 Dept. of Physics, ANITS, Sangivalasa, Visakhapatnam, A.P, India.

ABSTACT

Magnesium is known to highly influence the growth rate of yeast and also contribute to product formation. Saccharomyces cerevisiae NCIM 3288 was used as the experimental organism and the effect of Mg²⁺ supplementation on product yield were studied. The amount of Mg²⁺ taken up by the organism to from the optima yield of 84.5g/l from jaggery as the carbon source was analysed by PIXE (Proton Induced X-ray Emission). Simultaneously, the effect on invertase formation by the rapidly growing yeast was also estimated. It was observed that when the optimal concentration of Mg²⁺ was supplemented, the cells rapidly divided which is evident from the weight of the biomass, and these cells required more of invertase to hydrolyze the sucrose present in jaggery, therefore its content was found to be more when compared to the control.

* Author for correspondence

Dr.P.Mary Anupama

Sr.Assistnat Professor,

Email- palukuty@yahoo.com

Phone- 9885808345

Last author

T. Naveen Kumar

Email—naveenkumartelganagmail.com

Phone-9985043440.

CB7

Modulatory role of NO and antioxidant enzymes as adaptive mechanism(s) in chronic smokers

Padmavathi.P and N.Ch.Varadacharyulu

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur - 515 003, India.

Presenting author Email id: padma7yte@yahoo.co.uk.

Plasma total cholesterol (TC), triglycerides(TG), lipoprotein patterns (LDL-C, HDL-C and VLDL-C), nitrite and nitrate levels as well as red cell antioxidant enzymes were measured in twelve cigarette smoking (CS) male human volunteers (with past history of 7-10 years smoking, 10-15 cigarettes per day) to compare them with age and sex matched individuals who were healthy nonsmokers as controls to understand the effect(s) and mechanism(s) of smoking as preliminary study. The results of the study showed an increase in concentrations of nitrite and nitrate (NO₂&NO₃), total cholesterol, LDL cholesterol in plasma as well the contents of cholesterol and phospholipids in red cell membrane followed by a decrease in HDL-C with no significant change in VLDL-C and triglycerides. These changes indicated cardiac risk. Increased membrane as well as plasma lipid peroxidation (LPO) in smokers suggested free radical generation causing damage. Further more, increased activities of catalase, SOD, GPx and the content of GSH observed in the present study suggested a possible compensatory mechanism to prevent the free radical damage. Evidences reveal that NO might have played a role in these changes. Besides, changes in contents of red cell membrane cholesterol and phospholipids suggested an enrichment of membrane with phospholipids and cholesterol indicating alterations in physicochemical properties as well in structure, organization and function of the membrane.

CB 8

Neuroprotective Activity of Catechin and its Relevance to

Glaucoma Treatment

Venu Talla and Dorairajan Balasubramanian

Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad 500034.

Purpose: Glaucoma is a leading cause of irreversible blindness. It is characterized by progressive visual field defects and optic disc changes, which are attributed to retinal ganglion cell (RGC) death-a hallmark of glaucoma. The exact mechanism for initiation of RGC death in glaucoma is not known, but recent studies have shown that glaucomatous RGC death involves an apoptotic pathway, and is one of the earliest signs of the disease process. Recent reviews suggest that glutamate-mediated excitotoxicity, depletion of trophic factors due to high intra ocular pressure (IOP) and oxidative stress play major roles in glaucoma. Screening for a compound that can inhibit cell death induced by all the above mentioned factors will be of great importance. In the present study we report our results on catechin, which shows the protective effect on oxidative stress and glutamate-mediated excitotoxicity induced RGC apoptosis in cell culture experiments.

Methods: Rat retinal ganglion cell lines (RGC5) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FCS, at 37^oC in a 5% CO₂ incubator. Cellular morphology was assessed using light microscopy. RGC cell death was induced using either glutamate or hydrogen peroxide (H₂O₂), whose concentrations and time of incubation was optimized in order to get ~ 50% apoptosis. The percentage of cell death was quantified by MTT assay and apoptosis induction was assessed using Annexin V labeling and FACS analysis. The anti-oxidant property of catechin was assayed using ABTS assay. The protective affect of catechin on RGCs was analysed using MTT assay, Anexin V labeling and FACS analysis using a BD Aria instrument. The dose dependent effects of catechin were studied using MTT assay.

Results: 30 mM Glutamate and 500 M H₂O₂ upon o/n incubation (~12 hr) induced near 50% apoptosis in RGC5 cell lines. Light microscopic analysis of RGCs indicated that catechin does not show any cytotoxic affects even at 1 mM concentration. MTT assay, FACS analysis results indicated that catechin inhibits the oxidative stress and glutamate excitotoxicity-induced apoptosis of rat RGCs, in a dose dependent manner.

Conclusion: Catechin acts as a neuroprotective agent and might be useful in treating some of the neurological diseases like glaucoma, where the neuronal apoptosis is induced by excitotoxicity and oxidative stress. Further studies will be warranted to study its mechanism of action and effectiveness in animal models.

CB 9

Title: Evolution of a phosphotransferase of Bacillus anthracis from Histidine kinases.

Abid R. Mattoo, Souvik Maiti, Yogendra Singh.

Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi, 110007, India.

abid.mattoo@igib.res.in

Bacillus anthracis is a gram positive, spore forming bacterium which is the causative agent of anthrax. Sporulation in B. subtilis is regulated by the phosphorelay signal transduction pathway, which is activated by five histidine sensor kinases in response to stress signals. Comparison of the protein sequences of the phosphorelay components between B. subtilis and B. anthracis revealed high homology in the phosphorelay orthologs of Spo0F and Spo0A while Spo0B of B. anthracis showed 35% identity with the Spo0B of B. subtilis. Spo0B is an important component of phosphorelay, the pathway involved in the initiation of sporulation in Bacillus subtilis. Our bioinformatic, phylogenetic, and biochemical studies showed that Spo0B of Bacillus antharcis has evolved from citrate/malate kinases. During the course of evolution Spo0B has retained the characteristic histidine kinase boxes H, N, F, G₁ and G₂ and has acquired nucleotide binding domains, walker A and walker B of ATPases. Due to the presence of these domains, autophosphorylation and ATPase activity was observed in Spo0B of B. anthracis. The thermodynamic and binding studies of Mg-ATP to Spo0B using isothermal titration colorimetry (ITC) suggested that the binding is driven by favorable entropy changes and the reaction is exothermic, with the apparent K_d of 0.02mM. The value of dissociation constant (K_d =0.05mM) which was determined by the intrinsic fluorescence of trytophan of Spo0B was of similar value to that obtained by ITC studies. The purified Spo0B of B. anthracis also showed nucleoside diphosphate kinase like activity of phosphate transfer from nucleoside triphosphate to nucleoside diphosphate. These results indicate that Spo0B of B. anthracis has multiple enzymatic activities that may have roles both in vegetative growth as well as during initiation of sporulation.

CB 10

Title: Biochemical and molecular studies of Serine/threonine protein kinase PknK

Pawan Kumar¹,Dimple Ranawari², Vinay K Nandicoori², Yogendra Singh¹

1. Institute of Genomics and integrative Biology, Mall Road, Delhi, India 110007.

2. National Institute of Immunology, Aruna Asaf Ali Road, New Delhi, India 110067.

pawan.kumar@igib.res.in

Eukaryotic-like Ser/Thr protein kinases and phosphatases expressed by bacterial pathogens are often mediators of host/pathogen interactions. M. tuberculosis possesses eleven Ser/Thr protein kinases (STPKs) and one phosphatase. Recent genetic, biochemical and structural studies suggest that mycobacterial STPKs are implicated in the regulation of diverse processes including cell division and molecular transport and may even directly contribute to pathogenesis.

PknK is one of the soluble kinase amongst the 11 identified STPKs of Mycobacterium tuberculosis. In order to biochemically characterize PknK it was cloned, expressed and purified and its biochemical properties and functional significance were analyzed. The full-length kinase as well as the kinase domain was shown to auto-phosphorylate on serine and threonine residues. Furthermore, our preliminary data suggests that PknK may be involved in regulation of Transcription of MymA operon. Characterization of PknK and the regulation of MymA operon by PknK will be discussed.

CB11

Title: Elucidation of signal transduction pathway through Serine/Threonine protein kinase PknI in Mycobacterium tuberculosis.

Andaleeb Sajid, Yogendra Singh

Institute of Genomics & Integrative Biology (CSIR), Mall Road, New Delhi, 110007, India

andaleeb.sajid@igib.res.in

Protein phosphorylation is a principal mechanism for transduction of extracellular signals into cellular response. This is carried out by specific protein kinases and is coupled to dephosphorylation by protein phosphatases. Serine/Threonine protein kinases (STPKs) have been shown to be involved in the survival of pathogens within the host. PknI (Rv2914c) is one of the 11 STPKs of Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Till date no substrate has been identified for PknI. In Mycobacterium tuberculosis genome, PknI exists along with three conserved hypothetical genes (Rv2912c, Rv2913c & Rv2915c). The conservation of these genes across mycobacterial genera and the emergence of PknI in the cluster in Mycobacterium tuberculosis suggest a possible regulation of these proteins via reversible phosphorylation by PknI and formation of an operon. The three genes of PknI cluster viz. Rv2912c, Rv2913c & Rv2915c were cloned, expressed and purified. The significance of these genes in mycobacterial metabolism, their conservation and possible interaction with PknI will be discussed.

CB12

Title: Regulation of effector proteins by multiple serine/threonine kinases and phosphatase in Mycobacterium tuberculosis.

Meetu Gupta, Kirti Sharma, Vibha Tandon, Yogendra Singh

Institute of Genomics & Integrative Biology (CSIR), Mall Road, New Delhi, 110007, India

meetu.gupta@igib.res.in

Phosphorylation dependent signal transduction between a sensor kinase and its cognate response regulator triggers the regulation of plethora of genes. These kinases have been implicated in diverse control mechanisms including stress responses, developmental changes and host-pathogen interactions in several microorganisms. The genome of M. tuberculosis, the causative agent for tuberculosis, has shown the presence of 11 genes that code for putative Ser/Thr protein kinases (STPKs). The mycobacterial STPKs regulate diverse processes by phosphorylating distinct substrates including proteins implicated in regulating cell division and morphology, an ABC transporter, the mediators of metabolism and a transcriptional regulator EmbR. We have earlier demonstrated that PknH/EmbR pair of M. tuberculosis regulates embCAB operon encoding arabinosyltransferases, thereby affecting the ethambutol resistance and the LAM/LM ratio - an important determinant for averting host’s immune response. Here we discuss EmbR phosphorylation by multiple ser/thr kinases viz. PknA, PknB and PknH in vitro. The idea of one response regulator protein communicating with multiple sensor kinases will also be discussed in reference to other novel substrates identified for multiple STPKs.

CB 13

Title: Regulation of stress dependent transcription factors by serine threonine protein kinases of Mycobacterium tuberculosis.

Preeti Sachdeva,^1,2, Azeet Narayan¹, Vani Brahmachari², Yogendra Singh¹

1. Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi, 110007, India.

2. Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, 110007, India.

preetisach_22@rediffmail.com

Mycobacterium tuberculosis is a remarkable pathogen capable of surviving in various hostile conditions via a highly regulated spatio-temporal and niche-adapted gene expression. Establishment of this pathogen in host system and its long-term survival in nonreplicating state is ensured by the coordinated shutdown of active metabolism, upregulation of virulence pathways and activation of stress responses through transcriptional reprogramming. The crucial role of protein phosphorylation at serine-threonine residues in fine-tuning gene expression that regulate energy balance and stress resistance has been well established in prokaryotes. One of the examples include mycobacterial serine-threonine kinase, PknH, shown to have a positive regulatory effect on transcription of the arabinosyltransferase genes via phosphorylation of a transcription factor. Comparative genome analysis and contextual information helped us identify pathogen specific regulatory proteins involved in regulation of stress dependent transcription. Biochemical studies showed that these proteins are phosphorylated by mycobacterial serine threonine kinases. These findings throw a light on regulation of transcriptional events central to survival of tubercle bug in host by serine threonine phosphorylation mediated signaling mechanisms. The implications of these phosphorylation events will be discussed.

CB 14

Unusual stability of protective antigen of anthrax toxin

Raj K kurupati^1,2, Pratibha M Luthra¹ and Yogendra Singh²

1. Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, 110007, India

2. Institute of Genomics and Integrative Biology(CSIR), Mall Road, Delhi, 110007, India

krajkumar13@gmail.com

Anthrax toxin comprises of three nontoxic proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF) that self-assemble and forms a series of toxic complexes. The central component PA monomeric protein oligomerizes to form a membrane insertion heptameric pore for delivery of LF and EF. Our previous studies revealed the importance of conformational stability of anthrax protective antigen in establishing cytotoxicity. In this study, we have analyzed the structural stability of PA by limited proteolysis using subtilisin. The proteolysis showed formation of specific pattern of nicked PA fragments at various time intervals. Interestingly, the cleaved fragments were efficiently and rapidly religated in the presence of aqueous organic solvents like acetonitrile, tetrahedrofuron and trifluroethanol. This observation was specific for subtilisin protease as no other protease showed this kind of activity. Electrophoretic analysis of the subtilisin nicked PA revealed the retention of structural integrity under native conditions. This study highlights the structural integrity of PA as the introduction of nicks by subtilisin like proteases does not disrupt the tertiary conformation.

CB 15

Comparative genomic study of spo0E family of genes in prokaryotes and elucidation of the role of Spo0E in Bacillus anthracis

Gyanendra P. Dubey^1,2, Sharmila Basu-Modak², Yogendra Singh¹

1. Institute of Genomics and integrative Biology, Mall Road, Delhi, India 110007.

2. Department of Zoology, University of Delhi, India 110 007.

gp@igib.res.in

In bacteria, to sporulate or retain in the vegetative form depends on the amount of phosphorylated Spo0A, regulated by Spo0E family of phosphatases. Phylogenetic analysis revealed that spo0E family of genes from aerobic and anaerobic sporulating bacteria fall into two distinct clades. In pathogenic species of genus Bacillus, the spo0E multigene family of phosphatases (SMFP) exhibit high sequence similarity. Spo0A of Bacillus anthracis, dephosphorylated by Spo0E of Bacillus subtilis did not bind the consensus sequence (5′-TGNCGAA-3′; ‘0A-like’ boxes). As putative SMFP and ‘0A-like’ boxes exist in Clostridia, therefore, it suggests that these SMFP may regulate Spo0A in this genus. Over expression of Spo0E of B. subtilis, which has retained its synteny with BAS1251 (B. anthracis), resulted into sporulation deficient phenotype in B. anthracis. Thus, spo0E of B. subtilis can complement BAS1251 in B. anthracis and retains specificity towards Spo0A, irrespective of sequence variability during evolution.

CB 16

IN VITRO MODEL FOR TRANS-DIFFERENTIATION OF BM-HSCS INTO HEPATOCYTES: AN IDEAL SYSTEM FOR MECHANISTIC ANALYSIS AND AN ALTERNATE SOURCE OF HEPATOCYTES

Satish Khurana and Asok Mukhopadhyay, NII, New Delhi.

svkanand@gmail.com

End-stage liver diseases are treated by orthotopic liver transplantation. Supporting acute liver failure patients until their own liver repairs itself may negate the need for a liver transplant. Growth limitations of primary cells have spurred attempts to find out alternate sources of hepatocytes. Reports on trans-differentiation of adult hematopoietic stem cells have opened up a new frontier, which has potential to act as an alternate source of hepatocytes.
It was reported that liver damage acts as an induction for the bone marrow cells to convert into hepatocytes. We utilized this notion to establish an in vitro system for differentiating bone marrow cells (BMCs) into hepatocytes and used this system to characterize the subpopulation of BMCs having hepatic potential. We used ECM coated plates to culture the BM cells in the presence of serum from liver damaged mice (DLS). By culturing different sub-populations of BM cells we analyzed their ability to transdifferentiate. Lin^- fraction of BMCs was found to be more potent in this regard. We combined this study with in vitro migration experiments to find out which BM sub-population has the natural tendency to migrate in response to liver damage. We found out that these are Lin^-CXCR4⁺OSMRβ⁺ cells. By using single cell culture assay by limiting dilution we found out that these cells can be clonally expanded and can make hepatic colonies. The hepatic phenotype of the differentiated cells was determined by immunostaining for liver specific markers, albumin and cytokeratin 18. The expression of other liver specific genes was examined by RT-PCR. Cytochrome P450 (CYP) activity was assessed by using pentoxyresorufin (PROD) assay and the results showed that the cells were functionally comparable to the primary hepatocytes. The ultra-structure of the trans-differentiated cells was analyzed by transmission electron microscopy, which revealed characteristic small size of nuclei and developing endoplasmic reticulum, as found in primary hepatocytes. Thus, the cultured cells may be considered suitable for in vivo therapies in case of liver disorders or for use in bioartificial liver devices.
Next, we started studying the molecular mechanisms involved in this differentiation process. Our preliminary results showed enhanced expression of OSMRβ and HGFR on the trans-differentiating cells. Further analysis showed that OSMRβ is expressed before HGFR gets expressed and was thought to be important for the differentiation events to occur. OSMRβ expression was accompanied by HNF4α, a transcriptional factor known to be involved in hepatocyte differentiation process in the development of fetal liver. It was found that OSMRβ and HGFR were not expressed together. We hypothesized that while OSMRβ and HNF4α are involved in the differentiation of the hematopoietic stem cell into progenitor hepatocytes, HGFR and HNF1α are important for the proliferation of these precursor cells. We confirmed this hypothesis using clonally expanded Lin^-CXCR4⁺OSMRβ⁺ cells on single cell basis, which showed that the cells first differentiate into albumin expressing hepatocyte and start dividing only after that.
In conclusion, we developed an in vitro model for generating hepatocytes from BM and studied the mechanism of trans-differentiation. The preliminary results showed that we could get functionally active hepatocytes from the BM cells. Our results showed the involvement of OSMRβ and HGFR in sequential manner for differentiation and cellular proliferation.

CB 17: Glucose uptake in Bovine Retinal Endothelial Cell after exposure to Advanced Glycation End Products (AGE-BSA)

S. Bharathi , N. Angayarkanni, Venil.N.Sumantran, S. Ramakrishnan

Biochemistry Research Department, Vision Research Foundation, Sankara Nethralaya, Chennai-600 006, email: drak@snmail.org

Purpose: Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease. High glucose is capable of impairing retinal endothelial cell barrier function directly and through advanced glycation end products. These products have been shown to increase the cell permeability. Therefore the aim of our study is to determine the effect of advanced glycation end product on the glucose (labeled) uptake in bovine retinal capillary endothelial cells (BREC) in vitro.

Method: AGE was prepared incubating 50mg/ml BSA with 0.5M glucose-6-phosphate in 0.2M PBS, pH 7.4 at 37⁰ C for 6 weeks. Bovine retinal endothelial cells were isolated and cultured in proprietary media formulated for endothelial cells. For experiments 3000cells/petriplate (100mm² dish) coated with 1% gelatin were put in high Glucose (4.5g/ L) DMEM medium with 10% FBS and allowed to grow for 3 days, followed by exposure to 100mg/ml AGE-BSA, BSA and control (untreated), up to 6 days, with redosing every 48 hours. Glucose uptake study was done as described by Lawrence et al. Since BREC characteristically show a rapid glucose uptake, we chose to assess the glucose uptake in the media from 10sec to 10 min. The dot blot assay for the VEGF and PEDF analysis was done also in the lysates.

Results: Glucose uptake in the BREC lysates, was seen in the increasing order of the control, BSA and AGE-BSA exposure. This trend was also observed in the corresponding media, as rapid loss of labeled glucose, at a time point, as early as 10 sec. AGE-BSA action on BREC was also independently confirmed by demonstration of an increase in the VEGF/PEDF ratio in the lysate on day 6.

Discussion: This is the first report showing increased glucose uptake in the cells exposed to AGE-BSA The increased uptake of glucose may be due to the permeability changes caused by AGE.

CB 18: Signaling molecules involved in the apoptosis of lymphocytes by Chromium (III) picolinate

Jana Mahadevan¹, Sankaramanivel Sundararaj, Anantanarayanan Rajaram², Rama Rajaram¹

Biochemistry Laboratory¹ & Biophysics Laboratory², Central Leather Research Institute, Adyar, Chennai, 600 020. India.

janaa79@yahoo.com

Chromium (III) has been recognized as an essential trace element in human nutrition as it plays an important role in fat and glucose metabolism. Chromium (III) picolinate [Cr(III)(pic)₃] has become a popular nutritional supplement in recent times and it is shown to bring down the sugar levels in diabetics. However its effect on the immune system is still poorly understood. We had earlier reported the induction of apoptosis in lymphocytes by [Cr(III)(pic)₃] and preliminary evidence suggested that reactive oxygen species (ROS) may be involved in bringing the about the process. In order to investigate the role of ROS in apoptosis brought about by [Cr(III)(pic)₃], lymphocytes have been treated with antioxidants before addition of [Cr(III)(pic)₃]. On pretreatment with antioxidants, the apoptotic morphological features due to [Cr(III)(pic)₃] could be reversed as is also the fragmentation of DNA. The activation of downstream signaling molecule such as lck, fyn and lyn could also be reversed by pretreatment with antioxidants. The apoptosis induced by [Cr(III)(pic)₃] seems to involve the alteration of the mitochondrial permeability transition state as revealed by confocal microscopy. The significance of the participation of mitochondrial membrane potential in the apoptotic process induced by [Cr(III)(pic)₃] will be reported.

Immunology

Spinco Biotech Pvt Ltd, Bangalore

Invited talk

HIV/AIDS pathogenesis and novel antiviral gene therapeutic approaches

Akhil C. Banerjea

National Institute of Immunology, New Delhi

HIV-1 infection leads to widely varying outcome of the disease ranging from slow to fast progression of HIV/AIDS with very small number of individuals who show protection against HIV-1 infection despite exposure to infection and are called long-term non-progressors. Very small number of individuals show almost complete protection against HIV-1 infection due to a remarkable deletion of 32 base pairs in the open reading frame of CCR5 chemokine receptor genes. Several host genes have been identified that impact the progression of HIV-1. Predominant among them are the chemokine genes (RANTES, SDF-1, MIp-1 etc), the chemokine receptors (CCR5 and CXCR4 – also known as HIV-1 co-receptors) and the interleukins that directly influence the surface expression of the above two HIV-1 coreceptors besides controlling several key immunological reactions. DC-SIGN is an important surface receptor on the surface of dendritic cells that is known for its very early interaction with HIV-1 and other pathogens and variations in the neck-repeat regions have been linked with decreased susceptibility to HIV-1 disease progression. Individuals with 32 base pair deletion (32) in the CCR5 HIV-1 coreceptor continue to lead a healthy life, and therefore, it has been recognized as one of the most promising targets against HIV-1. Furthermore, some of the viral gene products may modulate the chemokine system. Therefore, a comprehensive antiviral approach that targets both classes of genes (viral and host), is highly desirable. We present the genetic analyses and functional characterization of some of the HIV/SIV/AIDS modifying genes from normal healthy North-Indians and monkeys and discuss the promising antiviral approaches against HIV-1 that include ribozymes, DNA-enzymes and small interfering RNAs that may complement the existing anti-retroviral drugs. Some of the promising ribozymes and small interfering RNAs have been placed in the fourth generation lentiviral gene therapy vectors. Upon introduction into the human hematopoietic stem cells, long term expression of the trans gene was observed. These stem cells matured into human macrophages and when challenged with R5 tropic HIV-1, impressive inhibition was observed. Hematopoietic stem cells that were transduced with retrovirus vector to express siRNAs against a conserved Tat/Rev region of HIV-1, were placed into the human Thy/Liver grafts in a SCID-Hu mice. These transduced cells matured into T-lymphocytes and showed remarkable protection against HIV-1 challenge.

Invited talk 2

A Journey into the world of HLA and Medicine

N.K. Mehra,

Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Major Histocompatibility Complex (MHC) harbors genes whose primary function is to govern immune responsiveness. MHC is the most polymorphic genomic region known in humans and serves as a very exciting minigenome model for studying susceptibility to a variety of diseases. We have observed enormous diversity in HLA class I and class II genes in the North Indian population. The presence of novel alleles and unique haplotypes in this population suggest concurrent gene conversion events that add to population survival advantage against vigorous pathogenic or environmental challenges encountered. For example, multiple DR3+ve autoimmunity favouring haplotypes have been identified in Asian Indians, some of which are unique to this population. Linkage analysis on extended MHC haplotypes have confirmed the importance of these haplotypes, most importantly the ancestral haplotypes (AH8.1 (HLA-A1-B8-DR3-DQ2). The AH8.1 is of interest not only because of its high frequency in most Caucasian populations, but also because it is associated with markers of immunological hyper reactivity and multiple autoimmune diseases including type 1 diabetes and fast progression to AIDS. Our studies have revealed that the classical Caucasian AH8.1 is really observed among the North Indians and has been replaced by a variant AH8.1v that differs from the Caucasian AH8.1 at multiple loci. The most common DR3 haplotype found in the Indian population is AH8.2 (A26-B8-DR3-DQ2) followed by AH8.3 (A24-B8-DR3), AH8.4 (A3-B8-DR3) and AH8.5 (A31-B8-DR3). The studies have suggested that AH8.1 and AH8.1v might have evolved from a common ancestor but with preferential divergence of AH8.2 over AH8.1 leading to survival advantage in the population. These studies have important implications in our understanding of disease pathogenesis and management, identification of high-risk individuals, and MHC based therapeutic approaches.

Invited talk 3

Functional Genomics of serine threonine kinases of M.tuberculosis

Dr Sujatha Narayanan

Deputy Director

Immunology Department

Tuberculosis Research Centre

Chennai

Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. Genomic analysis of Mtb revealed 11 predicted eukaryotic-like serine/threonine protein kinases (STPKs). The STPKs are implicated in development, stress responses and host-pathogen interactions. The downstream substrates, effectors, and other binding partners of the STPKs are only beginning to be identified. We have characterized two of the serine threonine kinases by creating gene disrupted mutants of pknE and pkn I. We assessed the intracellular survival, promoter regulation, differential gene expression invitro of serine threonine kinase pknE . Microarray Analysis comparing pknE mutant and wild type M.tuberculosis H37Rv infected THP1 cells monocyte reveal that it induces apoptosis. The promoter of pknE is expressed under nitrosative stress. The global gene expression studies indicate down regulation of metabolism, induction of apoptotic genes and immune response genes. These confirm that the STPK pknE has a role in bacterial survival inside the host especially under nitrosative stress. The pknI of M.tuberculosis has been found to have a role in cell division .

IM 1

Role of the N-terminal trx motif containing region of PknG, A eukaryotic like serine threonine kinase from Mycobacterium tuberculosis; in regulation of its enzymatic activity and identification of its downstream substrates.

Divya Tiwari and Vinay K. Nandicoori

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 India

e-mail:divya14@nii.res.in

PknG, one of the 11 kinases encoded by the M. tuberculosis genome is one of the virulence mediator molecules secreted within the host macrophages upon infection. It has been reported that an active form of PKnG is essential in M. tuberculosis for the modulation of phagosome-lysosome fusion which is indicative of implications that PKnG may phosphorylate some of the host factors or some of the mycobacterial proteins which may in turn modulate the host machinery {Walburger et al (2004) Science 304 1800-1804}. To date, there are no reports identifying substrates of PKnG either in mycobacteria or in the host macrophages. The recently published crystal structure of PknG reveals a regulatory role of the N-terminal region towards the kinase activity of PKnG. We have determined the phosphorylation sites on the N- terminal region of PKnG and the role of this phosphorylation in PKnG activity. We have also determined the role of the two conserved Cys in Trx motifs in regulation of PKnG activity in differential redox environments.

Cowley et al, showed that PknG deficient M. tuberculosis mutant accumulates glutamate and glutamine {Cowley et al(2004) 52, 1691-1702}. We are working towards elucidating the physiological role of PKnG in the regulation of this important metabolic pathway. One of the proteins with a crucial role in regulating a checkpoint in this pathway has been identified as an in vitro substrate for PKnG. We are in the process of identifying the phosphoryaltion sites and determining the in vivo significance of PknG mediated phosphorylation on the checkpoint regulation. We are investigating biochemical regulation of PKnG. and would like to probe the regulatory role, played by otherautophosphorylations in PKnG activity. Towards this we have mapped the activation loop phosphorylation site(s) on PKnG. Experiments are underway to determine all the autophosphorylation sites on full length kinase. The identification of autophosphorylation sites will be followed by an investigation into their regulatory role, if any, by mutating the relevant serine/threonine residues to alanine and studying the effect of the mutations on the activity of the kinase in vitro and when it is secreted inside the host macrophages upon infection.

IM2

Immunomodulation and signaling cascade by methylglyoxal

Aparajita Pal, Nivedita Bhattacharyya and Manju Ray

Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata 700 032, India; email palaparajita@yahoo.co.in

Methylglyoxal is a normal metabolite and has the potential to affect a wide variety of cellular processes. Methylglyoxal has growth inhibitory effect and can act specifically against malignant cells. Because malignancy generally depresses the immune system, so a drug that can stimulate the immune system may be able to destroy the tumor cell and other pathogens by its innate mechanism. Most of the known anticancer drugs suppress the immune system, which cannot clear the cellular debris. So a drug, which can stimulate the immune system, in addition to its ability to clear the debris can be effective in treating cancer. So in the present study we investigated whether methylglyoxal can enhance nonspecific immunity in host against tumor cells through macrophages, the important phagocytic cells within the body and also responsible to scavenge cellular debris. It had been observed that methylglyoxal increases the number of peritoneal macrophages and also elevated the phagocytic capacity of macrophages in both the normal and tumor bearing mice. The activation of macrophages was brought about by enhanced production of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). It had been observed that the production of both superoxides and extracellular nitrite are highly augmented in murine peritoneal macrophages treated with methylglyoxal in both the groups. The possible mechanisms for the production of ROIs and RNIs were due to stimulation of respiratory burst enzyme NADPH oxidase and iNOS respectively. Moreover our investigation includes the exact signaling cascade, which plays an important role in the process of enhancing the ROIs and RNIs. For this reason the expression of the various signaling molecules such as mitogen-activated protein kinases (MAPKs) namely p-38 and ERK were investigated. Secretion of various cytokines such as IL-12, IFN-g by activated macrophages in response to methylglyoxal treated tumor bearing mice also showed marked alteration in comparison with tumor inoculated mice that did not receive methylglyoxal.

IM 3

Anti-oxidant, anti-inflammatory and anti allergic effects of C-Phycocyanin isolated form Phormidium valderianum

Rama krishna BS, Nishant P Reddy, Sreedevi B, Jyotshna G, Reddy GV and Reddanna P

Department of Animal Sciences, School of Life Sciences, University of Hyderabad. Hyderabad – 500 046.

E-mail: ram_viro9@yahoo.co.in

C-Phycocyanin(C-PC), is a water soluble, non-toxic, biliprotein pigment found in majority of Blue green algae like Spirulina, Aphanizomenon, Phormidium. Previous studies from our laboratory have shown that the C-PC isolated from Spirulina platensis is a selective inhibitor of COX-2 and exhibits potent anti-inflammatory and anti cancer properties. Recently ABL Biotechnologies, Chennai., have identified an another rich source of C-Phycocyanin i.e Phormidium valderianum. In the present study we have evaluated the anti-inflammatory, antioxidant and anti allergic effects of C-PC isolated from Phormidium valderianum. Using conventional chromatographic methods purified the protein, and the highly purified C-PC was shown to exhibit potent antioxidant properties and these effects were found to be comparable to that C-PC from Spirulina platensis. Cyclooxygenases, the target for non-steroidal anti-inflammatory drugs (NSAIDs) exists in two forms: the constitutive Cycloxygenase-1 (COX-1) and inducible Cycloxygenase-2 (COX-2). COX-2 has been implicated in the mediation of various inflammatory disorders and mediation of variety of cancers. In this connection we have shown that C-Phycocyanin isolated from Phormidium inhibits COX-2 selectively over COX-1 and much more effectively than the C-PC isolated from Spirulina. C-Phycocyanin also showed anti-histaminic effects during the induced hypersensitivity in mast cells. These studies reveal the C-Phycocyanin isolated from Phormidium valderianum is selective COX-2 inhibitor with potent anti-oxidant and anti-allergic properties.

IM 4

COMPARATIVE STUDY OF PZE-6 ON THE DEVELPOMENT OF ADJUVANT ARTHRITIS IN RATS

Aparanji Poosarla, Raghava rao T and Rama Rao Athota

Department of Biochemistry, Andhra University, Visakhapatnam, Andhra Pradesh, India, apara_p@yahoo.co.in

Anti-arthritic effects of PZE-6, a freeze dried ethyl acetate fraction, purified from the roots of Plumbago zeylanica, on disease development was assessed in comparision with indomethacin and cyclophosphamide in rats with adjuvant-induced arthritis (AIA). PZE-6 fraction significantly suppressed the arthritis by decreasing clinical score. Hematological parameters, which includes hemoglobin concentration, total leukocytes count, differential leukocyte count and ESR (1^st hr)were found to be normalized in PZE-6 treated rats when compared to that of arthritic control rats. The effect of PZE-6 was studied on prevention of arthritis. Our results demonstrate that in PZE-6 treated rats, the edema in the right hind paw (inflammation) 60% less than the adjuvant controls on day21. The left hind paw (immune) was 42% less than that of controls. However, arthritic rats treated with drugs like, Indomethacin and cyclophosphamide anti-inflammatory and immunosuppressive drugs respectively could not show any inhibition of neither inflammation nor immune response. Thus PZE-6 exhibited potent anti immunoinflammatory responses.

IM 5

N-glycans in the biology of Taenia solium infections

Jayaraman T and A Oommen

Neurochemistry Laboratory, Department of Neurological Sciences, CMC, Vellore, India.

E.mail: ntr_jayaraman@yahoo.co.in

Taenia solium causes two infections in humans, taeniasis and cysticercosis. Taeniasis is an intestinal disease resulting from ingestion of larva while cysticercosis is a systemic infection from ingestion of eggs. Taeniasis is benign although is the cause that Taenia infections perpetuate. Cysticercosis of the central nervous system is a serious infection and a major cause of adult epilepsy in India. Both infections establish in immune privileged environments. Developmental specific glycosylated determinants govern the establishment of the different T solium infections. They are important to identify for developing disease specific immunodiagnostics.

The aim of this study was to identify and characterize antigenic glycans of T solium glycoproteins specific for taeniasis and cysticercosis respectively.

Blots of T solium adult worm and larval extracts subjected to SDS-PAGE were probed with antisera from taeniasis and cysticercosis patients. Distinct antigenic proteins were detected by each sera. Chemical oxidation and enzymatic
N-deglycosylation abolished the antibody binding of these proteins. Carbohydrate estimation of N-glycans isolated from these proteins indicated a predominance of fucose, galactose and lesser amounts of sialic acid in all of them.

The results indicate N-glycans specific for different stages of the life cycle of T solium that are similar in composition but uniquely antigenic for the stage of development. The discussion will explore possibilities of exploiting Taenia N-glycans as markers of taeniasis and cysticercosis in immunodiagnostic tests.

IM 6

Approach to develop a simple and rapid Immunochromatographic test for antigen and antibody detection using colloidal gold for diagnosis of tuberculosis.

Shweta H. Morey¹, R. S. Kashyap¹, H. j. purohit², g.m. taori¹ and H. F. daginawala¹

¹Biochemistry Research Laboratory, Central India Institute of Medical Sciences, 88/2 Bajaj Nagar, Nagpur-440010, India.

²Environmental Genomics Unit, National Environmental Engineering Research Institute, Nehru Marg, Nagpur-440020, India.

shweta.morey@gmail.com

Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. Our earlier studies have shown that early diagnosis of TB could be achieved using indirect ELISA using monoclonal antibodies (mAb) against the purified Ag 85 complex. In the present study we have extended the approach to develop a simple and rapid Immunochromatographic method using colloidal gold for antigen and antibody detection. The developed Immunochromatographic method showed a good sensitivity and specificity against selected positive (purified Ag 85 complex) and negative (other mycobacterial antigens) control. The developed kit for suspected/confirmed tuberculosis samples along with age and sex matched control samples is being evaluated. The results of the study will be presented in detail. The developed method will be useful in field work and remote places where sophisticated instruments are not available for initial screening of tuberculosis.

IM 7

EMPLOYMENT OF ALGINATE IMMOBILIZED UREASE FOR DETECTION AND QUANTITATION OF HAZARDOUS HEAVY METALS IN AQUEOUS MEDIA

OM PRAKASH, MAHE TALAT AND RAJESH KUMAR PANDEY

Department of Biochemistry, Faculty of Science, Banaras Hindu University

Varanasi – 221005, India

E-mail of the presenting author: oprakash01@yahoo.co.in

Presence of hazardous heavy metals in wastewater, industrial effluents, ground water and drinking water has gained considerable attention because of its impact on human life. Heavy metals are well known to inhibit the activity of enzymes and most of the time enzymes are specific to the inhibitors. Thus, enzymatic reactions have proved to be very promising tools to identify major pollutants, such as heavy metals, enabling a very accurate toxicity identification and evaluation. Owing to its pronounced sensitivity, urease (urea amidohydrolase, EC 3.5.1.5) has been considered as a model enzyme for application as a probe for heavy metal ions in aqueous media. Interestingly, different metals exhibit quite different behavior in their ability to act as urease inhibitor. Therefore, an attempt has been made to employ urease obtained from a rather non-conventional, readily available and inexpensive source, i.e., the discarded seeds of pumpkin (Cucumis melo). It was extracted and purified to apparent homogeneity (5.2 fold) by heat treatment at 48±0.1 ^oC and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp Activity 353 U/mg protein, A₂₈₀/A₂₆₀ = 1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric and cadmium ions on the activity of soluble as well as immobilized enzyme were investigated. Both exhibited a concentration-dependent inhibition in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition and required more amount of the inhibitor. There was no leaching of the enzyme over a period of 15 days at 4 ^oC. The inhibition caused by these ions was non-competitive. Time-dependent interaction of urease with Hg²⁺ and Cd²⁺ exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly and reminder in a slow phase. Binding of Hg²⁺ and Cd²⁺ with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed in light of exploring the possibility of detecting and quantitating the concentrations of these hazardous ions in aqueous media.

IM 8

Ex-vivo effects of 15-Hydroxyeicosatetraenoic acid on inflammation

Sreedevi B, Ramakrishna BS, Nishant P. Reddy, Reddanna P

Department of Animal Sciences, University of Hyderabad, Hyderabad, A.P. India. 500 046

E. Mail: sdevibhcu@gmail.com

Inflammation is a complex phenomenon, involving interplay of cellular and particulate mediators. The understanding on the molecular mechanisms of inflammatory reaction is still incomplete. Arachidonic acid is the biological precursor of classic eicosanoids such as prostaglandins, prostacyclin, thromboxanes, leukotrienes, and hydroperoxyeicosatetraenoic acids. They are generally associated with allergic reactions and inflammation. The first three groups of compounds are derived via the cyclooxygenase pathway, whereas the latter two classes are lipoxygenase metabolites.

15-Hydroxyeicosateraenoicacid is the product of arachidonic acid formed via the 15-Lipoxygenase pathway which is known for both pro and anti-inflammatory properties. It is reported to play a role in atherogenesis, psoriatic skin lesions, angiogenesis and arthritis. It is known to induce neutrophil migration which is important for progression of inflammation and also its regeneration. To better understand the effect of 15-HETE at molecular level of inflammation, the present study was designed to treat granulation tissue of carrageenan treated rat air pouch model of acute inflammation with 15-HETE under ex-vivo conditions. The model was characterized earlier for pro-inflammatory NF-κB activity and for the expression of NF-κB induced enzymes like iNOS, COX-2, PGES and the levels of their end products NO₂ and PGE₂, pro-inflammatory cytokine TNF-α, stress induced HSP-70 and pro-angiogenic VEGF at different phases of inflammation. All these pro-inflammatory parameters are induced during the progressive phase of the inflammation, with a decrease in the later phases of inflammation. 15-Lipoxygenase, on the other hand, is expressed in the resolution phase only with the formation of 15-HETE. The ex-vivo studies on the effect of 15-HETE on inflammation showed pro-inflammatory effect even at 10nM concentration. It increased NF-κB activity by 5.8 folds at 1mM and increased the other pro-inflammatory parameters in a dose dependant and time dependant manner. Ex-vivo studies suggest that 15-HETE and thus 15-LOX is pro-inflammatory in nature, at least in rat air pouch model of inflammation.

IM 9

Anti-inflammatory and Analgesic effects of defatted Sterculia foetida L seed cake

G.S.ShamSunder* S.Param jyothi.

Department of Biochemistry Gulbarga University, Gulbarga, India

Email: sham_shahabadkar@yahoo.com

Chloroform, ethanol, and water extracts of defatted seed of Sterculia foetida L were

studied for anti-inflammatory and analgesic activities in animal models. The tested

extracts produced significant inhibition of carragenan induced paw edema and

also exhibited central analgesic effect. The chloroform extract (CE) and water

extract (WE) showed a significant anti-edematogenic activity from the 1^st hr

indicating that the active principle in both the extracts is inhibiting both the

phases of carragenan induced edema. The ethanol extract (EE) was effective

in inhibiting the carragenan induced edema from the 2^nd hr indicating that the

active compound/s is inhibiting prostaglandin synthesis. All the studied extracts

showed central analgesic effect.

Medicinal Organisms

Invited Talk:

IDENTIFICATION AND PURIFICATION OF CYCLIC PEPTIDES FROM CLITORIA TERNATEA

LALADHAS K. P., BIOMOLECULAR RESEARCH UNIT,

DEPARTMENT OF ZOOLOGY, ST STEPHEN’S COLLEGE, PATHANAPURAM, KOLLAM, KERALA.

&

BALARAM P. MOLECULAR BIOPHYSIS UNIT,

INDIAN INSTITUTE OF SCIENCES, BANGALORE.

Kerala so called God’s own country is blessed with rich heritage of plant diversity and diversity of health traditions. The history of the drug discovery implies that the ethno-botanical approach is most productive and informative. The well established drugs available now in practice were developed after scientist began to analyze the chemical constituents of plants used by traditional people. Exploration of the medicinal uses of plants from rural people in remote parts of the world coupled with the scientific research methodologies, may be able to determine whether such plants exerts progressive therapeutic usages or not.

Clitoria ternatea, a plant from the family Fabaceae is a slender perennial climber used as an anti-inflammatory agent in folklore medicine. We have purified two novel peptides from this plant after HPLC fractionation using C18 reverse phase column. Masses of the peaks were analyzed in Matrix assisted lazar dissorption and ionization mass spectroscopy (MALDI ToF) and the peaks ionised with masses of 3074 and 3059 Da respectively. Reduced and carboxamidomethylation of the peptides showed the presence of three disulfide bonds and was linearised by tryptic digestion. Both the peptides are cyclic and bound with three disulfide linkages.

Invited talk

Phytoceuticals: the future of plant derived health aids

Dr. Suman PS Khanuja

Director

Central Institute of Medicinal & Aromatic Plants

PO CIMAP, Lucknow-226015

e-mail: khanujazy@yahoo.com and director@cimap.res.in

Plants are emerging as the largest utilizable repository of natural products with the potential to become novel drugs. Many of the commercially used pharmaceuticals already are products of plant secondary metabolism. Out of the 350,000 plant species identified so far, about 35,000 (some estimate up to 70,000) are used globally for medicinal purposes. However, it has been estimated that only 6% of all described species have been analyzed chemically and only a small fraction analyzed pharmacologically. Nearly 100 plant species are involved in 25% of all drugs prescribed in developed countries. The annual market value of herbal drugs used worldwide was estimated to be around US$ 45 billion in the late nineties and the value varies in different reports (60 to 100 billion $) presently. Bioprospection for novel molecules from plant resources and novel bioactivities for previously known phytomolecules is the major activity towards development of plant-derived drugs. The capability of plants to cure diseases is due to the in planta biosynthesis of secondary metabolites. These secondary metabolites may be classified into alkaloids, terpenoids, phenylpropanoids and the complexes of these metabolites (Croteau et al., 2000). All the natural/secondary product pathways originate as branch points from primary metabolism. However, the genetic maps of biosynthetic pathways of these compounds are still far from complete and the information on the regulation of these pathways is even lesser (Shasany et al., 2007).

Two unique phytomolecules possessing therapeutic activity that have been isolated at CIMAP are niaziridin (Khanuja et al., 2005, US Patènt No. 6,858,588; Arya, 2003) and oenostacin (Shukla et al., 2002, US Patent No. 6,365,197). However, most of the bioactive phytomolecules, being the secondary metabolites, are produced in quite low amounts in the plants. Genomic interventions are therefore required to engineer the metabolic pathways for the therapeutically useful phytomolecules, either in the plant itself, or in heterologous systems. It would be worthwhile to mention Catharanthus roseus and Mentha spp., as model prototype plants, in which maximum number of genes involved in secondary metabolism have been elucidated. In the former fifteen genes of the terpenoid indole alkaloid (TIA) biosynthetic pathway are known, whereas in the latter ten genes involved in the monoterpene biosynthesis have been characterized till date. Besides, the temporal and plant tissue and age dependant partitioning of pharmaceutically important TIAs has been carried out in C. roseus (Shukla et al., 2006, J. Exp. Bot. 57: 3921-3932). Similarly, in Papaver somniferum eight out of the seventeen steps involved in the conversion of tyrosine to morphine have been characterized and the genes are being elucidated. In Artemisia annua, the plant that produces very potent antimalarial drug compound ‘artemisinin’, the genes for the key enzymatic steps, ads and cyp71, in the artemisinin biosynthetic pathway have been cloned and characterized but a lot remains to be dissected out for the pathway.

Medicinal plants also provide functional foods or nutraceuticals, which are dietary components that provide health benefits beyond basic nutrition. Some examples of nutraceutical components are carotenoids (like β-carotene, lutein, zeaxanthin, lycopene), fiber (insoluble fiber, β-glucan, soluble fibres, whole grains), fatty acids (mono-unsaturated fatty acids, poly-unsaturated fatty acids like omega-3-fatty acids, ALA, DHA, and conjugated linoleic acid), flavonoids (anthocyanidins, proanthocyanidins, flavanones, flavonols, flavanols - catechins, epicathecins, procyanidins), isothiocyanates (sulforaphane), phenols (caffeic acid, ferulic acid), plant stanols/sterols, polyols (sugar alcohols – xylitol, sorbitol, mannitol, lactitol), prebiotic/probiotics (inulin, fructo-oligosaccharides, polydextrose, lactobacilli, bifidobacteria), phytoestrogens (isoflavones, lignans), soy protein, and sulfides/thiols (diallyl sulfide, allyl methyl trisulfide, dithiolthiones). Recently, targeted efforts have been made, to engineer plant metabolomes to favourably produce nutraceutical components in an economical manner. Typical examples of such genetic modifications to produce nutritionally superior edibles are introduction of AmA1 gene from Amaranthus into potato to produce protein-rich GM potatoes, long-lasting GM tomatoes, tomatoes with three-fold higher lycopene levels (an anti-oxidant that prevents onset of cancer and checks cholesterol levels), and Golden Rice that is a GM rice containing higher amounts of Vitamin A precursor b-carotene. Another dimension has been added to nutraceuticals by the discovery of immunostimulant and bioenhancer (for drugs and nutrients) components from plants with Ocimum and Moringa oleifera providing the examples, respectively. In all probability the future world will benefit immensely from the ongoing biotechnological advances in the area of medicinal plant genomics, which will ultimately catalyze the transformation from ‘farming’ to ‘pharming’.

Team acknowledgements: Drs. Ashutosh K. Shukla, MP Darokar, Anirban Pal, Ajit K. Shasany and DU Bawankule.

Invited Talk 3

Structural and Pharmacological Studies of Conotoxins from Indian Marine Snails

Siddhartha P. Sarma, Sujit K. Sikdar, P. Balaram and K.S. Krishnan

sidd@mbu.iisc.ernet.in

The conotoxins are a group of peptide based toxins that are produced by marine snails of the genus Conidae. The toxins have elicited tremendous interest among neuropharmacologists and neurobiologists because of the specificity with these toxins target sodium, potassium and calcium ion channels and also other pharmacologically important targets such as adrenergic, serotonin, NMDA and Nicotinic Acetyl Choline receptors. Thus the identification and characterisation of these peptides could serve as a valuable starting tool in the design of neuropharmacologically active drugs.

We have initiated a program to isolate and purify pharmacologically important peptides from Cone snails that inhabit the Indian coastal waters. It is estimated that ~ 77 species are unique to these waters. Physicochemical characterization of these peptides has relied almost exclusively on Mass Spectrometry and Multidimensional Nuclear Magnetic Resonance Methods with additional information from other spectroscopic methods. Electrophysiological methods have enabled us to identify specifically the ion channel targets of these peptides. The application of these methods to the charcterization of these peptides will be discussed.

Invited Talk 4

The Indian experience with Yersinia enterocolitica – a food- and water-borne enteropathogen

Dr. J.S. Virdi

Department of Microbiology, University of Delhi South Campus

New Delhi - 110 021. Email: virdi_dusc@rediffmail.com

Yersinia enterocolitica, a food- and water-borne enteric pathogen is regarded as an emerging pathogen. In India, the first outbreak of gastroenteritis due to this organism was reported in 1996 from Tamil Nadu. In our laboratory, the organism has been isolated from sewage water, pork, pigs (reservoir) and the stools of the diarrheic human subjects. All strains have been deposited with WHO Reference Laboratory at Pasteur Institute.

The Indian strains belong to several serotypes represented by biovar 1A.However genotyping using REP- and ERIC-PCR showed that these strains belonged to only two clonal groups indicating their limited genetic heterogeneity. Similar results were inferred from genotyping based on rrn and gyrB loci. The RFLP of beta-lactamase genes (blaA, blaB) also discerned two clonal groups. All these studies clearly showed that the serotype 6,30-6,31 strains isolated from sewage water were different from the serotype 6,30-6,31strains isolated from stools of diarrheic humans.

The detection of a large number of virulence-associated genes (inv, ail, virF, ystA, ystB, ystC, myfA, fepA, fepD, fes, hreP, ymoA, tccC , sat) in the Indian strains showed that these belonged to low virulence group. It was also found that virulence genes were distributed differentially among the two clonal groups. Studies are in progress to further discern genomic differences between the two clonal groups using suppression subtractive hybridization.

1. Bhagat N, Virdi JS (2007) Distribution of virulence-associated genes in Yersinia enterocolitica biovar 1A correlate with clonal groups and not the source of isolation. FEMS Microbiol Lett 266:177-183.

2. Sharma S, Mittal S, Mallik S, Virdi JS (2006) Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A.FEMS Microbiol Lett 257: 319-327.

3. Sachdeva P, Virdi JS (2004) Repetitive elements sequence (REP/ERIC)- PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups. FEMS Microbiol Lett 240: 193-201.

MO 1

Lipopolysaccharide stimulated oxidative stress in murine alveolar macrophages and protection accorded by Tinospora cordifolia (Willd.)

MEENAKSHI TIWARI AND POONAM KAKKAR

INDUSTRIAL TOXICOLOGY RESEARCH CENTRE

80,M.G. MARG,LUCKNOW , INDIA.

meenak_zzttm@yahoo.co.in

Tinospora cordifolia (Willd.) is a well known medicinal herb and is used for promoting longevity,in reducing hepatotoxicity,enhancing immunomodulatory responses,as a chemopreventive agent.These protective effects are by the virtue of its various chemical constituents including alkaloids such as berberine,bitter substances including tinosporin, tinosporic acid, tinosporol all of which contribute to its medicinal value. Studies were undertaken to explore the antioxidant potential of the extract of T.cordifolia (Willd.) on lipopolysaccharide stimulated murine alveolar macrophages isolated from Bronchoalveolar Lavage Fluid (BALF). Cytotoxicity studies were carried out in alveolar macrophages under two treatment schedules .In the pretreatment macrophages were first treated for one hour with varying extract concentrations (2.5µg,5µg,10µg per 1x10⁴cells) and quercetin (2.5µg per 1x10⁴cells) and incubated further with LPS (1 µg/ml) for 24 hours. The cytotoxicity was assessed by MTT assay and trypan blue exclusion assay. In post treatment macrophages were stimulated with LPS (1 µg/ml) for one hour followed by incubation with extract/quercetin for 24 hours and viability assessed. Modulation of antioxidants in treated cells was measured by assaying SOD activity, TBA reactive substance formation and nitric oxide quenching. At the subcellular level correlation between alteration in mitochondrial membrane potential and intracellular oxidant generation was studied. None of the doses of extract was found to be cytotoxic to macrophages. Stressed cells during pretreatment (2.5 µg) showed 86.36% and during post treatment (10 µg) showed 102.27% cell survival.The 5 µg dose of T.cordifolia during pretreatment was able to increase the Superoxide Dismutase (SOD) activity by 2.27 folds.The TBA reactive substance (TBARS) formation was reduced by 34% as compared to LPS stressed cells during pretreatment while it was reduced by 39.31% during post treatment.NO quenching capacity was increased by 15.73% during pretreatment while 31.03% enhancement was observed during post treatment as compared to LPS stressed cells. There was significant enhancement of GSH content as compared to stressed cells during pre and post treatment respectively as assessed by DTNB recycling assay .Mitochondrial membrane potential (MMP) was also evaluated using cationic fluorescent dye, Rhodamine 123 whose fluorescence intensity decreases quantitatively with respect to MMP.An increase of 27.16% and 29.14% in MMP during pre and post treatment respectively by 5µg extract concentration was observed.Intracellular oxidant production was evaluated by 2’,7’-dichlorofluorescindiacetate (DCFH-DA) and decrease of 29.46% and 28.44% in fluorescence as compared to LPS stimulated cells was observed. Effect on the expression of key enzyme of inflammation inducible Nitric oxide Synthase (i-NOS) which is involved in the release of nitric oxide was also seen by western blotting. The effect of the extract on the cytokine TNFsecretion a proinflammatory cytokine was also seen by the ELISA kit The results suggest strong antioxidant potential of the herb which can be used as a protective agent in formulations used for various free radical mediated respiratory disorders

Keywords:Lipopolysaccharide,antioxidant,TBARS,mitochondrialmembranepotential,alveolar macrophages.

MO 2

PRECLINICAL EXPERIMENTAL EVIDENCE FOR ANTI ULCER ACTIVITY IN AZADIRACHTA INDICA A. JUSS

R. Chatterjee^a , S. Kumar^b, M. Ray^a, N. B. Mondal^b, and S. Banerjee^b

^aDepartment of Biological Chemistry, Indian Association for the Cultivation of Sciences, Kolkata-700032, India; ^b Steroid and Terpenoid Division, Indian Institute of Chemical Biology, Kolkata-700032, India.

E-mail: chat_rus@yahoo.com

Modern lifestyle, junk food habits, occupation- related stress and strain induces gastric hyperacidity and ulceration which has become a very common human ailment today. Neem (Azadirachta indica A. Juss) is a tall woody versatile plant having a wide spectrum of medicinal properties. Various parts of the plant, such as leaves, bark, fruit, oil and root have been extensively used as traditional medicine to control various ailments. Based on the efficacy of various parts of the plant, the present study has been designed to explore the probable active chemical constituents from neem leaves for remedy of gastric ulceration. Gastric ulceration is multiaetiologic and two key reasons responsible for the disease has been targeted in this study viz; a) drug-induced ulceration and b) alcohol-induced ulceration for evaluating anti ulcer potential of the leaves. Experimental protocols were standardized to suit screening of crude leaf extract and fractions. Acute toxicity up to a dose of 1g/kg body weight of experimental animals did not show any mortality after 24 h. The fractionation scheme and the activity profiles in different experimental models to be presented in detail indicated that the leaf part is raising a strong prospect of developing a monoherbal anti ulcer preparation.

MO 3

Oxidative stress mediated cytotoxicity and protection accorded by medicinal plants

MADHULIKA TRIPATHI, BRIJESH KUMAR SINGH and POONAM KAKKAR

Herbal Research Section, Industrial Toxicology Research Centre,

P.O. Box- 80, MG Marg Lucknow

e-mail: madhulika_tripathi@yahoo.com

Abstract:

Nimesulide is a prescription non-steroidal anti-inflammatory drug (NSAID) with known analgesic and antipyretic activities. It acts by inhibiting the synthesis of prostaglandins as a consequence of blockade of the enzyme cyclooxygenase II (COX II). Side effects associated with the drug are hepatotoxicity, renal toxicity and it is contraindicated during pregnancy. Nimesulide may cause necrosis or apoptosis and oxidative stress is believed to participate in nimesulide induced hepatotoxicity but the molecular signaling responsible for nimesulide induced death in liver cells needs to be explored. Since it is an effective drug for pain management in debilitating conditions, it seems logical to explore the agents which can reduce its toxicity. In the present study three different plants were taken up and their hepatotprotective competence was checked in nimesulide induced toxicity. Extracts of Fumaria parvilflora Lam. (whole plant), Boerhaavia diffusa Linn. (roots) and Solanum nigrum Linn. (ripe fruits) were screened for their hepatoprotective efficiency in vitro. Antioxidant capacity of selected plants was evaluated using microassays for SOD mimetic activity, LPO inhibitory capacity, NO quenching and reduced glutathione content. Primary hepatocytes were treated with a selected dose of nimesulide (500µM) which caused 50% decrease in the cell survival. Different concentrations of plant extracts were taken and effective dose was determined using MTT activity and trypan blue dye exclusion test. Cytotoxicity was reduced by 54.59% in the cells pre-incubated with 5 µg of Fumaria followed by Boerhaavia and Solanum with which the recovery was found to be 13.19% and 10.26% respectively as compared to stressed cells. Cytoprotective action of Fumaria was comparable to Silymarin 56.62% which was used as a positive control.SOD activity was found to increase by 83% in cells pre treated with Fumaria which was higher than the SOD activity of Silymarin (75%) followed by Solanum and Boerhaavia in which the activity was found to be increased by 59% and 25% respectively as compared to stressed cells.Decrease in lipid peroxidation was not so significant in the presence of all the three extracts.The NO Quenching capacity was found to be enhanced by 45.06% as compared to the stressed cells in the extract (Fumaria) pre treated cells which was comparable to silymarin (46%).Flow cytometric analysis was done to measure reactive oxygen species generation (DCHF-DA dye), mitochondrial membrane potential (JC-1dye), chromatin condensation (Hoechst 33258). DNA fragmentation in primary hepatocytes pre and post incubated with plant extracts was also studied. ROS generation was found to increase by 1.3 folds with respect to control whereas membrane potential was also found be low in the cells treated with nimesulide alone. Fumaria decreased the ROS generation and increased membrane potential of mitochondria and reduced DNA damage when cells were pre incubated with it.

Key words: Hepatotoxicity, Nimesulide, Antioxidant, Chromatin condensation, DNA fragmentation, Mitochondrial membrane potential.

MO 4

Isolation, purification and characterization of dual COX-LOX inhibitor

from Terminalia chebula

Bharat Reddy D, Jyotsna Radhika G, Satish Sharan, Nalini Priya, Lakshmipathi V,

Reddanna P.

Department of Animal Sciences, School of Life Sciences, University of Hyderabad,

Hyderabad- 500 046, India

e-mail: dbharat2000@gmail.com

Terminalia chebula is called the "king of medicines" and is always listed first in the Ayurvedic meteria medica because of its extraordinary powers of healing. In Ayurveda, it is considered to destroy all diseases and eliminate all waste from the body. At the same time it is known to promote tissue growth and health. Its fruit powder is one of the constituent of Triphala, a well known Ayurvedic medicine. In the present study we have tested the activity of alcoholic extract of Terminalia Chebula against COX-1, COX-2 and 5-LOX, and observed potent inhibition of the same. In order to isolate the active principle, further studies were undertaken by employing RP-HPLC was performed in the alcoholic extract of Terminalia chebula. One of the compounds isolated from RP-HPLC showed potent inhibition against COX-1, COX-2 and 5-LOX. The IC₅₀ observed for the three enzymes were 15ug/ml, 0.92ug/ml and 2.1ug/ml respectively. The above results suggest that the active principle is a dual COX-LOX inhibitor and showed more specificity towards COX-2 than COX-1, and furtherly this compound was characterized by LC-MS, MALDI-TOF, IR and NMR. Anti-proliferative properties of the principle compound identified were evaluated by performing MTT assay (cell proliferative assay) in 4 different cancer cell lines HCT-15(colon), COLO-205(colon), MDA-MB-231(breast) and DU-145(prostate). GI₅₀ values of the active principle against these cell lines were 25ug/ml, 19ug/ml, 20ug/ml and 28ug/ml respectively, suggesting broad spectrum anti-cancer properties. Further in depth anti-proliferative studies were carried out in COLO-205 cell line. Western blot analysis of PARP cleavage, p53, HSP-70, PKC, C-myc, Bcl₂, BAX, Cytochrome-c and DNA fragmentation studies confirmed the anti- proliferative property of the compound. The above results indicate that the active principle is a potent anti-inflammatory and anti-proliferative agent.

MO 5

Title: Protective potential of antioxidant fractions of Euphorbia hirta L. leaves on oxidative damage to biomolecule.

Nilesh Kumar Sharma, Ramasare Prasad*

Molecular Biology and Proteomics Laboratory, Department of Biotechnology

Indian Institute of Technology Roorkee, Roorkee, Uttaranchal, India 247667

*E mail-rapdyfbs@iitr.ernet.in

Abstract

The aqueous crude extract of Euphorbia hirta L fresh leaves was extracted in boiling water. Crude extract was further partitioned with liquid-liquid partitioning method into n-hexane, diethyl ether, ethyl acetate, methanol, ethanol and water fractions. The results obtained from DPPH (2, 2’-diphenyl-1-picrylhydrazyl) free radical scavenging activity assay revealed that only ethyl acetate and methanol fraction showed most of the scavenging activity with EC₅₀ concentration 20 and 14 µg per ml extract fraction, respectively. The deoxyribose degradation experiment was studied using hydroxyl radical generated from iron mediated Fenton reaction. The results revealed significant hydroxyl radical scavenging potential with EC₅₀ 108 and 86 µg per ml extract fraction for ethyl acetate and methanol fraction, respectively. The ethyl acetate and methanol fractions didn’t demonstrate pro-oxidant activity in the absence of reducing agent. The preventive potential against oxidative injury to calf thymus DNA and plasmid was studied on 1% agarose gel using copper (II) and ascorbate reaction mixture as source of free radical damage. The densitometric analysis of DNA band intensity was performed to study the extent of protection against control DNA damage. The results showed significant inhibition potential against oxidative damage to DNA with EC₅₀ 120 and 93 µg per ml extract fraction for ethyl acetate and methanol fraction, respectively. The protective effect of ethyl acetate and methanol fraction on hypochlorite mediated oxidative damage to bovine serum protein was evaluated using native PAGE and spectrophotometer technique. The results revealed that both fractions ethyl acetate and methanol showed marked inhibition potential with EC₅₀ 220 and 158 µg per ml extract fraction, respectively. On the basis of above findings, we found these two fractions ethyl acetate and methanol from E. hirta leaves as good source of antioxidant constituents and promising protective agent against oxidative damage to DNA and protein.

Key Words. Free radical, antioxidant, scavenging, oxidative damage, hydroxyl radical

MO6

Title: Evaluation of antifungal activity of plumbago rosea

stem extract

Sunity*, Ramasare Prasad

Molecular Biology and Proteomics Laboratory, Department of Biotechnology

Indian Institute of Technology Roorkee, Roorkee, Uttaranchal, India 247667

*E mail-sunitdbs@iitr.ernet.in

Abstract

The antifungal activity of the ethyl acetate extract of the stems of Plumbago rosea was investigated by agar disc-diffusion assay and broth microdilution method against fourteen species of potentially human pathogenic fungi. (Cryptococcus neoformans, Sporothrix shenkii, Fusarium oxysporum, Aspergillus flavus, Aspergillus fumigatus, Trichophyton mentegrophytes, Microsporum gypseum, Curvularia lunata, Rhizomucor pussilus, Pseudalleshcheria boydii , Candida albicans and Candida tropicalis, Candida krusei and Phialophora verrucosa) Seperation of the crude extract by column chromatography yielded ten fractions of which f₄ and f₅ exhibited desired activity with f₅ being the most active. There was significant difference in MIC of the crude (1.23-156.3 µg/ml) and f₅ (.16-4.89 µg/ml) showing that the activity had considerably enhanced on purification. The result of this study support the use of this plant in traditional Ayurvedic and Yunani medicine to treat skin infections and scabies.

Keywords: Plumbago rosea, Antifungal activity, Disc-diffusion, Broth microdilution

MO 7

Tinospora cordifolia prevents fructose-induced insulin resistance in rats

Sreenivasa Reddy S^*, Ramatholisamma P and Saralakumari D

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur-515 003,

Andhra Pradesh, INDIA

sreenivasareddys@gmail.com

In an attempt to develop new substances for handling insulin resistance, the effect of aqueous extract of Tinospora cordifolia stems on insulin resistance and its related abnormalities induced by high-fructose diet were investigated in male wistar rats. Male wistar rats weighing about 175-200g were randomly divided in to four groups and treated for 60 days with starch diet (group-S), starch diet plus Tinospora cordifolia at the dose of 400 mg/day/kg (group-S+Tc), fructose (66% of diet) (group-F), and fructose diet plus Tinospora cordifolia (group-F+Tc). Body weight, plasma glucose, insulin and triglycerides were measured for every 15 d interval and insulin sensitivity was assessed at the end of experimental period by measuring glucose-insulin index which is the product of the areas under the curve of glucose and insulin during oral glucose tolerance test (OGTT). Relative to group-S, the group-F rats had increased body weight, plasma glucose, insulin and triglyceride and decreased insulin sensitivity; these changes were prevented by Tinospora cordifolia as seen in group-F+Tc. In conclusion our results suggests that oral administration of Tinospora cordifolia extract can prevent fructose-induced insulin resistance in rats and may be used as an adjuvant therapy to patients with insulin resistance.

MO 8

Protective action of Medicinal plants S. cinnamomifolia , T. chebula and I. frutesens on Streptozocin Induced Diabetic Rats

J. Kamakshamma ¹ U.Sivaiah² and G. Sudarsanam¹

1. Division of Medicinal Plants and Natural Products, Dept. of Botany,

Sri Venkateswara University, Tirupati- 517502 A.P.

2. Dept of Zoology, Sri Venkateswara University, Tirupati- 517502 A.P

A study to investigate the use of plant extract of the S. cinnamomifolia , T. chebula and I. frutesens and in promoting protective effect on experimentally induced diabetic rats.Diabetes is associated with insufficient secretion of Insulin or improper absorption of Insulin by the cells and affects the functioning of various organs of rat. This study reports the ethanol extract of S. cinnamomifolia , T. chebula and I. frutesens act as anti diabetic agent. In streptozotocin (STZ), induced diabetic rats. A Single dose of each plant extract individually and combindly administered intraperitonially and blood sugar level (BSL) was monitored at regular intervals.Streptozotocin induced diabetic rat exhibited high blood sugar accompanied by distinct histopathological changesin major tissues of rat. The present study was under taken to ascertain whwther the ethanolic extract of these plants possess any hypoglycemic effects in STZ, induced diabetic rats.Plant extracts shown a significant reduction in blood sugar level in rats. The plant extracts has longer activity, it may reduce the blood sugar level by potentiating the release of insulin from pancreatic islets. It is for the first time that the extract of S. cinnamomifolia, T Chebula and I Frutens is found to be an effective anti diabetic agent.