Poster Abstracts-Details

Membrane Transport

IT MT: Phosphorylation of Mitochondrial Voltage Dependent Anion Channel:

A Potential Tool to Control Cytochrome c Mediated Cell Death

Jyotirmoy Banerjee and Subhendu Ghosh

Department of Biophysics

University of Delhi South Campus

New Delhi 110021, India

Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also it plays a role in neuro-degenerative disorders like Alzheimer’s disease, etc. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules like cytochrome c etc., which are released into the cytoplasm at the onset of apoptosis. Through bilayer electrophysiological experiments we demonstrate that rat brain mitochondrial Voltage Dependent Anion Channel (VDAC), an outer mitochondrial membrane protein, when interacts with Bcl-2 family proteins Bax and tBid, its pore size increases. This leads to release of cytochrome c and other apoptogenic molecules into the cytosol causing cell death. Regulation of this tBid and Bax induced increase in pore size of VDAC is a significant step towards controlling cytochrome c induced cell death. We have shown through bilayer electrophysiological experiments that increase in VDAC conductance due to its interaction with Bax and tBid is reduced due to action of cyclic AMP dependent protein kinase A (PKA) in presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c mediated cell death in living cells.

MT 1

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.

MT 2

Interaction of insecticides with mammalian P-glycoprotein and their effect

on its transport function

K. Sreeramulu ¹ , Ronghua Liu ² , Frances J. Sharom*

Department of Biochemistry, Gulbarga University, Gulbarga-585 106, India

Department of Molecular and Cellular Biology, Science Complex Room 2255, University of Guelph,

Guelph, Ontario, Canada N1G 2W1

We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated Kd values fell in the range 4–6 µM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.

* Corresponding author.

E-mail address: fsharom@uoguelph.ca (F.J. Sharom).

Tel.: +1 519 824 4120x52247; fax: +1 519 837 1802.

MT 3

Effect of Emblica officinalis extract supplementation beneficially modulates the properties of biomembranes of alcohol receiving rats

Damodara Reddy.V and N.Ch.Varadacharyulu

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

Presenting author Email id: reddi_damodar@rediffmail.com.

Nearly 2 billion people worldwide consume alcohol and 76 million people suffering from alcohol induced diseases seeking treatment. Biomembranes are largely responsible for the characteristic actions of alcohol. Alcohol fluidizes biomembranes in general. However it is also known that decreased fluidity and altered composition are marked features in membranes from chronic alcoholics. Since the globe is turned towards plant medicine, we propose the fruit of Emblica officinalis for treating alcohol related diseases which opens up new avenues. This study chiefly investigates the changes induced by supplementation of Emblica fruit dry powder (EFP) in biomembranes [viz., RBC, synaptosomal, hepatic microsomal, mitochondria (liver & brain)] of chronic alcoholic rats. Anisotropic studies using the probe DPH (1, 6 diphenyl 1, 3 hexatriene) and fluorescent polarization studies using the probe pyrene suggested that EFP supplementation to alcohol receiving rats can reverse the fluidity changes to a large extent. Surprisingly all the membranes responded similarly to the treatments and indicated the restoration of native properties of these membranes upon Emblica treatment. Further a comparative evaluation of various phospholipids in various membranes of alcoholic rats revealed beneficially modulated role of Emblica supplementation as evidenced from changes in the contents of total phospholipids as well individual phospholipid classes viz., phosphotidyl choline (PC), phosphotidyl serine (PS) and phosphotidyl inositol (PI) in different membranes viz., synaptosomal, RBC and mitochondrial (liver & brain) uniformly. However, microsomal membrane showed an increase in concentration of PI, followed by a decrease in PS and sphingomyelin (SM) with no difference in the contents of PC and phosphotidyl ethanolamine (PE). Surprisingly Emblica supplementation has restored membrane properties of alcohol receiving rats. In order to alleviate the effects of alcohol with a therapeutic approach, the present study is designed.

Key words: Biomembranes; Emblica officinalis; Membrane Fluidity

MT 4

Ethanol effect on membrane glycosylation in rat intestine.

*Ravneet Grewal & Akhtar Mahmood

Department of Biochemistry, Panjab University, Chandigarh (INDIA)

Ethanol ingestion is well known to induce morphological and biochemical changes in intestine and is responsible for intestinal dysfunctions. Epithelial surface of enterocytes is rich in glycolipids and effect of ethanol ingestion on membrane glycolipids and glycoproteins has not well characterized. In the present study, rats were given 1 ml of 30% ethanol daily for 45 days. To evaluate glycosylation process of glycolipids, the incorporation of [¹⁴C]- mannose and [¹⁴C] glucosamine into brush border membrane of rat intestine was studied. Results on incorporation of labeled sugars revealed an enhanced (p<0.001)>14C]- mannose and [¹⁴C]- glucosamine was reduced significantly in native and delipidated membrane preparations in ethanol- fed rats compared to control animals. These findings suggest the role of membrane glycolipids in the pathogenesis of intestinal dysfunctions upon alcohol ingestion.

*Supported by Council of Scientific and Industrial Research (CSIR)

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

MT5

Impact of gender on alcohol induced changes in physicochemical properties of biomembranes

K. Rameshwar Reddy*, G.Kavitha, V.Damodara Reddy, N.Ch.Varadacharyulu

Department of Biochemistry, Sri Krishnadevaraya University

Anantapur - 515 003, India

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

Existing literature and emerging evidences suggest that there exists an impact of gender on effects of alcoholism. Studies clearly revealed that females respond differently to alcohol due to different hormonal milieu and other known reasons. Biochemical composition and physicochemical properties of biomembranes are largely responsible for the characteristic actions of alcohol. Present study investigates the changes in physical state as well lipid composition (cholesterol and phospholipids contents) of the membranes of female alcoholic rats in comparison to male rats. This study is aimed at understanding the impact of gender in alcoholic rat membranes. Membrane fluidity in male and female alcoholic rats was assessed by measuring the movements of fluorescent probes pyrene and DPH in mitochondrial (brain and liver) and microsomal (liver) membranes to compare them with respective controls. Each group consisted of 10 animals. Further the contents of cholesterol and phospholipids were determined in all the above rats. Results from the present study indicated that there is a decrease in membrane fluidity property when compared to respective controls as these changes are more pronounced in female rats indicating that the females are more prone to these changes. Changes in C:P ratio of mitochondrial and microsomal membranes of alcoholic rats in both sexes suggested a decrease in membrane fluidity. Changes in the C: P ratio are in correlation with fluorescent probe anisotropic studies.

Key words: Membrane fluidity, mitochondrial membranes, alcoholics, gender difference.

MT 6 (modified)

YLR099c encodes for a novel soluble lysophosphatidic acid acyltransferase in Saccharomyces cerevisiae

Ananda Kumar Ghosh, R.Geetha Menon and Ram Rajasekharan^*

Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India

Plasma membrane is a lipid bilayer, the composition of which not only maintains a proper homeostasis between the external and internal environment of the cell but also protects the cell from the other environmental hazards. The key players which constitute the membrane structures are phospholipids and proteins. Dynamic interplay between membrane phospholipids and proteins maintain proper cellular function in both normal and stressed cellular environments. Organic solvent stress and its tolerance have been widely studied in prokaryotes such as Pseudomonas. However, mechanisms pertaining to tolerance towards organic solvent have been unclear. In order to understand the role of membrane phospholipids in organic solvent tolerance in eukaryotes we chose Saccharomyces cerevisiae, Pichia pastoris and Rhodotorula glutinis as our model system. Following isooctane exposure Pichia and Rhodotorula showed a decrease in phospholipid content. In contrast, Saccharomyces showed an increase in phospholipid content upon exposure to the solvent for 12 hrs. YLR099c (ICT1) in Saccharomyces has been reported to be maximally expressed during isooctane stress. In silico analysis revealed the absence of any transmembrane domains in Ict1p. Domain analysis showed that it has a hydrolase / acyltransferase domain with a distinct lipid binding motif and a lysophospholipase domain. Phylogenetic tree of Ict1p suggested that the protein is evolutionarily conserved from Caenorhabditis elegans to humans. Based on the above information we wanted to ascertain the role of ICT1 in phospholipid metabolism of S. cerevisiae. Phospholipid analysis of ∆ict1 strain using [P³²] orthophosphate surprisingly showed a drastic reduction of phosphatidic acid (PA) and a significant reduction of all the major phospholipids as compared to wild type. To understand the biochemical function of Ict1p, the gene was cloned and expressed in Escherichia coli. Ict1p was affinity purified using Ni-NTA column chromatography. The purified recombinant enzyme was found to acylate lysophosphatidic acid (LPA) to PA in a time and enzyme concentration dependent manner. However, no phospholipase activity was found. Kinetic analysis revealed a Km of 66.7 μM for LPA and Vmax of 7.33 nmole/min/mg. The enzyme was found to be specific for LPA and did not accept any other acyl acceptors. Lysophosphatidylcholine was found to drastically reduce the LPA acyltransferase activity of Ict1p. Specific activity of Ict1p for unsaturated acyl CoAs like oleoyl and palmitoyl CoA was found to be two fold higher than that of stearoyl CoA. Therefore, our studies show that isooctane tolerance in Saccharomyces is contributed by accumulation of major membrane phospholipids. Increased biosynthesis results from enhanced expression of ICT1, which in turn enhances the synthesis of PA, the major precursor molecules for all the membrane phospholipids. Here we report for the first time identification, cloning, expression and characterization of a soluble LPA acyltransferase.

Microbiology

Invited Speeches:

1. Emerging infections diseases: Ochrobactrum infection in gastritis patients

Yogesh S. Shouche ^1,*, Mahesh S. Dharne ¹, Sri Prakash Misra ², Vatsala Misra ³, Milind S. Patole ¹, Hitendra Munot ¹, Santosh Tiwari ⁴, Niyaz Ahmed ⁵, Aleem Khan ⁴, Chitoor M. Habibullah ⁴, Manisha Dwiwedi ², Arjun Lal Kakrani ⁶

^* Corresponding and presenting author

¹Molecular Biology Unit, National Centre for Cell Science, Ganeshkhind, Pune- 411 007, India, Departments of ² Gastroenterology and ³ Hepatology, Moti Lal Nehru Medical College, Allahabad, India, ⁴ Deccan Medical College, Kanchanbaugh, Hyderabad, ⁵ Centre for DNA fingerprinting and diagnostics, Hyderabad and ⁶Department of Medicine, B.J. Medical College, Pune, India

During the course of multicentric studies on diversity of H. pylori bacteria in Indian patients of Southern, Northern and Western parts, some unusual non-H. pylori bacteria were isolated especially from Non ulcer dyspeptic cases. These bacteria were cultured in the laboratory and in depth analysis was done using various genotypic (multilocus sequence typing with 16S rRNA, 23S rRNA, RNA Polymerase subunit D, Recombinase gene subunit A, DNA-DNA hybridization) and biochemical (API-20NE, BIOLOG, antimicrobial susceptibility testing) methods. This study revealed their identity as Ochrobactrum intermedium. In order to prove the pathogenecity of these bacteria, in vitro (HeLa and CaCO-2 cells) and in vivo (BALB/c, C57/BL6 and IL10 knock out mice) studies were conducted. The O. intermedium isolates showed adherence and invasion in cell lines studied. The histological analysis revealed presence of short rods in case of long term infection model and disruption of lamina propria of epithelial lining of stomach, indications of superficial ulcerogenic symptoms. We conclude that the presence of opportunistic pathogens like Ochrobactrum sp. may create serious consequences and ailments in the treatment of H. pylori infections. The excess antibiotics and therapies used to eradicate H. pylori may indirectly lead to growth of such bacteria creating complex situation. Diet and nutritional factors also play important role in development of such complications.

2. Glycosomal targeting of Hexokinase by an N-terminal PTS-2 peptide in Leishmania.

P. K. Umasankar, R. Y. Parikh, S. V. Surve, Y. S. Shouche, A. Jardim^*, M. S. Patole [National Centre for Cell Science, University of Pune Campus, Pune. ^*Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Canada.]

Leishmania is an evolutionarily primeval kinetoplastid pathogen and a distinct cellular feature relating evolution with this parasite is that it contains glycosomes, a microbody organelle that is similar to the peroxisomes in animals and plants. Glycosomes compartmentalize a large number of key metabolic pathways that include purine salvage, pyrimidine and ether-lipid biosynthesis. Energy generation is one of the important functions of glycosomes as it contains enzymes for glycolysis and fatty acid oxidation. All the glycosomal enzymes are synthesized in cytosol on free ribosomes and transferred to glycosomes. Recent studies have demonstrated that the correct targeting of proteins, especially of glycolytic enzymes, to the glycosome is crucial for parasite viability. We have studied targeting of hexokinase enzyme by cloning GFP-hexokinase fusion proteins. GFP fusion protein, having complete Leishmanial hexokinase sequence at its N-terminal end, was targeted to glycosomes. Similar translocation of GFP was observed, when only first 12 amino acids of the Leishmanial hexokinase, that includes putative PTS2 like domain, were fused with GFP. On the contrary, GFP fusion protein with hexokinase sequence devoid of first 12 amino acids, having PTS2 like domain, was found to be localized in cytoplasm and not translocated to glycosomes, signifying that PTS2 like domain is necessary and sufficient to target proteins to glycosomes. However, PTS2 sequence from Leishmanial hexokinase was not able to target EGFP efficiently to peroxisomes in mammalian CHO cell line. This Leishmanial specific PTS2 like domain and machinery involved in targeting of proteins to glycosomes can be used as a potential drug target.

3. FASCINATING MICROBES DEFEAT THE HOST: WHOSE WORLD IT IS ANYWAY!!

Sandeepa Eswarappa M., and Dipshikha Chakravortty

Department of Microbiology & Cell Biology, Indian Institute of Science, Bangalore 560 012.

Pathogenic micro-organisms have evolved many strategies to counteract the antimicrobial peptides, which they encounter upon entry into host systems. The ‘sapABCDF’ operon in Salmonella encodes an ABC transporter which is involved in counteracting these antimicrobial peptides. In addition to this, Salmonella enterica serovar Typhimurium genome has another putative operon consisting of yejA, yejB, yejE and yejF genes which encode for a putative ABC transporter. Gene products of these two operons have significant sequence identity. We hypothesized that the transporter encoded by yejA, yejB, yejEyejF genes might also be involved in counteracting antimicrobial peptides. In this study we have deleted the yejF gene which encodes the ATPase component of the ABC transporter. This and yejF strain showed increased sensitivity to antimicrobial peptides like protamine, melittin, polymyxin B and human defensin HBD- 2 and was compromised in its capacity to proliferate inside epithelial cells but not inside macrophages. In mouse model the yejF strain displayed decreased virulence as evident by the increased survival of the mice infected with the yejF strain as compared to those infected with the wild-type strain. These findings suggest that YejF is a virulence factor involved in counteracting antimicrobial peptides.

4. Metagenome: An abstract document of biogeochemical processes

Hemant J Purohit

Environmental Genomics Unit,

National Environmental Engineering Research Unit (CSIR),

Nehru Marg, Nagpur – 440 020 (MS) India

E Mail: hj_purohit@neeri.res.in, hemantdrd@hotmail.com,

Microbial communities are the main driving forces in the biogeochemical processes and in abstract form it constitute the major part of the ecological metagenome. With the changing environmental conditions this available genetic resource, in any ecosystem always under continuous dynamics. The knowledge of and for all these different events is stored in the metagenome. For survival it undergoes lateral, horizontal transfers and/or also rearrangements as the stress are experienced by the system. Theses processes can be presented and studied in time series analysis as changes in microbial communities.

In this paper, using the complexities of the available microbial diversity using different effluent treatment plant (ETP), as scenarios has been discussed. The data shows that these treatment plants have diverse microbes, both culturable and unculturable. The extracted total metagenome with the type of organic loading gives challenge for exploring the metabolic pathways that are never been predicted. The simple tools that can analyze total metagenome and their applications will be discussed for monitoring ETPs. The issue of environmental impact assessment using metagenome approach will be discussed with the case studies that provide the inputs for action plan to restore the base line data of contaminated sites.

5. Fungal dimorphism: Biochemical and molecular studies with respect to the ammonium assimilation, polyamine synthesis and the morphological transition

M.V.Deshpande

Biochemical Sciences Division

National Chemical Laboratory

Pune-411008

E-mail: mv.deshpande@ncl.res.in

The dimorphic fungi have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by the change in the environmental conditions. The reversible growth phenomenon has made dimorphic fungi as a useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in, plant, animal and insect too, fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition. The non-pathogenic zygomycetous fungus Benjaminiella poitrasii also exhibits temperature, pH glucose, metal ion like Zn⁺⁺, and ethanol/ myo-inositol dependent budding yeast- hypha reversible transition. As the cell wall metabolism plays a pivotal role in this transition, the biochemical correlation of different enzymes involved in nitrogen metabolism directly or indirectly, in the cell wall chitin synthesis, with dimorphic behavior of B. poitrasii parent and mutant strains has been studied extensively. The form –specific expression of two distinct NADP-dependent glutamate dehydrogenases (NADP-GDH, EC 1.4.1.4) and one NAD-dependent glutamate dehydrogenase (NAD-GDH, EC1.4.1.2) and their correlation with ornithine decarboxylase (ODC, EC 4.1.1.17), a key enzyme which regulates polyamine level in the cell has been studied. The role of nitrogen metabolizing enzymes in the dimorphic behaviour of B. poitrasii and other fungi such as Yarrowia lipolytica, Myrothecium verrucaria and Metarhizium anisopliae will be discussed.

MB-1

Biochemical nature of oil contaminated soil : an approach for bioremediation

Runita Rani Singha and Neelima Saikia

North-East Institute of Science & Technology

(Formerly Regional Research Laboratory)

Jorhat, Assam, India, E-mail : neelimasaikia@yahoo.co.in

Crude oil is a complex structure of hydrocarbons with numerous elements and metals. In oil fields during many operations crude oil is spilled over the neighboring areas. This spilled crude oil changes the biochemical nature of the soil. They cause serious threat to the flora and fauna of drill sites. Once the soil is contaminated with oil, no vegetation occurs in those contaminated areas. For natural plant succession the soil takes years together. Therefore, an approach was made to reclaim such soil with bioformulation for development of vegetation.

For our study we collected soil samples from abandoned drill sites area of Sivasagar district, Assam, India. Our study showed that soils are highly alkaline which is not suitable for seed germination. Oil content present in the sample is also recorded nearly 17%. In such a high oil containing soil vegetation usually never occurs. Amount of wax recovered from the contaminated soil is 10% which showed that the Assam crude oil is waxy in nature. Essential elements including heavy metals are detected in crude oil contaminated soil samples.

Biological nature of soil showed that beneficial microbes like nitrogen fixers, phosphate solubilizers, sulphur oxidizers and cellulose degraders and their activities are recorded less in such contaminated soil. As per the bio-chemical nature, soil was treated with microbial bioformulation which is a culture of three types of bacteria isolated from soils of Assam, India. Biochemical changes of soil was recorded every three months interval after the soil samples were treated with bioformulation. Total count study of soil beneficial bacteria showed remarkable changes in improvement of biological nature of soil. Soil pH which was an important factor for not germinating common seed in that soil was reduced to 7.5-6.2 after six months of treatment. Seed germination test further confirmed the reclamation of soil through application of bioformulation.

MB 2

In vitro evaluation of antimicrobial activity of an endophytic fungus isolated from a medicinal plant.

Sharbani Mazumder*, Dip Kr. Gogoi and Tarun C. Bora.

Biotechnology Division, North East Institute of Science & Technology (CSIR)

Jorhat-785006, Assam, India

e-mail: sharbanimazumder @yahoo.co.in

ABSTRACT:

Natural products associated with plants and their endophytic bioactivity has a great potential in the search for antimicrobial compounds as the endophytes are constantly exposed to intergeneric-genetic exchange with the host plant. Some of the compounds discovered from endophytic microorganisms so far are taxol, paclitaxel, cryptocandin, cryptocin, ambuic acid and many others that contribute a lot to the pharmaceutical market.

In this study, bark samples of medicinal plants were collected from the rain forest of Gibbon Wild Life Sanctuary, Jorhat and North East Ecology Park, NEIST, Assam and then endophytic fungi were isolated by standard method. Out of 51 endophytic fungi isolated from medicinal plants e.g. Azadiracta sp., Calotropis sp., Asparagus sp., Rauwolfia serpentina, four fungi were found to be more potential in preliminary screening. The crude cultivation medium extract of endophytic fungi were tested in vitro against both human and plant pathogens by Agar well diffusion method. The culture broth extract of isolate YMP showed antimicrobial activity, almost against all the test organisms, Candida albicans, Saccharomyces cerevisiae, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Therefore the active compound of YMP was solvent extracted and partially purified by preparative TLC using standardized running solvent system ethyl acetate and petroleum ether (1:3). Out of six major fractions recovered, the fractions with R_f values 0.56 and 0.46, respectively were found to be active against the tested organisms. The TLC fraction with R_f value 0.56 produced highest inhibition zone diameter against Bacillus subtilis (22 mm).The preparative HPLC separation fraction with RT 24.45 min produced no inhibition zone against test organisms.

Further identification and detailed chemical characterization of the active molecule may be a novel addition to the existing list of bioactive molecules from endophytic flora of this mega biodiversity hot zone.

* Presenting author

MB 3

Studies on control of biogenic hydrogen sulfide production in oil field produced water

Palakshi Bordoloi*, A.K Sarmah, B.K Gogoi, A.C.Kakoty , R.L.Bezbaruah

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

*email- palakshi2006@yahoo.co.in

email- rlbez@yahoo.com

Sulfate reducing bacteria (SRB) are a group of anaerobic bacteria that reduce sulfate (SO₄^2-) to sulfide (S^2-) during dissimilatory anaerobic respiration. In petroleum industry large volumes of deaerated water from underground reservoirs get contaminated with SRB as it is rich in nutrients. In the present study different biocides were tested for control of SRB in oil field producing water. Among the various biocides tested, paraformaldehyde, formaldehyde, cetyl trimethyl ammonium bromide (CTAB), Cetyl Pyrimidium Chloride (CPC) and silver nitrate showed effective control of SRB. In case of silver nitrate the concentration may be reduced to 5 ppm. The experiments were done for 28 days at the incubation temperature of 50^oC. Directed manipulation of naturally occurring in situ denitrifying microorganisms present in the produced water was found to be effective for the control of SRB.

MB 4

Reduced sensitivity among Gram-positive bacteria to barrel-stave pore-forming antimicrobial peptide alamethicin is due to decrease in its interactions with the changed membrane phospholipids.

S. K. Sood, Isha Raj^*, Ronda Venkateshwarlu^* and H.S. Thippeswamy^*

Animal Biochemistry Division, National Dairy Research Institute, Karnal – 132 001, Haryana, India. E-mail: sood@ndri.res.in

*Isha Raj, Ronda Venkateshwarlu and H.S. Thippeswamy contributed equally to the study

The number of isolated drug-resistant pathogenic microbes has increased drastically over the past decades, demonstrating an urgent need for new therapeutic interventions. The diverse population of non-toxic, non-immunogenic and highly selective pore forming antimicrobial peptides (AMPs) has long been looked upon as natural alternatives. Among these, AMPs such as YGNGV-motif containing bacteriocins from LAB, alamethicin from fungi Trichoderma viridae, cercopins from insects and defensins from mammals forms voltage dependent pores in barrel-stave manner. As expected, resistances against YGNGV motif containing bacteriocins have been reported. However, these AMPs may provide interesting template for antimicrobial activity optimization. The process of optimizing peptide antimicrobial activity and specificity requires information about mechnism of resistance. It has been indicated that the mutants with reduced sensitivity to YGNGV motif containing bacteriocins have altered phospholipid composition. Since the barrel-stave type pore forming AMPs follow the same mechanism for their action, therefore, it can be hypothesized that the resistance against these AMPs may also be because of a common mechanism. If a common mechanism of resistance is found, then a common strategy to fight the resistance against these AMPs may also be developed. Therefore, to ascertain, if a change in phospholipids is also a mechanism of resistance against other barrel-stave type pore forming AMPs, we selected mutant strains of three Gram +ve pathogenic bacteria i.e. Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis showing approximately five times reduced sensitivity to alamethicin. Thin layer chromatography (TLC) revealed that new phospholipids with different head groups appeared in mutants from S. aureus and E. faecalis, which might affect peptide-membrane electrostatic interactions. Upon quantification of individual phospholipids, it was found that there were significant differences in the individual phospholipid content between a sensitive strain and the mutant with reduced sensitivity among all the species. Gas Liquid Chromatographic analyses of the total cellular lipid extract and TLC resolved phospholipids showed that there were significant differences between fatty acid saturation level and chain length from sensitive and resistant mutant in S. aureus. E. Faecalis revealed differences in saturation level and B. cereus,in vitro colorimetric assay to measure peptide-membrane interactions, it was confirmed that there was a decrease in interactions of alamethicin with biomimetic membrane prepared from total cellular lipids from each mutant with reduced sensitivity. Therefore, it may be concluded that the reduced sensitivity to alamethicin among these G+ve bacteria is due to decrease in peptide-membrane interactions. Therefore, alteration in membrane composition and consequently peptide-membrane interactions may be a common mechanism of resistance among G+ve bacteria against barrel-stave pore-forming AMPs. reveled an increase in long chain fatty acids in mutant with reduced sensitivity as compared to the sensitive strain. Therefore, in all mutants, there was a change in membrane fluidity, which might affect peptide-membrane hydrophobic interactions. Subsequently, using an

MB 5

Influence of various parameters on chromium bioremediation efficiency

of Bacillus cereus

Tuhina Verma^1*, Pramod W. Ramteke² and Satyendra Kumar Garg¹

¹Department of Microbiology, Dr R.M.L. Avadh University, Faizabad

224 001, India

²Department of Biotechnology, Allahabad Agricultural Deemed University, Allahabad 211 007, India

^{*tuhinaverma2000@yahoo.co.in}

Chromate Cr (VI) as environmental pollutant enters into the ecosystem from leather industry, metal finishing, electroplating, etc. It is non-biodegradable and thus bioaccumulate in the food-chain. However, chromium is amenable to bacterial bioremediation. Six bacterial strains resistant to high chromium concentration were isolated from treated tannery effluent of Central Effluent Treatment Plant (CETP), Unnao (India). The extent of reduction of toxic Cr (VI) to less toxic Cr (III) was different for these isolates. One, of the six isolates identified as Bacillus cereus, was resistant to 200 mg Cr (VI)/ml, and reduced 70% Cr (VI) during aerobic growth. The Cr (VI) reduction potential was assessed under varying growth conditions by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometer. A linear relationship was observed among initial Cr (VI) concentration, increased cell density and Cr (VI) reduction. Among the heavy metals tested, presence of Ni²⁺ and Cd²⁺ significantly effected Cr (VI) bioreduction. Further, chromate reduction was optimal over a broad pH range of 5.5 to 9.0 and temperature range of 25 to 40^oC. The Cr (VI) concentration of uninoculated broth (control) remained unaltered throughout, indicating that medium components were unable to reduce Cr (VI). Therefore, B. cereus can meet the challenge of bioremediating Cr (VI) from a milieu of ions at broad temperature and pH range.

MB 6

TOTAL SYNTHESIS OF CYCLOSPORINE O

Vommina V. Sureshbabu.^*, Tantry Subramanyam J., and G. Chennakrishnareddy

Department of Studies in Chemistry, Central College Campus, Bangalore University, Dr. B. R. Ambedkar Veedhi, Bangalore – 560 001, India. e.mail:hariccb@rediffmail.com

Cyclosporines produced by secondary fungal metabolites of cylindrocarbon lucidum booth and tolypocaldium inflatum gams, and are the family of extensively studied N-methylated cyclic undecapeptides. Their most important biological activities includes the T-cell specific immunosuppression. The finding of a potential anti-HIV activity of Cs has evoked much more interest for the design of more selective cyclosporines active against HIV. The nonimmunosuppressive analogues of cyclosporines inhibit HIV replication in newly infected lymphocytes by inhibiting the binding capacity of P55-GAG protein to cyclophilin. Cyclosporine O which does not contain MeBmt at position 1 but still has marked immunosuppressive activity. The presence of norvaline at position 2 also makes it less nephortoxic than that of the parent CsA. The development of efficient methods for the coupling of highly hindered N-methyl amino acids possess greater challenge. Herein, we report the solution phase synthesis of cyclosporine O by the step by step assembly employing Fmoc-amino acid chloride/zinc dust and Bsmoc-amino acid fluoride/KOAt. All the ten intermediate peptides from dipeptide to undecapeptide pertaining to both Fmoc as well as Bsmoc series are isolated as crystalline solids and are characterized by ¹H NMR, mass & HPLC techniques. Thus, an efficient and reproducible method for the synthesis of CsO in solution phase by step by step elongation of the peptide chain has been achieved.

MB 7

Characterization of bacteria with potential for Simultaneous Nitrification and Denitrification

Anshuman A. Khardenavis*, Atya Kapley, Hemant J. Purohit

Environmental Genomics Unit,

National Environmental Engineering Research Institute (NEERI)

Nehru Marg, Nagpur-440020 (India)

* Presenting author Email: aa_khardenavis@neeri.res.in

Simultaneous nitrification and denitrification (SND) via common intermediate nitrite has been the most feasible and economical method of biological nitrogen removal from high nitrogen containing wastewaters, which has been known to occur naturally in biomass films or activated sludge flocs and has been employed successfully on a full scale in SHARON (single reactor system for high ammonia removal over nitrite) process.

In the present study, we isolated bacteria capable of performing SND, from activated sludge treating chemical and dye industry wastewater followed by studies on enzymatic activity. The 16S rDNA sequence based phylogenetic analysis revealed that the eight isolates shared sequence similarity of 98-100 % to Diaphorobacter nitroreducens NA10B (type strain JCM 11421). Genetic variance between the eight different strains was confirmed by RAPD, which showed different band patterns in the different strains. Strains HPC 805 and 815 showed exponential growth from 24 to 48 hours in denitrification medium followed by stationary phase thereafter (maximum O.D. of 2.25). The remaining strains showed slower growth in the following order HPC 821>HPC 856 > HPC 848 > HPC 847 > HPC 820 > HPC 834.

Whole cell experiments revealed nitrite accumulation upto 72 h followed by decrease on further incubation indicating the presence of both nitrate and nitrite reductase activities. Maximum nitrate reductase activity of 0.39 U mL^-1 was observed in cell free extract of HPC 856 (specific activity of 0.075 U mg^-1 protein) followed by 0.17, 0.09 and 0.01 U activity in case of HPC 805, HPC 815 and HPC 821 respectively. Nitrate reductase activities were very low in case of the remaining isolates. Reduction in the concentration of nitrite from reaction mixture in presence of cell free extract of all the isolates indicated the existence of nitrite reductase activity in all the Diaphorobacter strains. The percent conversion of total available nitrogen (N) (7.5 mM NH₄⁺-N + 20.9 mM NO₃^- -N=28.4 mM NO₃^-) to nitrite varied between 17-70 % for the different strains and the absence of ammonium and nitrate indicated incorporation of the remaining amount of supplied nitrogen (30-83 %) into the newly synthesized bacterial biomass.

Conventionally the process of SND requires the presence of presence of organic matter or substrates such as acetate, methanol, ethanol, citrate etc. as electron donors. Studies on effect of different substrates such as sodium acetate, ethanol and molasses waste on SND resulted in highest COD removal of 85-90% in case of sodium acetate, followed by 80-90% for molasses waste and 55-75% for ethanol. The corresponding nitrite released from nitrification of ammonium and denitrification of nitrate supplied in medium was found to be 95-115 mg L^-1, 2.5-3.5 mg L^-1 and 55-115 mg L^-1 for the three substrates respectively.

Further studies have to be carried out for SND by these bacteria in presence of alternative electron donors such as high organic containing wastewaters especially effluents from distilleries and dye industries.

MB 8

Microbial Redox Processes in a Geothermal Ecosystem of Tantloi in the Jharkhand State of India

Preeti Jain, ¹ Diti Chattterjee Bhowmick, ¹ Bijay Bal, ² Subrata Pal ¹

Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, ¹ and Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, ² India

E mail: ditichatterjee@yahoo.co.in. e mail: jain_ju@yahoo.com

Microbially mediated redox processes like oxidation and reduction of iron, sulfur play important roles in the biogeochemical cycling of a variety of elements in surface water and stream sediments. Both mesophilic and thermophilic bacteria have been described which can utilize inorganic compounds of Fe and S as electron donors. Within sulfur and sulfide-rich environments, sulfur oxidizing bacteria (SOB) as well as sulfur reducing bacteria (SRB) play major role in energy production and the maintenance of microbial community and are crucial in certain industrial waste-treatment systems. Biological oxidation of Fe(II) may be the predominant process leading to the formation of Fe(III) oxides in superficial aquatic sediments. Wherever biologically generated ferrous ion encounters an aerobic environment a potential habitat for iron-oxidizing bacteria is created. In turn, by producing Fe hydroxides, these iron-oxidizing microbes generate an excellent substrate for Fe-reducing prokaryotes to utilize under anoxic condition.

A geothermal ecosystem located at Tantloi in Jharkhand State of India has been discovered to be an interesting habitat for microbes of diverse nature.

An attempt has been made to enrich and study the bacterial consortia from the above ecosystem with respect to reduction and oxidation of iron, sulfur and, wherever possible to isolate the microorganisms from the enriched consortia. A consortium of microorganisms enriched in iron-reducing medium (iron oxyhydroxide [FeOOH] as an iron source) at 65°C under anaerobic condition was found to reduce Fe(III) to Fe(II). Fe(III) reduction by an enrichment and pure cultures were quantitatively estimated by ferrozine colorimetric method. Chromium [Cr(VI)] reduction was determined by Diphenyl carbazide method (DPC). In order to show the presence of γ-proteobacteria in the sediment soil, a fluorescence in situ hybridization (FISH) was performed with a probe of gamma proteobacteria. On the other hand, microorganisms enriched in LB-medium under partially aerobic conditions oxidized iron in iron-oxidizing medium at pH 7.0.

Furthermore, samples could also be enriched at 65°C under aerobic conditions in sulfur oxidizing medium using thiosulfate as electron donor and in sulfate reducing medium under anaerobic conditions. Pure colonies of bacteria capable of oxidizing sulfur were also isolated from such enrichment cultures. Cells from the different enrichment consortia were examined by epifluorescence microscopy after DAPI staining which indicated presence of bacteria with diverse morphologies.

The information about the microorganisms involved in redox processes in Tantloi Hot Spring as revealed by consortium studies points toward the unexploited biotechnological resource which could be employed for various industrial applications as well as in bioremediation of metals, radionuclides and organic contaminants. In addition to that, isolation of the microorganisms involved in the above processes will help in elucidation of the molecular mechanisms responsible for redox cycles of different metals at high temperatures.

MB 9

Antifungal activity from an alkalotolerant actinomycete sp A-03-116

Amit Kumar¹, Bhairavi J.¹, ManiMaran U.¹, Saravanan A¹, Srinivasan, M. C.^1▲ ,Sachin P², Bhattacharya A²., Pore V². and MV Rele^1*

¹Division of Biochemical Sciences and ²Division of Organic Synthesis

National Chemical Laboratory, Pune-411008, India

E-mail: ab.kumar@ncl.res.in

Since the early 1970s, both the medical and agricultural communities have battled with the sharp rise in the incidence of fungal diseases and with the parallel evolution of pathogen resistance to antimicrobial agents. It has been noted that fungi have been the most neglected pathogens, clearly demonstrated by the fact that Amphotericin B, a polyene antibiotic discovered as early as 1956, is still used as the ‘gold standard’ for antifungal therapy. The need for new, safe and more effective antifungal has become a major challenge today, particularly due to the dramatic rise in the incidence of life threatening systemic fungal infections in immunocompromised patients. Furthermore, in the field of agriculture, public pressure to reduce the use of chemical fungicides has increased. Concerns have been raised about both, the environmental impact and the potential health hazards related to the use of these compounds.

Biological control offers an attractive alternative strategy to synthetic fungicides. The alkalotolerant actinomycete strain A-03-1160 exhibiting antagonistic effect against several phytopathogenic fungi was isolated from Sambhar Lake, Rajasthan. In preliminary results in shake flasks, it was found that the actinomycete culture caused complete degradation of several fungi such as Mucor, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Alternaria solani, Fusarium moniliforme, Curvularia fallax, Curvularia lunata, Claviceps purpurea, Helmintosporium, etc. indicating that the organism could be used as a potential biocontrol agent. Coating the seeds of Cicer arietinum with the spores afforded protection against fungal contamination during germination.

The antifungal activity was found to be largely extracellular. The crude filtrate and the mycelial extract were devoid of 1, 3 glucanase and chitinase activities. The culture filtrate and the mycelia were extracted with ethyl acetate. The residue after extraction showed antifungal activity when tested against several fungi including plant pathogens. The minimum inhibitory concentrations (MICs) of the crude compound against different fungi are being determined. The actinomycete does not appear to be pathogenic to the Gram seeds. No detrimental effect was seen on the seedlings.

^▲ Retired scientist

MB 10

Molecular studies on the alpha-amylases from the rice pest Scirpophaga incertulas (Walker)

Poonam Sharma, Amit Kumar, Geetha S., Suresh C. G., Meenakshi Rele and Lalitha Sunil Kumar*

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

E-mail: poonam_sma@yahoo.com

Abstract:

Scirpophaga incertulas, commonly known as yellow stem borer, is a predominant monophagous pest of rice. Though rice transgenics expressing the Bt toxins have shown 100% mortality of yellow stem borer larvae, it is possible that this pest could also develop resistance to Bt as has been observed in other insects. Hence, in order to attain durable resistance, it is important to pyramid different insecticidal genes into the same variety such that the product of each gene acts on a different physiological and biochemical process in the larvae. Inhibitors to the alpha-amylases which are the major digestive enzymes are ideal candidates for genetic engineering. As a first step towards studying the specificity of interaction between the alpha-amylases and the inhibitors, we have identified and cloned the amylase gene which is 1506bp long, with a complete open reading frame encoding a protein of 502 amino acids, including the signal peptide. The different classes of inhibitors from a local variety of wheat, specific for the alpha-amylases of this rice pest, have been isolated and characterized. These results will be presented.

MB 11

A flexible ring cleavage option in Stenotrophomonas HPC69 isolate

Vinita Verma*, Atya Kapley and Hemant J. Purohit

National Environmental Engineering Research Institute,

Nehru Marg, Nagpur 440020 (India)

Presenting author* Email: vinita12may@gmail.com; Corresponding author: hj_purohit@neeri.res.in

In aerobic degradation of aromatic compounds by microorganisms, catechol plays a key role, since it is a ring cleavage substrate in which an exceptionally large number of peripheral pathways converge. Catechol formed as an intermediate is subjected to its ring fission by catechol 1, 2- dioxygenase (C12O) or catechol 2, 3- dioxygenase (C23O). C12O is an intradiol-type enzyme that cleaves the C-C bond at the1, 2- (ortho) position of catechol to make cis-cis-muconic acid, which is further degraded by the β-ketoadipate pathway. TheC23O, an extradiol-ring fission enzyme, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde by breaking the C-C bond at the 2, 3- (meta) position, which is part of the α-ketoadipate pathway. We have isolated a bacterium Stenotrophomonas HPC69 (AY996983) from contaminated soil with petroleum products that has catabolic potential towards phenol, p-cresol, catechol, methyl-catechol and hydroquinone. It could degrade phenol upto 4mM and p-cresol upto 3mM (individually) in 72h. HPC69 was identified by partial sequencing of 16S rDNA. The ring cleavage option of the isolate has been demonstrated by available genotype and enzyme activity. Genetic analysis by PCR using different catabolic primers showed catabolic loci of pheB and xylE (that code C12O and C23O enzyme respectively) revealing presence of both ortho and meta pathways of aromatic ring cleavage. PCR result was confirmed by enzyme assay done spectrophotometrically. Culture demonstrated ortho cleavage of catechol (C12O activity) during growth in p-cresol and meta cleavage of catechol (C23O activity) during growth on phenol. Results show that isolate had both C12O and C23O activity depending upon culture conditions. HPC69 also carried aromatic ring hydroxylating dioxygenase (ARHDO) gene which gave PCR product with ARHDO primer. ARHDO play important roles in the cycling of organic carbon in many environments. Thus, genes coding for this enzyme are useful markers for bacteria that are able to use aromatic hydrocarbons as growth substrates. Cloning of genes of loci pheB, xylE and ARHDO and their characterization are future targets.

MB 12

Decolorization of Basic Yellow-2 dye by Klebsiella sp.

Munmun Sinha¹, Atya Kapley, Hemant J. Purohit^*

Environmental Genomics Unit,

National Environmental Engineering Research Institute (NEERI)

Nehru Marg, Nagpur-440020 (India)

¹Presenting author Email: m_sinha213@yahoo.co.in

*Corresponding author Email: hemantdrd@hotmail.com

Basic Yellow-2 (syn. Auramine O) is a well-known, synthetic textile dye, which is widely used in industries to impart golden yellow color. It belongs to the diarylmethane group of Triphenylmethane (TPM) dyes and the presence of ring structure along with high solubility in water, makes it persistent in the environment. The parent compound of this dye is benzophenone, which is a reported carcinogen.

In, the present study, we have isolated 20 bacterial cultures from contaminated soil and various effluent treatment plants, belonging to the genus Klebsiella, and determined their diversity and capability to decolorize basic yellow-2 dye. This genus has earlier been reported to decolorize other dyes like methyl red and reactive violet 5.

The phylogenetic study based on 16s rDNA sequence revealed the percentage similarity among the isolates. The genetic variance was supported by Randomly Amplified Polymorphic DNA (RAPD) analysis. Screening of all the isolates for the decolorization of Basic Yellow-2 dye was done and three isolates namely, HPC68, HPC70 and HPC157 were found to cause effective decolorization of dye at 100mgL^-1 in the presence of 2mM sodium succinate as secondary carbon source under aerobic condition. The cultures sustained maximum up to 500mgL^-1 of dye.

The results based on UV Visible Spectrophotometric scanning showed decline in peak height at 430nm, revealing maximum color removal up to 97% in 9 days by HPC 70 followed by HPC68 (96%) and HPC 157(94%). Biomass increase was low and the addition of a carbon source was found to be mandatory for sustained growth of the culture. Among acetate, citrate, pyruvate, succinate and glutamate as carbon source, only succinate (2mM) favored decolorization.

Fourier Transform Infrared Spectrophotometeric (FTIR) analysis showed the disappearance of major peaks for aromatic ring and the emergence of new peaks belonging to secondary amines and dimethyl groups, suggesting the ability of the cultures to convert the chromophoric group of dye into non-chromophoric group by rearrangement of bonds.

The study reflects the occurrence of genetically diverse Klebsiella sp. in vast number within polluted environment, supporting their capability to resist stress conditions. The potential of certain isolates to adapt to environmental conditions and show effective involvement in the decolorization of Basic Yellow-2 dye is a novel finding. This phenomenon can be of great interest in future for dye industry wastewater bioremediation.

This phenomenon can be of great interest in future for dye industry wastewater bioremediation.

MB 13

Scanning electron microscope (SEM) studies of biosurfactant producing bacillus strain

VeenaKumaraAdi** and Karanth NGK*

**Bapuji Institute of Engineering and Technology Davangere,

*FPIC Dept CFTRI, Mysore

** Email: drveena.adi@gmail.com

The molecular targets of bioremediation are mixtures of recalcitrant, persistent and toxic chemicals. A heavy spill of oil/pesticide during transportation/usage is often heard of. During this mishap, only certain localized small piece of land is exposed to a very high tune of pesticides. The fate of micro flora and fauna is serious and is of ecological concern. It is not judicial to waive off the potential of the pollutant. Therefore there is an urgent need that less expensive and more efficient remediation approaches should be initiated. Here we describe the results of a scanning electron microscopic study (SEM) on the biodegradation of the solid substrate, wheat bran fortified with an OP pesticide, Fenthion in the laboratory scale using Bacillus subtilis FE-2 stain. The bacillus strain known to emulsify and degrade Fenthion was successfully grown on an agricultural waste wheat bran in spite of the heavy pesticide coat and disintegrate (degrade the solid substrate) Wheat bran was fortified with 0.4% fenthion as acetone solution and inoculated with 5% inoculum of bacillus. The series of SEM photographic images represent the gradual influence of the organism in attacking the bran coated with pesticide.

MB 14

Purification and properties of extreme halotolerant, alkaliphilic and thermostable α-amylases from Chromohalobacter israelensis TVSP 101

Prakash. Sangolgi ¹, Malashetty Vidyasagar ¹, Vineet Mahajan², Shouche Yogesh², Angela Corcelli³, K. Sreeramulu^1*

Extreme halotolerant α-amylases secreted by the halophilic bacterium Chromohalobacter israelensis TVSP 101 when the culture reached the stationary growth phase. These enzymes were purified to homogeneity by ethanol precipitation and subsequent butyl sepharose hydrophobic interaction chromatography and sephacryl S-200 gel filtration chromatography. The apparent molecular masses of the amylases were 72 and 62 kDa respectively by SDS-PAGE. The purified amylase I had a specific activity of 36.7 and amylase II 36.2 U mg^-1 of protein. The optimum pH and temperature of the enzymes were 9.0, at 65^oC. The enzymes were stable and had a broad NaCl profile (0-30%) with an optimum of 5% NaCl for amylase I and 15% NaCl for amylase II. A broad optimum was observed for amy I between 0 to 25% with 5% NaCl optimal, while Amy II activity was highest at 15% NaCl. When amy I and II were dialyzed against distilled water, amy I remained stable over a period of one week, where as amy II was stable up to 3 days and lost its activity. The enzymes exhibited high thermal stability retaining 65% of the activity after 1h at 75^oC. The enzymatic activities of both Amy I and II were completely lost by Hg⁺ ions at 1mM concentration. Both the amylases exhibited similar pattern of starch hydrolysis after 1 h incubation.

*Corresponding author: Dr. K. Sreeramulu

Fax: +91 8472 245927

Tel. No: +91 8472 263290

E-mail: ksramu@rediffmail.com

MB 15

Simultaneous saccharification and Fermentation of aow root starch for ethanol production

Mary Anupama,P¹, Vivek Babu.K², Naveen Kumar ,T³, Guru Rajesh. ⁴⁵

M.N.V.S.Satyanarayana. ⁵ 1,3,4,5- Dept. of Biotechnology, ANITS, Sangivalasa, Visakhapatnam.

2, Dept. of Chemical Engineering, A.U, Visakhapatnam. Email- drkandimalla@yahoo.com

Ethanol production from the starch of most widely cultivated rhizomatous plant, Maanta aundicea (L.) was attempted. The rhizome of this West Indian Manta Plant yields starch called arrowroot starch that finds its application in biscuit manufacture and also in the preparation of infant food. In the present study, simultaneous saccharification and fermentation of this starch using Saccharomyces cerevisiae was attempted. Under optimal conditions of experimentation, the ethanol produced is 60gm/l. When optimization was carried out using response surface methodology, for the critical parameters, temperature, pH and amylase to yeast ratio, the final ethanol content has improved by 11.6% w/v.

MB 16

Xanthomonas compestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline-alkali soils.

N.S. Darmwal and Adhyayan Sharan

Department of Microbiology

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

E-mail: dr_nsdarmwal@yahoo.co.in

Abstract

A total of 198 bacterial strains were isolated from various niches of saline-alkali soils, out of which 85 strains were able to solubilize phosphate on plates at 30°C. The strain RMLU-26, identified as Xanthomonas compestris was the most efficient with its ability to solubilize P, subjected to N-methyl-N’-nitro-N-nitrosoguanidine (NTG) for mutagenesis. The P solubilizing ability of X. compestris is reported for the first time. The wild type and mutant strains of X. compestris revealed a differential response to various stress factors (high pH, temperature, and salt concentration). Mutant strain revealed maximum P solubilization (67.1%) at 30°C & pH 8.0 while the wild type strain showed maximum solubilization (41.9%) at 35°C & pH 7.0. Percent P­₂O₅ solubilization by both strains revealed a steep decline in Tricalcium phosphate solubilization with an increase in NaCl concentration from 0.5 to 10% along with a concomitant drop in pH of the medium from 8.0 to 4.5 in wild type and 4.0 in mutant strain. However, a 1.5 to 2-fold increase in ‘P’ solubilization was observed in the mutant strain when compared to the wild type strain in the presence of NaCl. The overall improved tolerance of the strains to alkalinity and salinity could be due to accumulation and/or secretion of specific solute (Xanthan).

MB 17

PRODUCTION AND OPTIMISATION OF HALO-TOLERANT ALKALINE PROTEASE BY AN ALKALOPHILIC BACILLUS HALOURANS PPKS2.

Prakash P and Sreeramulu K*

Department of Biochemistry, Gulbarga University, Gulbarga-585 106, India

An extremely alkaliphilic bacillus halodurans sp ppks-2 was isolated from rice mill industries for production alkaline protease. The strain is Gram positive, spore forming, non-motile, catalase and oxidize positive. Alkaliphilic identified as bacillus halodurans according to physiological properties and 16s r RNA sequence analysis. The bacterium was designated as PPKS-2, good growth of strain was observed at pH 11. It grows also at 12 and 13.pH. The crude extracellular alkaline protease produced by isolate showed the maximal activity at pH, 11 and 60 ⁰C. A high level of alkaline protease activity was obtained in presence of glucose as carbon and gelatin as nitrogen source. The nearly similar growth and production of enzyme in cheap sources like Soya bean (1% w/v), and meat (1% w/v). The organism was halotolerant as it retained its 80%of original activity at 14% NaCl at 37 ⁰ C.

Corresponding author: Dr. K. Sreeramulu

Fax: +91 8472 245927

Tel. No: +91 8472 263290

E-mail: ksramu@rediffmail.com

MB 18

GENETIC DIVERSITY BETWEEN TWO GENUS OF PENICILLIUM

Malabika pandit and Pranab Roy

Dept.Of Biotechnology University of Burdwan, Burdwan----713104

N.C,Diagonistic Centre,NC-32\1 Arjunpur(N),Kolkata----700059, INDIA

e.mail-------Malabika_p@yahoo.com

Heritable variation is the raw material is the raw material both of evoution and artificial selection and is ultimately therefore the basis for all present day biodiversity. Genetic diversity is a part of Biodiversity. Here we study Genetic Diversity of Penicillium genus isolated from soil from East Kolkata Region.

Our objetive in this study is to identify the genetic diversity between P.Notatum and P.crysogenum.For this Purpose we have estimated different parameters, which can differentiate both the specis of penicillium.

Differences between individual organisms have two causes variation in the genetic material which all organisms possess and which is passed generation to generation and variation caused by environmental influence on each individual organism.Here we isolated both the specis of Penicillium and tried to identify the differences of parameters like Dna sequence , GC- content and LDso values etc. between two species.Sequence was determined by Amino acid analyzer,GC content was estimated using Caesium chloride by Density Gradient Method and the dose LDso(no. of microorganisms that will kill 50% of the animal ina test series have been determined by dose response curve).

It was found though a little difference of the sequence and GC-content However there is a great difference of LDso values between two specis.

Acknowledgement: Authors are gratefully acknowledging Mr. Niloy Chaterjee for his Laboratory Facilities.

Molecular Genetics

Invited talk 1

Molecular Genetics of Primary Microcephaly: Indian Scenario

Arun Kumar¹, M. Rao¹ and S. C. Girimaji²

¹Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore; ²Department of Psychiatry, National Institute of Mental Health and Neurosciences, Bangalore.

Microcephaly (small head) is defined as a condition in which the head circumference of an affected individual is >3 SD below the population age-related mean. The small cranial capacity results from underlying hypoplasia of the cerebral cortex rather than abnormal development of the overlying skull. Primary microcephaly (MCPH; OMIM 251200) is a distinct subtype that is defined by the absence of associated malformations and of secondary or environmental causes. It is inherited as an autosomal recessive trait. Patients with MCPH have mild to severe mental retardation without any other neurological deficits. It has an incidence of 1/30,000 to 1/250,000 live births in western populations. The actual incidence of MCPH is not known in India, but it could be higher in south Indian states of Karnataka, Andhra Pradesh, Kerala and Tamil Nadu where ~33% of marriages are consanguineous. It is a genetically heterogeneous disorder with six known loci: MCPH1-MCPH6. So far, genes for MCPH1 (BRIT1), MCPH3 (CDK5RAP2), MCPH5 (ASPM) and MCPH 6 (CENPJ) loci have been identified. We have ascertained a total of 43 families with MCPH from the states of Karnataka, Andhra Pradesh and Tamil Nadu. We have carried out linkage analysis of a majority of these families using PCR-based microsatellite markers from the candidate regions of six known MCPH loci. Our analysis showed that the ASPM gene is a major cause of MCPH in Indian families. The presence of unlinked families to any of the known MCPH loci in our dataset suggested the involvement of a seventh locus for this disorder. A genome-wide screening of unlinked families has led to the identification of a novel seventh locus (MCPH7). DNA sequence analysis in three MCPH5-linked families has identified one known and two novel mutations in the ASPM gene in a homozygous state. The results of our study will be presented and discussed.

Invited talk 2

Macromolecular uptake in Drosophila pericardial cells requires Rudhira function

Debjani Das, Rajaguru Aradhya T.C., Ashoka D. and Maneesha Inamdar

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India.

The vertebrate reticuloendothelial system (RES) functions to remove potentially damaging macromolecules, such as excess hormones, immune -peptides and -complexes, bacterial- endotoxins, microorganisms and tumor cells. Insect nephrocytes are thought to be functionally equivalent to the RES. We examined Drosophila pericardial cells (PCs) and analyzed their function, morphology and gene expression patterns. We demonstrate the nephrocyte function of PCs in live animals. We also develop and use live- cell assays to show that PCs take up soluble macromolecules in a Dynamin- dependent manner and insoluble colloids by a Dynamin- independent pathway. We identify rudhira (rudh), a novel gene expressed specifically in PCs. Loss- and gain- of- function experiments show that rudh regulates Dynamin- independent uptake in PCs. We also show that cardiac function is unaffected in larvae in which PCs have been removed post- embryonically by inducing cell- death. Late removal of PCs, even after the third larval instar, results in viable and fertile adults with a reduced lifespan and increased sensitivity to toxic chemicals. Thus, while embryonic PCs may have a role in cardiogenesis as suggested by others, they do not appear to have a later role in cardiac function as reported. Our study establishes important functions for Drosophila PCs, describes methods to identify and study them, provides a genetic handle for further investigation of their role in maintaining homeostasis and cardiac function and demonstrates that they perform key subsets of the roles played by the vertebrate RES.

Invited talk 3

Association of Taq1B polymorphism in CETP gene and C514T polymorphism in hepatic lipase gene with lipids and lipoprotein classes

R.Lakshmy, M.Sharma, H.P.S.Sachdev, G.R.Chandak, K.S.Reddy, S.K.Bhargava

AIIMS, New Delhi.

Cardiovascular disease (CVD) is projected to be the leading cause of death in India by the year 2020 and alarmingly 50 % of these deaths are projected to occur among young and middle aged individuals. Nearly 30% of the patients with premature CVD have normal lipid values. Recent studies have suggested that lipoprotein particle size may be an additional risk factor for atherosclerosis. Compared to large buoyant LDL particles a predominance of small dense LDL particles is associated with an increased risk. Constituents of lipoprotein metabolism like lipoprotein lipase (LPL), hepatic lipase (HL) and cholesterol ester transfer protein (CETP) contribute to formation of small dense LDL. Both CETP and HL are important determinants of plasma HDL and LD subclass distribution. Levels of HL and CETP are genetically determined and genes encoding HL and CETP are candidate genes for CAD. The HL gene promoter polymorphism C514T and Taq 1B polymorphism in intron 1 of CETP gene have been associated with the levels of HL and CETP respectively. In a large cohort of 1000 subjects we looked at the C514T polymorphism in HL gene and Taq1B polymorphism in CETP gene. In addition lipids, lipoprotein subclasses and other cardiovascular risk factors were determined. Association of genotypes with lipids, LDL sub fraction and other cardiovascular risk factors were assessed.

The T allele frequency of HL gene was 0.22 in the cohort. Subjects with TT genotype had significantly higher triglyceride levels and lower HDL cholesterol levels. Allele frequency of Taq1B: B1 was 0.47 in the population. B1B1 homozygote was associated with lower HDL in males. Differences were also apparent in LDL particle size with genotypes. The data on genotypes and their association with lipids and other cardiovascular risk factors would be presented.

Invited talk 4

Evolution of Cellobiose Utilization in E. coli

Aashiq H. Kachroo¹, Aswani K. Kancherla², Nongmaithem S. Singh³, Umesh Varshney³ and S. Mahadevan<sup>1

1</sup>Department of Molecular Reproduction, Development and Genetics, ²Molecular Biophysics Unit, ³Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012.

Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel+) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. By large scale mutant isolation and characterization, we show that the Cel+ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N-N?-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single amino acid substitutions. Introduction of chbR from two Cel+ mutants resulted in activation of
transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel+ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and chitobiase, upon induction, could recognize, transport and cleave cellobiose, respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-beta-glucosidase under a heterologous promoter. Biochemical characterization of one of the mutants, ChbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of Cellobiose-6-phosphate as an inducer compared to the wild type regulator. These results show that acquisitive evolution of E. coli towards utilization of cellobiose under laboratory conditions occurs by the genetic modification of the regulation of the chb operon under selective pressure.

MG 1

Cloning and developmental expression of a putative transferrin cDNA from the spruce budworm, Choristoneura fumiferan.

Dinakar R. Ampasala^{1, 2*}, Peter Krell², Arthur Retnakaran¹ and Qili Feng¹

¹Great Lakes Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1219 Queen Street East, Sault Ste. Marie, Ontario, Canada, P6A 2E5. ²Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. ^*To whom correspondence should be addressed: Tel: +01-519-253-1044;

E-mail: ampasaladr@yahoo.com.

Insect transferrins are suggested to be involved in iron transport and in innate immunity, probably by binding ferric ions to prevent pathogens and parasites from utilizing them. A spruce budworm (Choristoneura fumiferan) transferrin cDNA (CfTF) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. The cloned cDNA was 2221 base pairs in length and coded a predicted protein of 681 amino acids with a molecular mass of approximately 76kDa. At amino acid level CfTF shared higher than 70% homology with transferrins isolated from Muduca sextaBombyx mori. and Like other insect transferrins, CfTF retains most of functional iron-binding site at N-terminal lobe. Northern blot analyses indicated that transferrin mRNA is present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth to sixth instar larval stages. The highest levels of CfTF expression were detected in the fat body, whereas relative low levels of the expression were detected in the epidermis and no expression was found in the midgut tissue. The expression of CfTF mRNA could be induced by bacteria but not by fungi. Expression of CfTf mRNA was suppressed by iron load. Experimental methods and results will be described and discussed.

Keywords: Transferrin; Iron-binding protein; Innate immunity; Antimicrobial protein; Spruce Budworm; Choristoneura fumiferana.

MG 2

A 22bp inverted repeat sequence found in the signal coding region of organophosphate degrading gene is involved in down-regulation of organophosphate gene expression in E. coli

J. P. Pandey, Purushotham Gorla, Bramanandam Manavathi^# and Dayananda Siddavattam*

Dept. of Animal Sciences, # Dept. of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad – 500 046, India. Email: jpp_hcu@yahoo.co.in

Abstract:

Organophosphate hydrolases (OPHs), involved in hydrolytic cleavage of structurally diverse organophosphates are coded by a plasmid borne, highly conserved organophosphate degrading (opd) gene. An inverted repeat sequence found in the signal coding region of the opd gene was found to be responsible for inducing a stable stem loop structure with a DG of –23.1 kcals/mol. This stem loop structure has shown significant influence on the expression levels of organophosphate hydrolase (OPH) in E. coli. When the signal coding region comprising the inverted repeat sequence was deleted a ~3 fold increase in the expression levels of OPH was noticed in E. coli BL21. Mutations in the inverted repeat region, especially at the third position of the codon, to a non‑complimentary base destabilized the inverted repeat. When such opd variant, opd’ was expressed, the expression levels were found to be similar to expression levels coded by the construct generated by deleting the signal peptide coding region. Deletion of signal peptide did not influence the folding and activity of OPH. Though high level induction has resulted in accumulation of OPH as inclusion bodies, modulation of expression levels by reducing the copy number of the expression plasmid, inducer concentration and growth temperature has produced majority of the protein in soluble and active form. Taken together these results suggest that inverted repeat (IR) sequence plays critical role in the expression of opd gene.

MG 3

Molecular mechanism of membrane targeting of parathion hydrolase in Brevundimonas diminuta MG

Purushotham Gorla*, J.P. Pandey*, Bramanandam Manavathi^# and Dayananda Siddavattam*

*Department of Animal Sciences, # Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad – 500 046, India. Email: itsurpurushotham@yahoo.co.in

Abstract:

Organophosphates are neurotoxic chemical agents widely used as pesticides and herbicides for productive agriculture. These organophosphates are hydrolysed by bacterial hydrolases called organophosphorus hydrolases (OPH EC 3.1.8.1). Though OPH protein has been shown to associate with membrane in Flavobacterium sp. ATCC27551 and Brevundimonas diminuta MG, the molecular mechanism behind its membrane targeting is not known. Here we report that precursor form of OPH contains a signal peptide with 29 amino acids and carries an invariant motif (TRRVVLK) within this signal peptide which showed homology to the recently discovered sequence motif (S/T-RR-XFLK) of twin arginine transport (tat) pathway in higher eukaryotes. Since twin arginine motifs play a key role in tat secretion pathway, we have substituted the second consecutive arginine to alanine in OPH and cloned in a broad host range expression vector with C-terminal His-tag. Cell fractionation combined with western blot analysis showed the accumulation of twin arginine mutant OPH in cytosolic fraction while the wild type OPH was found in membrane fraction indicating the involvement of RR motif in membrane targeting. In addition, we have also noticed a protein band with low molecular weight than the mature form of OPH suggesting a putative second translational start site in coding region of opd gene and are consistent when expressed either in E. coli or B. diminuta. Furthermore, we have also noticed approximately a 5 kDa increase in size of OPH when it is expressed in B. diminuta than in E. coli indicating the possible posttranslational modifications in OPH protein. Together these results suggest that twin arginine motif as well as unknown posttranslational modifications might play potential role in membrane targeting of OPH in B. diminuta.

MG 4

Activation modulation of renal glucocorticoid receptor during postnatal development of chicken

Daniel Nongbri and R. Sharma

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

Presenting author E-mail: dannongbri@yahoo.com

The formation of the glucocorticoid receptor (GR) complex initiates a two step process that ultimately leads to the regulation of specific gene transcription. The first step after steroid binding is termed as activation (or transformation) and involves a conformational change which exposes the receptor’s DNA binding site. The second step is the binding of the activated receptor to specific regions on the chromatin. Activation can be achieved in vitro by increasing the temperature, ionic strength, pH and dilution of steroid bound cytosol. Modulation of activation process was studied in the kidney of male chicken (Rhode Island Red breed) at two postnatal ages, 0- and 30-day. Studies on the modulatory effect on heat and salt activation of GR by various modulators were made. The inhibitors used were sodium molybdate, sodium tungstate, cadmium chloride and oleic acid. The magnitude of inhibition was judged using DNA-cellulose as well as purified nuclei. Molybdate was found to be a more potent inhibitor compared to tungstate. Cadmium and oleic acid were also found to be potent inhibitors of both heat and salt activation of GR. However, the magnitude of activation modulation by these inhibitors remains the same at the two ages studied, indicating that the process of activation modulation do not get altered at the ages studied.

(Financial support to DN as SRF from CSIR (F.No.9/347(160)2K3-EMR-I) is gratefully acknowledged)

MG 5

Induction of heat shock protein (hsp16) in Caenorhabditis elegans by the synthetic pyrethroid, Cypermethrin

Shashikumar. S and Rajini P.S.

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

Mysore-570020

E-mail: shashikmr12@gmail.com

Living organisms exhibit a universal response to the presence of external stressors by expressing a set of proteins called as heat shock proteins. Induction of heat shock response has now been recognized as a biomarker for the presence of external stressors. Pesticides are known to induce cellular and toxic effects in living organism, exerting stress. Many reports demonstrate induction of stress responses by pesticides, leading to transcriptional activation of heat shock promoters. Synthetic pyrethroids have gained importance worldwide due to their low mammalian toxicity and high insecticidal activity and consequently the environmental occurrence of synthetic pyrethroids has become widespread. Synthetic pyrethroids elicit neurotoxicity by interacting with sodium channels on axons of central and peripheral nervous system. In the present study, we evaluated the potential of Cypermethrin, a synthetic pyrethroid to induce heat shock response employing transgenic strain of C. elegans, PC-72, carrying a reporter LacZ gene under the control of homologous heat shock (hsp16) promoter. The evaluation of induction of heat shock response in wild type animals is tedious and requires either metabolic labeling or immunoblotting techniques. The transgenic strain of C. elegans (PC-72) offers itself as a convenient and reliable model for studying heat shock response both qualitatively and quantitatively by the virtue of b-galactosidase gene placed under hsp-16 promoter. In situ staining for b-galactosidase activity with X-Gal revealed nonspecific expression of b-galactosidase throughout body in worms exposed to varying concentrations of cypermethrin and heat shock of 30^oC (positive control), while CdCl₂ (0.03mg/ml), which was included as chemical positive control, induced expression of b-galactosidase only in pharynx region. Quantitative assessment of heat shock response was carried out by spectrophotometric assay for b-Galactosidase with ONPG as substrate after worms were exposed to varying concentration of Cypermethrin (5, 10 and 15mM) for different intervals (4, 12, 24, and 36h). Maximum b-galactosidase was evident in worms subjected to heat shock while the b-galactosidase activity in DMSO exposed group was comparable to that in control. An increase of 16-145% was found CdCl2 treated worms in a time dependent manner. At all the concentrations of cypermethrin tested, maximum b-Galactosidase activity was observed after 12h of exposure (8, 9 and 10 fold increase by 5, 10, and 15mM cypermethrin when compared to control). These data suggest that Cypermethrin induces strong heat shock response in transgenic strain of C. elegans and therefore, the transgenic starin-PC-72 can be exploited as a convenient and reliable model for studying induction of heat shock response by xenobiotics.

MG 6

Acetylation dependent regulation of multifunctional human

histone chaperone NPM1

Jayasha Shandilya, Swaminathan Venkatesh, Shrikanth S. Gadad,

Nagashanker G and Tapas K. Kundu

Transcription and Disease Laboratory, Molecular Biology and Genetics Unit,

Jawaharlal Nehru Centre for Advanced Scientific research, Bangalore-64, INDIA

jayasha@jncasr.ac.in

Eukaryotic genome is organized into a highly compact but dynamic chromatin structure composed of nucleosomes as basic structural unit. Among the different protein machineries which confer chromatin fluidity and precise organization, histone chaperones have emerged as one of the most important group of proteins amongst them. Histone chaperones are cellular proteins rich in acidic residues and have the ability to directly interact with histones. Among the plethora of various histone chaperones present in the cell involved in replication coupled nucleosome disassembly and ordered histone deposition, there are emerging evidences for roles of these chaperones outside the S phase of the cell cycle which mainly transcription and repair related chromatin reorganization. Nucleophosmin (NPM1 also known as B23) is a ubiquitously expressed, highly dynamic, multifunctional nucleolar protein having histone chaperone activity. Besides, it also has roles to play during ribosome biogenesis, centrosome duplication and nuclear–cytoplasm shuttling. Its ability to activate transcription from the chromatin template in an acetylation dependent manner has been recently reported by our laboratory. The fine tuning of NPM1 function and cellular localization is modulated by its various cellular interacting partners and its post translational modification status like phosphorylation, acetylation and sumoylation. Among the various post translational modifications acetylation of NPM1 is implicated in its transcriptional activation potential. Using antibodies against acetylated NPM1, its cellular distribution and physiological significance is being addressed. Apart from this, NPM1 is known to get upregulated under various DNA damaging stresses as well as in tumors of different histological origin. Preliminary results have indicated at the possible connection of acetylation status of NPM1 with cancer manifestation and stress response.

MG 7

Human transcriptional coactivator PC4: a p53 responsive gene plays pivotal role in DNA repair

Kiran Batta and Tapas K. Kundu

Transcription and Disease laboratory, Molecular Biology and Genetics Unit,Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore-560064, India.

E mail : kiranbatta@jncasr.ac.in

Abstract: The tumor suppressor protein p53 regulates expression of several genes, which mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Recently, we have shown that human transcriptional coactivator PC4 is a unique activator of p53 function. Here we report that PC4 is a p53 inducible gene. We have found p53 acts an activator of PC4 transcription. Both PC4 mRNA and protein levels increase in response to stimuli that result in p53 induction. Interestingly, we found that PC4 stimulates DNA end joining independent of the source of ligase and the nature of DNA ends. PC4 helps in the association of DNA ends through its C-terminal domain. We show that PC4 coimmunoprecipitates with XLF and Ku proteins and shows significant colocalization with NHEJ proteins. By performing episomal plasmid reactivation assays we found that PC4 enhances DSBs rejoining in vivo independent of its transcriptional regulatory functions. In conclusion, above results suggest that PC4 is a down stream target for p53 and plays important role in DSB repair by promoting the association of DNA ends.

MG 8

ASSEMBLY AND DISASSEMBLY OF TOBACCO STREAK VIRUS

Chhavi Mathur & H. S. Savithri.

Department of Biochemistry, Indian Institute of Science, Bangalore, India.

chhavi@biochem.iisc.ernet.in

Tobacco Streak Virus (TSV) is the type member of Ilarvirus genus (Bromoviridae). It was first noticed to be a serious problem in India during an outbreak on sunflower in parts of Karnataka in 1997. TSV forms quasi-isometric, non-enveloped, spherical virions and has a tripartite single-stranded positive sense RNA genome. Each RNA is encapsidated in separate virions having different sizes, sedimentation co-efficients and nucleic acid content. The coat protein is encapsidated by RNA3 through a subgenomic RNA, sgRNA4, which is encapsidated along with RNA3. Genome activation by coat protein is an important feature of TSV.

The mechanism of assembly and disassembly of this virus has not been investigated thus far. Therefore, the coat protein of TSV was cloned and overexpressed in bacterial system. Sucrose density gradient, gel filtration, glutaraldehyde crosslinking and electron microscopic analysis of the protein showed the formation of virus-like particles (VLPs). The VLPs were found to dissociate into dimers in the presence of high calcium chloride concentration. Removal of calcium chloride from the buffer resulted in reassembly of coat protein subunits into VLPs as analyzed using sucrose density gradient, gel filtration and electron microscopic studies. Cellular RNA was found to be non-specifically encapsidated in the VLPs. Interaction of nucleic acid with coat protein is being analyzed.

MG 9

Characterization of MutL, a mismatch repair protein from Neisseria gonorrhoeae

Chiranjeevi.B and  Viswanadham D

             Department of Biochemistry, Indian institute of science, Bangalore, India.

chiru@biochem.iisc.ernet.in

The mechanism of DNA methyl directed mismatch repair (MMR) has been most extensively studied in Escherichia coli. Repair is directed to the daughter strand at the replication fork by virtue of the transient absence of d(GATC) methylation in newly synthesized DNA. Repair is initiated by binding of MutS to a mismatch, and MutL is recruited to the heteroduplex in MutS- and in ATP dependent manner. Assembly of the MutL-MutS-heteroduplex ternary complex is sufficient to activate d(GATC) endonuclease activity of MutH.

Neisseria gonorrhoeae is a gram-negative diplococcus bacterium, which is pathogenic to humans who are its only natural host.  Its pathogenic potential critically depends on the phenomenon of phase variation of a number of surface expressed molecules. Implications of MMR in generating

phase variation in Neisseria gonorrhoeae are under active investigation. Proteins of the MutS and MutL family have been found in the most sequenced organisms and, therefore, the principal mechanism of mismatch recognition and repair are believed to be evolutionarily conserved. Sequencing of the complete genome of Neisseria gonorrhoeae revealed that it has homologues

of the genes that encode MMR enzymes, MutS and MutL but lacks MutH endonuclease, as do humans. It is possible that another component in Neisseria gonorrhoeae carries out the MutH strand specificity function or that the mechanism of strand discrimination is different.

Towards the characterization of Neisseria gonorrhoeae MMR proteins, we first compared the Ngo MutL DNA sequence with E.coli MutL and found that while the amino terminal region was conserved, the carboxyl terminal was highly divergent and both these domains in Ngo MutL are separated by a linker region.  We have cloned, over expressed and purified Ngo MutL to homogeneity and characterized the MutL protein. Interestingly we found that MutL protein of Neisseria gonorrhoeae has DNA nicking activity which is required for the initiation of MMR. In addition, we cloned the carboxyl terminal domain (CTD) of Neisseria gonorrhoeae MutL and found that it has a robust DNA nicking activity.  Characterization of these proteins showed that CTD is more thermal stable than the full length MutL. Both the full length MutL and CTD occur as dimers in solution as judged by molecular size exclusion chromatography, chemical cross linking, dynamic light scattering and analytical ultracentrifugation.

Despite of the importance of MutL in MMR the only available information on the function of the protein is the fact that it is recruited to the heteroduplex in MutS- and in ATP dependent manner. Our demonstration of nicking endonuclease activity of Ngo MutL, thus provides a novel context to view its function in MMR.

MG 10

MODULATING LEAF SIZE BY ALTERING THE ACTIVITY OF TCP4, A TRANSCRIPTION FACTOR IN Arabidopsis thaliana

Kavitha Sarvepalli and Utpal Nath (kavitha@mcbl.iisc.ernet.in)

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

A variety of body forms seen in the living world, are generated by two universal cellular processes – division and expansion. In plants, especially, where the presence of a rigid wall precludes cell migration, the development of all lateral appendages, such as leaves, depends entirely on these two processes. Many genetic mechanisms have evolved to lay down the precise timing and location of cell growth in a leaf.

One of the key growth regulators in Antirrhinum is CINCINNATA (CIN), belonging to the TCP family of genes that encode plant-specific transcription factors. In a developing leaf, cell divisions occur in a basipetal fashion – the cells at the base of the leaf divide for a longer duration of time than those at the top. In the cin mutant, the normal pattern is disrupted. Excess divisions occur throughout the lamina which results in larger leaf size. As CIN is expressed in the actively dividing regions of the leaf and functions as a growth-repressor, it has been suggested that CIN controls the growth potential of cells in response to an unknown growth-arrest signal. In absence of CIN function, the perception of the arrest signal is lost, hence more growth. On the other hand, heightened CIN activity in presence of the arrest signal is expected to retard the growth. This possibility can be tested by observing the effect of a hyperactive form of CIN protein in transgenic Antirrhinum plants. However, as transgenic technology in Antirrhinum remains technically challenging, we are testing the hypothesis in the model plant Arabidopsis thaliana, by manipulating the function of CIN orthologue, TCP4. Like CIN, TCP4 is involved in regulation of leaf growth. However, unlike CIN, the loss-of-function phenotype of TCP4 is mild because of functional redundancy of TCP4 with two other TCP family genes.

We have generated a hyperactive TCP4 which showed >20-fold higher activity than the wildtype protein, as tested by reporter gene assay in yeast. We introduced the hyperactive version under the control of TCP4 endogenous promoter in one of the mutant lines of TCP4. In five out of ten independent transgenic lines, leaf size was reduced to varying degrees. The strongest transgenic line showed up-regulation of the expression of a direct downstream target of TCP4 at the RNA level, which indicates presence of hyperactive TCP4 function. This suggests a correlation between the level of activity of TCP4 and the final size of the leaf. Thus, leaf size in Arabidopsis appears to be dependent on the level of TCP4 activity in the cells.

MG 11

Characterization of an Adenine/cytosine methyltransferase from Helicobacter pylori 26695.

Arun Banerjee

Department of Biochemistry, Indian Institute of Science, Bangalore, India.

arun_ban11@rediffmail.com

DNA methylation is catalyzed by a group of enzymes called DNA methyltransferases, which catalyze the transfer of methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. DNA MTases are further classified based on the position of the methyl group transfer on bases in DNA: exocyclic amino MTases and endocyclic MTases.

Helicobacter pylori is a Gram-negative, spiral-shaped, flagellated, fastidious bacterium with remarkable adaptation to its human host. H.pylori was recognized as a definitive carcinogen (type I) by the international agency for research on cancer (IARC, 1994), mainly on the basis of large sero-epidemiologic case-control studies and animal models. H.pylori is the infectious agent responsible for the second highest number of infection associated cancers. The H.pylori genome contains an unusually high proportion of restriction-modification (R-M) systems. With the abundance of R-M systems in H.pylori, till date, maximum numbers of Type III R-M systems are also found to be present in H.pylori. Three Type III R-M systems are present in strain 26695 and two in J99 strain. Using genomic microarrays of H.pylori it was observed that in response to acid exposure there was 3-fold over expression of hp0593 gene (a putative Type III adenine methyltransferase gene) at pH 4.5 than at physiological pH. The putative HP0593 protein sequence analysis also revealed the presence of both DPPY motif (present in N⁶-adenine MTase) and PCQ motif (present in C-5 cytosine methyltransferase).

Towards biochemical characterization, hp0593 gene was cloned, confirmed by sequencing. The HP0593 protein was purified both in native condition and with histidine tag to homogeneity. The protein was confirmed by mass spectrometry and western blotting. Further the presence of DPPY motif was confirmed by formic acid cleavage assay. Fluorescence spectroscopy analysis showed the AdoMet binding to the HP0593 protein. Radioactive methylation assay was carried out to show that HP0593 protein is a methyltransferase. The Oligomeric status of protein was determined by molecular size exclusion chromatography and it was found to be monomer.

MG 12

N Mediated Antitermination Mechanism In Lambdoid Phages

Anoop Cheeran, Ghazala Muteeb and Ranjan Sen

Transcription antitermination is an important mechanism that controls the regulation of gene expression. Transcription antitermination by N protein of Lambdoid phages involves specific interactions of the C-terminal domain of N with the elongation complex. The interacting surface of N on elongation complex is unknown. The H19B-N specific mutations in RNA polymerase subunits, together with specific cleavage patterns generated near the active site Mg²⁺ of the RNA Polymerase of an N modified stalled elongation complex suggest that the H19B-N protein acts close to the active center of RNA Polymerase and it involves stabilization of the weak RNA:DNA hybrid at the terminator and destabilization of the interactions of terminator hairpin in the RNA exit channel. It is not known whether the interaction surface on elongation complex for H19B-N defines a generalized interaction surface for all the N proteins, especially so for the λ N, whose C-terminal domain is very different from H19B-N.The λN protein can be 107 amino acid or 133 amino acid long, although the upstream start is in large stem loop that constitutes an RNase III substrate and the in vivo processing of the RNA removes the first AUG from the 5’ end of transcript. However, comparision of the two lengths of this protein in their antitermination property shows slight variation in their requirement for host factors, in vitro. Moreover, the studies of N-Rho interactions throw a new light on mechanism of antitermination at the factor dependent terminators.

Presenting Author: Ghazala Muteeb, Junior Research Fellow, Lab of Transcription Biology, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad-500076, India. ghazalamuteeb@gmail.com, ghazala@cdfd.org.in

Corresponding Author: Dr. Ranjan Sen, Lab of Transcription Biology, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad-500076, India.

MG 13

Biochemical characterization of M.EcoPI: a type III DNA methyltransferase

Sameera. P

Department of Biochemistry, Indian Institute of Science, Bangalore, India

sam_dame@mrdg.iisc.ernet.in

A major defense mechanism found in bacteria against bacteriophage invasion is Restriction-Modification (R-M) system. R-M systems can be classified into four groups: Type I, II, III and IV based on their cleavage pattern, co-factor requirements etc.

Type III R-M enzymes are multifunctional proteins that exhibit both restriction and modification activities. Restriction is done by Res and Mod subunits and modification (methylation) by Mod subunit of the enzyme. Mod subunit of EcoPI (Type III DNA methyltransferase) methylates DNA at the internal adenine in the sequence 5’ AGACC 3’. This methylation is carried out in the presence of S- adenosyl methionine (AdoMet) and Mg²⁺. The Mod subunit is 646 amino acids long with a conserved DPPY motif required for its catalytic activity at the N-terminal and FXGXG motif required for AdoMet binding at the C-terminal. DNA recognition and binding is done by the Target Recognition Domain (TRD) situated at the center.

The Mod subunit of EcoPI was cloned into pET28a, which is an N-terminal His tagged vector. Its expression was checked in E.coli BL21 strain and purification was done through Ni-NTA affinity chromatography. Steady state and Single turn over experiments have been performed and kinetic parameters such as K_m, V_max and K_cat was determined.

The putative metal binding motif in the Mod subunit of EcoPI, made it essential to study the effect of metal ions on the methylation activity of the enzyme. Methylation assays were performed in presence and absence of Mg²⁺. Methylation reaction was enhanced in presence of Mg²⁺. Methylation assay was performed using different divalent metal ions. In presence of Fe²⁺, Co²⁺ and Ca²⁺ methylation activity was comparatively more than in presence of Mg²⁺.

Sequence comparison of M.EcoPI with another member of this group, M.EcoP15I, showed 90% similarity between the N- and C-terminal amino acids of both the proteins and a highly variable middle portion which corresponds to TRD. Attempts are being made to construct hybrids within the TRD of M.EcoPI by introducing some portion of M.EcoP15I TRD by mega primer PCR amplification method. This will enable to locate the region involved in DNA recognition and might provide an idea about its specificity.

Pharmacology and Nutrition

Invited Speech PN 1

Role of vitamin A in the regulation of obesity in WNIN/Ob rat model

Dr.A.Vajreswari

Deputy Director, NIN, Hyderabad

Abstract

Obesity is a multi-factorial disorder, resulting out of an imbalance in energy homeostasis leading to accumulation of excess energy as fat. It is increasing at an alarming rate even in developing countries. Vitamin A, an important micronutrient, with unusually wide range of vital biological functions (eg. morphogenesis, vision, embroyonic development, reproduction and immune function etc. in mammalian system) is a known regulator of adipose tissue growth. To understand the possible role of chronic vitamin A supplementation on body weight regulation and obesity, 7 months old, 24 male lean and obese rats of WNIN/Ob strain were taken, broadly divided into two groups; each group was subdivided into two subgroups consisting of 6 lean and 6 obese rats and received diets containing either 2.6mg or 129mg vitamin A/kg diet for two months. Chronic feeding of diets with high doses of vitamin A (high but non-toxic) resulted in significant reduction in body weight gain, adiposity index and retroperitoneal white adipose tissue weight (RPWAT) (without affecting food intake) in obese rats compared to their lean counterparts. Irrespective of the phenotype, this treatment significantly elevated the hepatic free and total retinol levels without affecting serum retinol level. Interestingly, chronic dietary challenging with high doses of vitamin A resulted in augmented expression of uncoupling protein1 (UCP1) gene in brown adipose tissue (BAT) of obese rats, whose basal expression was found to be low. Surprisingly, obese rats receiving normal levels of vitamin A-diet showed high serum HDL-C and lower hepatic SR-BI expression levels compared to lean counterparts. Furthermore, vitamin A supplementation resulted in over-expression of hepatic SR-BI receptors (protein & gene) with concomitant reduction in serum HDL-C levels in obese rats. In addition, vitamin A feeding resulted in significantly increased RPWAT apoptotic index, Bax protein expression and decreased expression of Bcl2 in the lean rats. However, no such changes were observed in the RPWAT of the obese rats subjected to identical treatment. In conclusion, high vitamin A supplementation reduces body weight gain in obese phenotype and decreases adipose tissue mass in both phenotypes, perhaps through different mechanisms.

Invited speech PN2

Iron and zinc cross-talk at the site of absorption: Implications in intervention programmes

Dr. K. Madhavan Nair, Assistant Director, Micronutrient Research Group, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania,

Hyderabad-500 007, India. Email: nairthayil@hotmail.com

Abstract:

Iron and or zinc deficiencies are serious public health concerns around the globe, particularly in pregnant women and children. Therefore, therapeutic supplementation of iron/zinc has been in practice to correct respective deficiencies. However, iron is a potential oxidant and therefore, it is necessary to understand the ill effects of excess iron supplementation, especially, at the site of the absorption and the effect of zinc and other nutrients on such process. We addressed these issues using iron and zinc - deficient rat models and the caco-2 human intestinal cell model. We have demonstrated that supplementation of excess iron leads to increased oxidative stress and loss of mucosal functional integrity as assessed by mucosal marker enzymes and ultra structure studies. Further we demonstrated that co-supplementation with either antioxidant nutrients or zinc abrogates the adverse effects of iron. Based on the plasma appearance after feeding, it is clear that zinc negatively interacts with iron absorption. Therefore, it is possible that decreased intracellular labile iron pool in the presence of zinc leads to decreased oxidative stress and related downstream events. Interestingly, we noticed that zinc but not iron inhibits each other’s absorption and cellular zinc status appears to modulate the iron absorption through regulation of apical iron transport protein, divalent metal ion tranporter-1 (DMT-1). Further, zinc inhibited the cytosolic aconitase, a 4Fe-4S cluster containing bifunctional protein IRP-1 that regulates the translation of several iron binding and transport proteins thus contributing to iron homeostatics in the gut. We further studied the mechanism of cytoprotective effects of zinc using human intestinal cell line model caco-2 cells. Inducing oxidative stress increased IRP-1 activation, DMT-1 expression, iron uptake, and leading to intracellular oxidative stress and apoptosis while co-treatment with zinc inhibited all the above intracellular events in caco-2 cells. Thus, there appears to be modulation of aconitases/IRP switch by zinc. Further understanding of this will help in deciphering the molecular cross-talk between iron and zinc.

(Financial support from CSIR and DBT and contributions from the research scholars and other and colleagues are acknowledged).

Invited Speech PN 3

Programming for adiposity & insulin resistance in WNIN rat offspring : role of maternal micronutrient deficiencies.

M. Raghunath , National Institute of Nutrition, Hyderabad.

Metabolic syndrome, a cluster of cardiovascular risk factors such as diabetes, hypertension, dyslipidemia and obesity, is a major health problem in India. Causes for Insulin resistance (IR), the common underlying feature are not yet understood. Maternal undernutrition retards intrauterine growth and programs the fetus to metabolic changes which predispose the offspring to adiposity, IR and associated diseases in adult life. Most studies so far, have considered maternal protein and calorie deficiencies but not of micronutrients (MN). Considering widespread prevalence of micronutrient deficiencies and low birth weight in India, we have investigated whether or not maternal MN deficiencies programme the WNIN rat offspring for high body adiposity, IR and associated diseases in their later life.

Our initial studies have shown that maternal and peri/post natal restriction of vitamins (VR) or minerals (MR) increased body fat % and plasma triglycerides in offspring while lean body mass (LBM), fat free mass (FFM) and insulin secretion to a glucose challenge were decreased. Both VR and MR modulated adiponectin and leptin levels whereas only VR altered oxidative stress and antioxidant status in offspring. Rehabilitation from parturition but not weaning corrected maternal VR induced changes in offspring whereas maternal MR induced changes appeared irreversible.

Considering the irreversibility of maternal MR induced changes, the effect of maternal deficiency of trace elements: Mg, Mn, Cr & Zn (which modulate insulin at several levels) is being studied. Maternal Mg & Cr deficiencies irreversibly increased body fat % and visceral adiposity in offspring and was associated with altered adipokine expression, synthesis and transport of fatty acids. Mg deficient offspring had transient fasting hyperinsulinemia while it was persistent in Cr deficient offspring. Maternal Mg deficiency decreased insulin response to a glucose challenge, muscle mass and its basal glucose uptake in offspring, Cr deficiency increased them. That only some changes due to maternal Mg & Cr deficiency were corrected by rehabilitation albeit from parturition but not weaning, indicates the importance of Mg & Cr nutrition during lactation. Maternal Mn and Zn deficiencies per se did not affect body composition in but rehabilitation altered it and the changes were seen early but were transient. However maternal Mn deficiency per se caused transient hyperglycemia, hypoinsulinemia , impaired glucose tolerance, altered lipid metabolism in offspring and rehabilitation had varied effects on different parameters. Interestingly maternal Cr, Mn and Zn deficiencies affected male and female offspring variably, in that some changes were seen in males but not females.

PN 1

Influence of mulberry (Morus indica L) leaves on gluconeogenesis in streptozotocin-diabetic rats

B. Andallu *, Vinay Kumar A.V ⁺ and N.Ch.Varadacharyulu^**

^*Dept. of Home Science, Sri Sathya Sai University; **Dept. of Biochemistry, Sri

Krishnadevaraya University, Anantapur-515001, Andhra Pradesh and ⁺ Vinay Kumar A.V, St.Joseph’s College of Engineering, Chennai

Influence of mulberry (Morus indica L.) leaves on gluconeogenic substrates and gluconeogenic enzymes in streptozotocin (STZ)-diabetic rats was investigated. Treatment with dried mulberry leaf powder at 25% of the diet for a period of 8 weeks was found to be beneficial to STZ-diabetic rats as evidenced by controlled hyperglycemia. In addition, mulberry leaves countered (reversed) the alterations in gluconeogenic substrates in STZ-diabetic rats as indicated by significant reduction in serum pyruvic and lactic acid levels, a significant increase in proteins and a significant decrease in free amino acid, urea, and creatinine levels in blood, and decreased urinary excretion of urea and creatinine. Anomalies in the activities of hepatic gluconeogenic enzymes associated with impaired glucose homeostasis in STZ-diabetic rats were ameliorated by the mulberry leaves indicating that control over hyperglycemia and associated complications in the diabetes by mulberry leaves is by way of regulation of gluconeogenesis. With respect to all the parameters, mulberry leaves were more effective than the oral hypoglycemic drug glibenclamide.

PN2

IN VITRO AND IN VIVO ANTIOXIDANT ACTIVITY OF MULBERRY (MORUS INDICA L.) LEAVES

B. Andallu*, S.Mahalakshmi*, A.V. Vinay Kumar**

*Department of Home Science, Sri Sathya Sai University, Anantapur, India.

**Dept. of Biotechnology, St. Joseph’s Engineering College, Chennai, India

The study investigated the free radical scavenging activity of mulberry leaves in vitro and antioxidant activity in vivo. Erythrocyte membrane of human volunteer was used as peroxidation model system in vitro while aged human volunteers served as subjects for in vivo assessment. Ethanolic extract of mulberry leaves (Morus indicaL.) was tested for the antioxidant activity by in vitro assays using butylated hydroxyl toluene as positive control. Mulberry leaf extract displayed scavenging activity against DPPH, nitric oxide and superoxide radicals and also inhibited FeSO₄-induced lipid peroxides and hydroperoxides in erythrocyte membrane in a concentration dependent manner. This is supported by the in vivo experimental data i.e. significantly decreased lipid peroxidation in erythrocytes and plasma; significantly elevated levels of non enzymatic antioxidants (b carotene, vit.A, C & E, ceruloplasmin,etc.) in serum and significantly elevated activity of enzymatic antioxidants (catalase, superoxide dismutase and glutathione-s-transferase) and reduced glutathione (GSH) in erythrocytes in aged human volunteers treated with mulberry leaf powder (5g/day) for 45 days. In conclusion, mulberry leaves possess antioxidant properties and the effect in vitro as well as in vivo is a result of coordinated play/ synergistic action of antioxidant phytochemicals and nutrients present in the leaves.

Key words: Antioxidant activity, mulberry, lipid peroxides, DPPH radical, scavenging activity, non enzymatic antioxidants.

PN3

Effect of ecological variation on antioxidant capacity and chemical constituents of Pinus roxburghii Sarg

DWAIPAYAN SINHA and P. KAKKAR

Herbal Research Laboratory,

Industrial Toxicology Research Centre

Post Box. 80, M.G.Marg.

Lucknow-226001

India.

E-mail: dwaipayansinha@yahoo.co.in

Pinus roxburghii Sarg is a common gymnosperm that grows along the slopes of outer Himalayas starting from Kashmir in the west to Bhutan in the east. It is traditionally used as curative agent for various diseases such as ulcer, snake bites etc.In this study, an attempt has been made to quantify the chemical constituents as well as evaluate the antioxidant potential of the bark extracts.Pinus roxburghii Sarg barks were collected from three different Himalayan regions of India namely Himachal Pradesh, Uttarakhand and Darjeeling.50% ethanolic extract of barks were subjected to quantitative estimation of phenolics, flavonoids,Tannins and proanthocyanidins.The extracts were also tested for their antioxidant potential.SOD mimetic activity, inhibition of lipid peroxidation, NO quenching capacity, ABTS⁺ decolourisation assay,hydrogen peroxide scavenging assays were used to assess antioxidant potential. Proanthocyanidins(0.345 gram equivalents of leucoanthocyanidin Kg^-1 of bark) and flavonoids ( 1.097 gram equivalents of quercetin Kg^-1 of bark) contents were highest in Himachal Pradesh sample.Total phenolic content was found to be highest in Uttarakhand sample (12.73 gram of Gallic acid equivalent Kg^-1 of bark) whereas tannin content was highest in the Darjeeling sample (7.88 grams of tannic acid equivalent Kg^-1 of bark).Darjeeling sample also showed highest percent inhibition of NO release.(64.79%) which was even higher than that of the same amount of catechin and quercetin taken as positive control.Uttarakhand sample showed highest SOD mimetic activity (373units.min^-1.mg^-1 extract), LPO inhibitory capacity (97.3%) and Trolox equivalent antioxidant capacity (8.27mM.mg^-1 dry bark) which was higher than positive control catechin by 85.6%, 41.7% and 54.7% respectively .Hydrogen Peroxide decomposing capacity was highest in Himachal Pradesh sample (15.92µM. mg^-1 of extract at 60 seconds) which was higher than catechin by 331.43%.Thus, variation in antioxidant capacity of Pinus roxburghii Sarg. bark from different ecological zones as tested against different free radical species was observed.It is noteworthy that Pine bark from Uttarakhand was found to have highest antioxidant capacity as well as total phenolic content

PN 4

Structural features of arabinoxylans from Indian wheat varieties having differences in chapati-making quality.

Revanappa S.B and Salimath. P.V.

Department of Biochemistry and Nutrition,

Central Food Technological Research Institute,

Mysore, 570 020, Karnataka, India.

revanappasb@gmail.com

Arabinoxylans are the major non-starch polysaccharides present in cell walls of cereal grains. These polymers have attracted considerable amount of interest because of their physico-chemical properties. More specifically, they are shown to affect water distribution and rheological properties of dough, loaf volume of bread and retrogradation of starch. Degree of substitution and distribution of arabinosyl residues along the xylan backbone are of great importance as they affect the property of arabinoxylans to interact with each other and /or with other polysaccharides and therefore alter physical and functional properties of these macromolecules. Structures of arabinoxylans in different varieties of wheat are known to vary and have a relation to bread making quality. Arabinoxylans were extracted from four different varieties of wheat such as DWR-162, GW-322 (good chapati making quality) and MACS-2496, HD-2189 (poor chapati making quality) with barium hydroxide and purified by a combination of alcohol precipitation and glucoamylase digestion. Structural features of the purified arabinoxylans were elucidated by a combination of methods such as methylation analysis, ¹³C and ¹H NMR, FT-IR, periodate oxidation and optical rotation measurements. The results indicated xylan backbone in β- (1-4) linkage, which are substituted at O-3 or at O-2 and O-3. Completely branched xylosyl residues were also observed in arabinoxylans of wheat varieties. The wheat varieties known for good chapati making quality have highly branched arabinoxylans compared to poor chapati making varieties. Differences in the structural features of arabinoxylans among these varieties could be responsible for their physicochemical properties, which might in turn lead to variations in their chapati making qualities.

PN5

Dietary calcium and lipid metabolism

SAGWAL, R. and KANSAL, V.K.

National Dairy Research Institute, Karnal, India.

e-mail: renusv@gmail.com

Effects of dietary calcium levels on body weight, body fat, lipid metabolism and blood lipid profile were studied in male albino rats fed for 101 d on diet varying in calcium level / source. Four groups, each comprised of 8 animals, were (1) low calcium, 0.2%; (2) moderate calcium, 0.6%; (3) high calcium, 1.2% and (4) high dairy calcium, 1.2% of which 50% of calcium was obtained from skim milk powder. Average feed intake and body weight gain were comparable in the four groups. Dietary calcium levels had no significant influence on the deposition of fat in inguinal, epididymal, retroperitoneal and mesenteric fat pad mass. Also, there was no significant difference in adiposity index and BMI values among four dietary groups. Low calcium group had significantly lower faecal fat excretion than the other three high calcium groups. The excretion of faecal matter increased by 31.3%, 49.2% and 57.5% on moderate calcium, high calcium and high dairy calcium diets, respectively, when compared with that on low calcium diet. FAS activity on high dairy calcium diet was reduced to less than 50% of the values recorded on low calcium, moderate calcium and high calcium diets. While there was no difference in Carnitine palmitoyltransferase activity in epididymal fat pad and skeletal muscle among four groups, liver mitochondrial β-oxidation rate decreased significantly by 77.6% and 88.1% on high calcium and high dairy calcium diets, respectively, in comparison to that on low calcium diet. The concentration of total cholesterol in plasma decreased by 18.6%, 13.2% and 27.4% and VLDL+LDL-cholesterol by 23.0%, 21.0% and 42.3% on moderate calcium, high calcium and high dairy calcium diets, respectively, when compared with low calcium diet. The HDL-cholesterol to total cholesterol ratio increased by 13.2%, 15.8% and 36.8% on moderate calcium, high calcium and high dairy calcium diets, respectively, in comparison with low calcium diet. The concentration of plasma triacylglycerols also decreased significantly on moderate calcium and high calcium diets when compared with low calcium diet. The atherogenic index decreased by 18.4%, 30.0% and 50.0% on moderate calcium, high calcium and high dairy calcium diets, respectively, when compared with low calcium diets. To conclude, dietary calcium has hypocholesterolemic effect and the dairy calcium is more efficacious, but it has no effect on body weight and fat pad mass in rats.

P N6

ISOLATION OF A NOVEL INSECTICIDAL COMPOUND FROM THE ROOTS OF DECALEPIS HAMILTONII FOR STORED GRAIN PROTECTION

Y.Rajashekar and T.Shivanandappa

Dept. of Food Protectants & Infestation control

Central Food Technological Research Institute

Mysore 570020

Email:- rajacftri@yahoo.co.in

There is a great interest in newer natural biopesticides for stored grain protection. We have isolated a insecticidal compound from the edible roots of Decalepis hamiltonii. The organic extract showing insecticidal activity against stored grain insects was fractionated on a silica gel column and the purity was ascertained by RPHPLC. The molecular characterization was done by LCMS and NMR spectroscopy. Bioassay of the compound for insecticidal activity was done by the filter paper technique with a exposure period of 24 hr against Sitophilus oryzae and Rhizopertha dominica. The minimum lethal dose(100% mortality) of the compound was 0.063mg/cm². Its effectiveness as grain protectant is being studied. This is a novel natural insecticidal molecule reported for the first time.

PN7

Effect of curcumin on protein carbonyls of cerebral hemispheres of mice as a function of age

Preeticia Dkhar and R.Sharma

Department of Biochemistry,North Eastern Hill University,Shillong-793022

Presenting author E-mail: preet123_dkhar@yahoo.com

Curcumin, the yellow pigment from the rhizome of Curcuma longa is a widely studied phytochemical which has a wide variety of biological actions: anti-inflammatory and antioxidative. Curcumin is considered a promising drug for the treatment of many diseases, most of which are related with the aging process. It can ameliorate a series of pathogenetic conditions that share an inflammatory or oxidative bases such as cardiovascular diseases, Alzheimer’s disease, sarcopenia and type II diabetes among others. Protein carbonyls are formed as a consequence of the oxidative modifications of proteins by reactive oxygen species. Protein carbonylation has various implications in health and diseases. In the present study, the effect of curcumin on age-related carbonyl content of cerebral hemispheres in mice was investigated. Protein carbonyls of cerebral hemispheres have been found to be higher in 6-months old mice as compared to 1-month old mice. Adminstration of curcumin (90 mg/kg body weight, intraperitoneally) for 3 consecutive days decreases the carbonyl content in 1- and 6-months old mice. These results suggest that the formation of protein carbonyls can be reduced by curcumin administration.

PN8

Isolation of the active principles with potent inhibition of 5- and 15-Lipoxygenases and Cyclooxygenase-2 from Terminalia bellerica: Studies on molecular characterization and kinetics of inhibition

T. Chandramohan Reddy, A. Aparna, Satish Sharan, V. Laxmi Pathi, P. Reddanna

Department of Animal Sciences, School of Life Sciences

University of Hyderabad-500046, India

e-mail: tcmr2008@yahoo.co.in

Terminalia bellerica commonly called as ‘Bihara fruit’’, is one of the combination of Triphala, a well known Ayurvedic medicine. Terminalia bellerica is rich in protein (40 percent) and oils (35 percent), and is particularly high in the omega 3 essential fatty acid, linoleic acid. It has strong antimicrobial properties and its compounds exhibit anti-HIV-1, antimalarial, and antifungal properties. In the present study we have tested the activity of ethanolic extract of Terminalia bellerica against 5-Lipoxygenase (5-LOX), 15-Lipoxygenase (15-LOX), Cyclooxygenase-1(COX-1) and Cyclooxygenase-2 (COX-2). We have observed potent inhibition of 5- and 15-LOXs and COX-2 but showed no inhibition of COX-1. The ethanolic extract also showed potent free radical scavenging activity of DPPH in a dose dependent manner. In the light of the above, further studies were undertaken to isolate active principles present in the fruit pulp of Terminalia bellerica by RP-HPLC. We have observed that one of the isolated compounds showed potent COX-2 inhibition (1.0mg/ml) but very less inhibition with COX-1 and no inhibition with 5- and 15-LOXs and another isolated compound showed potent inhibition of 5- and 15-LOXs (IC-50 values are 3.75mg/ml and 9.25mg/ml respectively) and no inhibition with COX-1 and 2. These active fractions isolated from RP-HPLC were further characterized based on LC-MS, NMR, MALDI-TOF and IR analysis and also studies were undertaken on the kinetics of inhibition of the enzymes by these isolated active fractions.

PN 9:

Protective effects of Vitamin E and Selenium on Fluoride induced alterations in albino mice

U.Sivaiah, A.Jayasankar, N.J. Sushma, J.Vasantha Sena and K.Jayantha Rao

Division of Toxicology and Pathobiology, Dept. of Zoology,

Sri Venkateswara University, Tirupati- 517502 A.P.

The present study is designed to find beneficial effect of Vitamin E and Selenium on Fluoride induced hematological parameters in three generations of mice , five different treatments like control (Group I), Fluoride treated (Group II), Fluoride and Vitamin treated (Group III), Fluoride and Selenium treated (Group IV), and Fluoride, vitamin E and Selenium treated (Group V). The RBC, WBC, Platelet count, Hemoglobin content, packed cell volume along with MCV. MCH, MCHC levels were investigated. During the experiment group III, IV&V animal show more it indicates the protective effect of Vitamin E and Selenium during the experimentation.

PN 10

COMPARATIVE STUDY OF MONOCROTOPHOS AND AZADIRACHTIN ON THE BLOOD OF ALBINO MICE.

U. Sivaiah ¹, N. John Sushma ² and K. Jayantha Rao^1*

¹Department of Zoology, Sri Venkateswara University, Tirupati-517 502.

²Department of Biotechnology, Sri Padmavathi Mahila University, Tirupati-517 502

ABSTRACT

Monocrotophos is a fast acting systemic and contact insecticide of short persistance. It is presently used as an effective insecticide in agricultural and animal management practices on a wide scale. The research and development cost of biopesticides is much less as compared to chemical pesticides. Recent awareness about the hazards of persistent synthetic pesticides to environment and their high cost has generated the need of intensified research on pesticide of plant origin. Natural products offer better degree of selective toxicity and may form ideal substitute for synthetic insecticides. The blood parameters are probably the more rapid and detectable sensitive indicators of variations under intoxication stress and useful in assessing health status of the animal. In the present study an attempt has been made with sublethal doses of Monocrotophos and Azadirachtin. Adult albino mice were divided into four groups having 6 animals each, the second, third fourth group animals exposed to sublethal doses (single, double, multiple) of pesticide has revealed a significant changes in blood of albino mice. Pesticide poisoning on various haematological parameters were observed.

The decreased levels of RBC count, haemglobin (Hb), Packed cell volume (PCV) and mean corpuscular volume (MCV) along with an increase in mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and White blood cells (WBC) count were observed in control and experimental animals. The degree of these changes was more at multiple dose exposure of pesticides. The results indicated that the Monocrotophos was more toxic than Azadirachtin. These elevation indication of toxi potential of a administered pesticide.

PN 11:

COMPARATIVE ASSESSMENT OF MONOCROTOPHOS AND AZADIRACHTIN ON THE PROTEIN METABOLISM OF ALBINO MICE.

U. Sivaiah ¹, N. John Sushma ² and K. Jayantha Rao.K¹*

¹Department of Zoology, Sri Venkateswara University, Tirupati-517502.

²Department of Biotechnology, Sri Padmavathi Mahila University, Tirupati-517502

Biological control of pests or biopesticides has been suggested as an effective substitute for chemical. It is nothing but the control of harmful pests by using other insects, pests, plants or any such living body. There is urgent need for safer, naturally occurring, environmentally friendly pesticides and new strategies to reduce resistance problems. In the present study an attempt has been made with monocrotophos, a synthetic OP pesticide and natural bio-pesticide azadirachtin by different doses of intraperitonial administration to observe the significant variations in protein metabolism and associated enzyme systems. Adult albino mice were divided into four groups having 6 animals each, the second, third and fourth group animals exposed to sublethal doses (single, double, multiple) of pesticides has revealed a significant changes in brain, liver, kidney and muscle tissues of albino mice. The relative effect of monocrotophos and zadirachtin was determined on the protein metabolism and associated enzyme system of mice exposed to single, double and multiple doses. Protein profile in the tissue can be considered as a diagnostic tool in assessing the physiological status of tissue or animal as a whole. The changes in protein metabolism is directly proportional to the amount of dose administered to the mice. These elevation in all parameters of present study indicates the toxic nature of pesticides and it also suggests that monocrotophos was more toxic than azadirachtin.

PN 12

Electrochemical Determination of Anti Protozoal Drugs Metronidazole and Ornidazole at b - Cyclodextrin Modified Carbon Paste Electrode

C. Narasimha Rao¹’, K. Balaji¹, M. Suman¹, P. Venkateswarlu¹*

1. Dept. Chemistry, S.V.U. College of Mathematical and Physical Sciences C.N. Rao²

2. Dept. Zoology, S.V.U. College of Biological and Earth Sciences S.V. University, Tirupati: cnrassvu@yahoo.co.in

Abstract

A simple, precise, inexpensive and sensitive voltammetric method has been developed for the determination of Anti Protozoal Drugs Metronidazole and Ornidazole. Cyclic voltammetry (CV) and Differential pulse voltammetry (DPV) techniques were used to determine Anti Protozoal Drugs Metronidazole and Ornidazole at a bare carbon paste Electrode (CPE) and a b - Cyclodextrin modified carbon paste electrode (CDMCPE). Compared with a bare CPE, CDMCPE gave the highest sensitivity. The effect of other parameters like pH of the supporting electrolyte, accumulation time, accumulation potential on the adsorptive stripping response were studied. A linear calibration was obtained from 2.4 x 10^-7 to 4.2 x 10^-5 M and 3.2 x 10^-7 to 2.8 x 10^-5M with a detection limit of 2.1 x 10^-7M and 2.9 x 10^-7M for Anti Protozoal Drugs Metronidazole and Ornidazole respectively. The method was applied successfully for the direct determination of Anti Protozoal Drugs Metronidazole and Ornidazole in pharmaceutical formulations and biological fluid samples.

Keywords:

Metronidazole, Ornidazole, pharmaceutical formulations, cyclic voltammetry, differential pulse voltammetry, clay modified carbon paste electrode.

Plant Biology

Invited talk:

A new pathway to engineer to maximize crop productivity in stress conditions

Narendra Tuteja, International Centre for Genetic Engineering & Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi – 110 067, Email: narendra@icgeb.res.in

Abiotic stress is an increasing threat in reducing agricultural productivity worldwide. The high salinity stress impairs crop production on at least 20% of irrigated land. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. Helicases are one of the smallest molecular motors of biological system, which harness the chemical free energy of ATP hydrolysis to catalyze the opening of energetically stable duplex DNA (called DNA helicases) or unfold the secondary structures in RNA (called RNA helicases) and thereby are involved in almost all aspect of nucleic acid metabolism. Mostly all the helicases contain some conserved signature motifs including DEAD-box, which act as an engine to power DNA unwinding. In plants the role helicases in abiotic stress is just begening to emerge. Here we present the isolation of a pea DNA helicases (PDH45) and its role in high salinity stress tolerance. PDH45 is homologous to eIF4A, involved in translation initiation, localized in nucleus and cytosol. It contains multiple activities including ATP-dependent DNA and RNA unwinding, ATP-binding and DNA-dependent-ATPase. The transcript of PDH45 genes was found to upregulated under high salinity stress. PDH45 enzyme is found to be unipolar, active at only basic pH, expressed more in root than shoot and not regulated by ABA treatment. The over-expression PDH45 in tobacco and rice plants confers high salinity tolerance without yield loss, thus suggesting a new pathway to engineer to restore crop yield in sub-optimal conditions. The discovery of salinity stress-induced helicase should make an important contribution to our better understanding of DNA and RNA metabolisms and stress signaling in plants. The possible mechanism of salinity tolerance will be discussed.

References:

1. Tuteja, N. Mechanisms of high salinity tolerance in plants. Methods in Enzymology: Osmosensing and Osmosignaling, Vol. 428, Oct. 2007 (In Press).
2. Tuteja, N. ABA and abiotic stress signalling. Plant Signaling & Behaviour, 2: 135-138, 2007.
3. Vashisht, A. A. and Tuteja N. Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance. J. Photochem. Photobiol.: Biology, 84, 150-160, 2006.
4. Mahajan, S. and Tuteja, N. Cold, salinity and drought stresses: An overview. Arch. Biochem. Biophys. 444, 139-158, 2005.
5. Vashisht, A., Pradhan, A., Tuteja, R., and Tuteja N. Cold and salinity stress-induced pea bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C. Plant J. 44, 76-87, 2005.
6. Sanan-Mishra, N., Pham, X. H., Sopory, S. K., Tuteja, N. Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natl. Acad. Sci. USA102, 509-514, 2005.
7. Tuteja, N. and Tuteja R. Prokaryotic and eukaryotic DNA helicases: essential molecular motor proteins for cellular machinery. Eur. J. Biochem. 271, 1835-1848, 2004.
8. Pham, X. H., Reddy, M. K., Ehtesham, N. Z., Matta, B., and Tuteja, N. A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity. Plant J., 24, 1-13, 2000.
9. Tuteja, N. Plant cell and Viral Helicases: essential enzymes for nucleic acid transactions. Crit. Rev. Plant Sci., 19, 449-478, 2000.
10. Tuteja, N. & Tuteja, R. DNA helicases: the long unwinding road. Nature Genet. 13, 11-12, 1996.

Invited talk:

Enhancing crop productivity in saline soils

Ajay Parida, M. S. Swaminathan Research Foundation (MSSRF), III Cross Street, Institutional Area, Taramani, Chennai - 600 113, Email: ajay@mssrf.res.in

Salinity is a significant limiting factor to agricultural productivity affecting about 9 x 10⁸ ha, worldwide. Poor quality of irrigation water and current unsustainable irrigation practices has significantly resulted in salinisation and other form of land damage. About one-third of all irrigated land is affected by salt due to secondary salinisation and it is estimated that 50% of the arable lands will be salinised by the year 2050. The problem of salinity is most acute in the coastal regions where it threatens the very sustainability of the agricultural system. Improving or maintaining yield potential of the crops under increased salinisation is of greater significance for the future With a view to identify and isolate novel genetic combinations offering resistance to coastal salinity, MSSRF has initiated work on mangrove species. Mangroves are salt tolerant plant communities occupying the coastal estuarine regions of the tropics. They serve as a vital link between terrestrial and aquatic ecosystems and provide livelihood and ecological security for the coastal communities. MSSRF was the first to propose the concept that mangroves can be invaluable donors of breeding crop genotypes adapted to coastal salinity through recombinant DNA technology. Identification and isolation of novel genetic combinations with implications to abiotic stress were undertaken from the widely distributed mangrove species, Avicennia marina and the wild rice Porteresia coarctata. Enriched gene libraries constructed from these two species are used for identification and isolation of stress tolerant genes. Many novel genetic combinations have been identified, sequenced and characterised from these libraries. Efforts for identification of unique genes in mangroves have also been undertaken using large-scale genome sequencing and differential expression analysis. Some of the isolated and characterized genes from mangroves were characterised and analyzed for their expression levels in varying saline conditions. Methodologies for construction of vectors for transformation and transformation systems have been established in Tobacco, Rice, Blackgram and Mustard. An integrated approach to gene isolation to development of transgenics in locally adapted cultivars and integration of pre-breeding with participatory breeding will help to strengthen the stability and sustainability of the fragile coastal ecosystem on the one hand, and the productivity and profitability of the coastal farming systems, on the other.

References:

1. Prashanth SR, Sadhasivam V, Parida A. Over expression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia marina in indica Rice var Pusa Basmati-1 confers abiotic stress tolerance. Transgenic Res. 2007 Jun 1; [Epub ahead of print]
2. Jithesh MN, Prashanth SR, Sivaprakash KR, Parida A. Monitoring expression profiles of antioxidant genes to salinity, iron, oxidative, light and hyperosmotic stresses in the highly salt tolerant grey mangrove, Avicennia marina (Forsk.) Vierh by mRNA analysis. Plant Cell Rep. 25(8), 865-876, 2006.
3. Mehta PA, Sivaprakash K, Parani M, Venkataraman G, Parida AK. Generation and analysis of expressed sequence tags from the salt-tolerant mangrove species Avicennia marina (Forsk) Vierh. Theor Appl Genet. 110(3), 416-424, 2005.
4. Parida AK, Das AB. Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf. 60(3), 324-349, 2005.

Invited talk:

Auxin-induced genes and the F-box protein encoding genes in rice and their probable role in regulating plant development

Jitendra P. Khurana, Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi - 110021, Email: khuranaj@genomeindia.org

The hormone auxin exerts pleiotropic effects on growth and development in plants throughout their life cycle. The molecular mechanism of auxin action (though not completely understood) has been largely unraveled through molecular genetic analysis of Arabidopsis mutants. Advances in genomics afford new opportunities for investigating these pathways in crop plants. We have identified and analyzed early auxin-responsive gene families in rice (Oryza sativa), including GH3 (12 members), Aux/IAA (31 members) and SAUR (58 members), by a reiterative database search and manual curation, and analyzed them phylogenetically. One of the Aux/IAA family members, designated earlier as OsiIAA1, has been characterized in some detail by our group. The increased accumulation of OsiIAA1 transcripts in auxin-treated rice coleoptiles even in the presence of, cycloheximide, suggested that OsiIAA1 is a primary auxin response gene. The antibodies raised against His₆-OsiIAA1 recombinant protein could detect the OsiIAA1 protein in the plant extract only in the presence of a proteasome inhibitor, MG132, indicating that OsiIAA1 is rapidly degraded by proteasome complex. Also, the proteasome inhibitor MG132 stabilized the purified His₆-OsiIAA1 protein to some extent in the cell free extracts of rice coleoptiles. Among the four conserved domains present in the Aux/IAA proteins, domain II has been implicated in protein turnover. One of the Aux/IAA genes encoding a protein harboring incomplete domain II turned out to be highly stable and the transgenic lines were insensitive to auxin applied exogenously. Recently, TIR1, an F-box protein, has been identified as one of the auxin receptors in Arabidopsis. The F-box proteins in general perform diverse functions in regulating cellular metabolism and various aspects of plant development. We have identified 687 potential F-box proteins in the rice genome and classified them in ten subfamilies based on their domain organization. The whole-genome microarray analysis revealed that the expression of several F-box protein encoding genes is developmental stage specific and is regulated by environmental cues, such as light and abiotic stress. The results presented here provide comprehensive view of the auxin-regulated genes and F-box protein encoding genes in rice, which will be fruitful in further elucidation of their precise role in regulating growth and development in plants.

References:

1. Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S, Tyagi AK, Khurana JP.Plant Physiol. 143(4), 1467-1483, 2007. F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress.
2. Jain M, Kaur N, Garg R, Thakur JK, Tyagi AK, Khurana JP. Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct Integr Genomics. 6(1), 47-59, 2006.
3. Jain M, Kaur N, Tyagi AK, Khurana JP. The auxin-responsive GH3 gene family in rice (Oryza sativa). Funct Integr Genomics. 6(1), 36-46, 2006.
4. Thakur JK, Tyagi AK, Khurana JP. OsIAA1, an Aux/IAA cDNA from rice, and changes in its expression as influenced by auxin and light. DNA Res. 8(5), 193-203, 2001.

Invited talk:

Signal transduction in guard cells: components during stomatal closure in abaxial epidermis by abscisic acid or methyl jasmonate or bicarbonate

Agepati S. Raghavendra, Department of Plant Sciences, School of Life Sciences,

University of Hyderabad, Hyderabad - 500 046, Email: as_raghavendra@yahoo.com

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis thaliana wild type, and its selected mutants. Some of the experiments were performed on epidermal strips of Pisum sativum and Nicotiana glauca. Both MJ and ABA promoted H₂O₂ production in wild-type guard cells, while diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, prevented ABA- and MJ-induced stomatal closure. MJ-induced stomatal closure was suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Further, MJ induced alkalization in guard cell cytosol and MJ-induced stomatal closure were inhibited by butyrate. The kinetics of cytosolic pH changes and reactive oxygen species (ROS) production revealed that the alkalization of cytoplasm preceded ROS production during the stomatal response to both ABA and MJ.

The MJ induced increase in NO of guard cells and stomatal closure in Nicotiana glauca were prevented to a large extent by cPTIO, a NO scavenger, and L-NAME, an inhibitor of NO synthase (NOS), suggesting that guard cells generate NO in response to MJ and that a NOS like activity is partially involved in such NO production. Time course experiments revealed that on exposure to MJ, the rise in guard cell pH peaked at 15 min, while NO production peaked at 18 min and the ROS production much later at about 30 min. Butyrate, a weak acid that reduces the cytosolic pH, restricted the NO increase and stomatal closure induced by MJ. In contrast, EGTA (Ca²⁺ chelator) or nicotinamide (the blocker of Ca²⁺ release) reversed the stomatal closure in presence of MJ. We suggest that during MJ-induced stomatal closure, changes in pH are upstream of NO production while calcium signaling is located downstream of NO effects.

Bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis as well as H₂O₂ production were restricted by exogenous catalase or DPI. The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase was involved during bicarbonate induced ROS production in guard cells. The bicarbonate induced increase in NO of guard cells, or stomatal closure was prevented partially by c-PTIO and L-NAME, indicating that guard cells generated NO on exposure to bicarbonate, with at least a partial involvement of NOS. Time course experiments revealed that on exposure to bicarbonate or ABA, the rise in guard cell NO production peaked within 10 min. We conclude that H₂O₂, NO and cytosolic pH are all essential secondary messengers during stomatal closure in response to not only ABA or MJ but also bicarbonate.

References:

1. Kolla, V.A., Vavasseur, A. & Raghavendra, A.S. H₂O₂ production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis. Planta 225, 1421-1429, 2007.

2. Kolla V. A. and Raghavendra A. S. Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum. Physiol. Plant. 130, 91–98, 2007.

3. Vavasseur A. and Raghavendra A.S. Tansley Review: Guard cell metabolism and CO₂ sensing. New Phytol. 165, 665-682, 2005.

4. Suhita, D. Raghavendra A.S., Kwak J.M. & Vavasseur A. Cytoplasmic alkalization precedes ROS production during MJ & ABA-induced stomatal closure. Plant Physiol. 134, 1536-1545, 2004.

PB 1

Plant regeneration and genetic transformation in petiole tissue of Populus deltoides Marsh.

Aditi Saraswat*, A. K. Thakur, A. A. Khan and D. K. Srivastava

Department of Biotechnology

Dr Y. S. Parmar University of Horticulture and Forestry

Nauni Solan - 173230

Himachal Pradesh, INDIA.

Plant regeneration and genetic transformation studies were carried out in petiole tissue of Populus deltoides. The plant material was procured from the nursery raised in the glass house of the Department. Plant regeneration studies were carried out using petiole explants. For shoot regeneration, the best medium was found to be MS + 0.25 mg/l IAA + 0.50 mg/l BAP + 15 mg/l AdS. Shoot elongation occurred on MS medium containing 1 mg/l GA₃. Root regeneration was obtained on MS medium supplemented with 0.25 mg/l IAA. The plantlets were able to regenerate within two months. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of callus/tissue (petiole) and to select transformed callus during transformation experiment. Kanamycin is a potent inhibitor of protein synthesis. Kanamycin sensitivity (10 – 50 mg/l) was checked by the fresh weight of the explants which was measured at an interval of 7 days. It was observed that the fresh weight decreased on increasing the concentration of kanamycin (0 – 50 mg/l). Maximum increase in fresh weight was observed in the normal MS (control) medium. A negative correlation was observed between different concentrations of kanamycin used versus fresh weight of explant(s)/callus at different interval of time. For genetic transformation, disarmed Agrobacterium tumefaciens LBA4404 strain containing a reporter -glucuronidase (gus) gene in binary vector pBI 121 system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for co-cultivation experiment to transfer gus and npt-II genes in poplar cells. After co-cultivation only the transformed cells were able to grow on selective shoot regeneration medium (50 mg/l kanamycin + 500 mg/l cefotaxime) whereas control explants died on the selective medium. Transformation experiment could be scored as early as 2 weeks after selection. Most of the calli obtained on the selective medium were GUS positive. Putative transgenic shoots were obtained, which were able to grow on the selective medium containing 50 mg/l kanamycin.

* Author presenting paper

PB 2

Reversal of heat induced alterations in photochemical activities in wheat primary leaves

Bindu Prasuna.A and S.D.S. Murthy

Department of Biochemistry, S.V.University, Tirupati-517502, India

Chloroplasts isolated from elevated temperature treated 8-day-old continuous white light grown wheat primary leaves lost the ability to photooxidise water. Also the ability of ascorbate to donate electrons to photosystem II declined. However, a significant increase in reduced dichlorophenolindophenol-supported photosystem-I-mediated methyl viologen photoreduction activity was observed. The plants stressed at 45⁰ C and 47⁰ C were subsequently grown at 25⁰ C and the partial photochemical activities were measured in chloroplasts isolated from the plants at 24 h intervals. The post stress alterations observed are (1) a significant restoration of water oxidation capacity in 45⁰C and partial restoration in 47⁰ C treated leaves. Ascorbate supported photochemical activities recovered more or less in similar fashion; (2) reversal of enhanced photosystem I activity in both 45⁰ C and 47⁰ C treated leaves. These results suggest that the restoration in water oxidation capacity is possible in 45⁰ C treated leaves and is limited by the severity of heat stress in 47⁰ C treated leaves. Restoration of water oxidation capacity vis-à-vis to the reversal of heat enhanced photosystem I activity also indicates the existence of possible endogenous control for repair of alterations during the post stress.

PB 3

Black tea quality in relation to catechin profile and the enzyme activity

Santanu Sabhapondit*, Nogen Gogoi, B.D. Baruah, Pradip Tamuly, and Mridul Hazarika

Department of Biochemistry, Tocklai Experimental Station, Tea Research Association

Jorhat, Assam, santanusabhapondit@yahoo.com

Catechin profile of six different cultivars along with three extreme varieties was studied during the tea-harvesting period in Assam. The six different cultivars selected were Tocklai released vegetative clones namely TV1, TV7, TV9, TV23, TV26 and the garden series clone T3/E-3 based on their varietal differences. The three extreme varieties were Assam, China and Cambod. The samples were collected fortnightly from the experimental gardens of Tocklai Experimental Station. The study also included the variation in the activity of the enzymes PPO and PO responsible for the oxidation of catechins. A significant correlation was observed in the formation of theaflavins (TFs) and thearubigins (TRs) with that of enzyme activity and catechin profile. A good correlation was observed with the HPLC profile of ethyl acetate soluble pigments (mainly TFs) and black tea quality, the formation of which is regulated by the enzymes PPO and PO. There is significant increase of Peroxidase (PO) activity at the end of the withering, which ultimately determines the pigment profile of made tea. A significant variation in the HPLC profile of ethyl acetate soluble pigments of black tea liquor among the cultivars was observed.

* Corresponding author, address as above.

PB 4

Formation of biochemical quality parameters of tea with respect to wither and fermentation temperature during processing

Deepika Baruah, Lakshi Prasad Bhuyan*1, Pradip Tamuly*2 and Mridul Hazarika*3

Department of Chemistry, Bahona College, Jorhat, Assam

*1,2,3 Tocklai Experimental Station, Tea Research Association, Jorhat, Assam bhuyanlp@yahoo.com

Tea is a popular beverage, quality of which depends upon certain processing parameters. The formation of quality parameters such as theaflavins (TF), thearubigins (TR), brightness, volatile flavoury constituents (VFC) etc. of curl, tear and crush (CTC) black tea, manufactured in environmental control manufacturing (ECM) system by changing the temperature in withering and fermentation stages was studied. Two –step withering technology i.e. chemical withering followed by physical withering (CPW) and only physical withering (PW) was undertaken in three temperatures to follow the variations of black tea quality parameters. Quality of made tea can be enhanced by restricting moisture loss in the initial hours of withering (CPW). Low withering temperature (22°C) produced tea with more TF, TRs and brightness. Generation of some of the VFC like trans-2-hexenal, benzaldehyde, terpeneol, methylsalicylate, and β-ionone was more at low temperature whereas flavoury constituents like linalool oxide-II, benzyl alcohol and α-ionone were increased at high temperature (35°C). Well marked variation of biochemical parameters were also observed at different fermentation temperatures. TF and brightness were found higher at temperature 26°C and 22°C whereas TR1 and total colour (TC) are more at temperature 30°C. Formation of geraniol, linalool, benzyl alcohol, β-ionone, hexanal and trans-2-hexenal were increased at temperature 34°C and their variations reflected in the overall quality of the finish product.

# Corresponding author

PB 5

Stress linked enzymic as well as biochemical changes related to quality during tea processing.

Binoti Devi Baruah, Santanu Sabhapondit, Dipta Jiwan Hazarika, Pradip Tamuly

and Mridul Hazarika

Department of Biochemistry, Tea Research Association, Tocklai Experimental Station.

Jorhat – 785 008, Assam. binoti_devibaruah@yahoo.com

A study on changes in the level of oxidative enzymes such as PPO and PO under applied stress during processing and their effect on made tea quality was taken up. In addition to this variation of enzyme activity (PO and PPO) due to the application of UV-light during withering is also studied. Similarly catechin profile at the different hours of withering were also analysed simultaneously under the above conditions. The cultivars selected were TV1, TV22, S3/A-3 and T3/E3. A significant variation of enzyme activity (PO and PPO) during withering was observed due to the stress applied during processing. The withering process can be hastened by application of mechanical pressure in the leaf before withering. From the results it is observed that quality of the made tea could be improved to a certain extent by exposing the green leaf to UV-light during withering which is reflected by the increase in the level of theaflavin and thearubigin content in the made tea of the experimental sample.

PB 6

Peptide design and development of polyclonal antibodies for light harvesting complex I of CHLAMYDOMONAS REINHARDTII

Y. Venkateswarlu, N. Sreedhar, S. Rajagopal

Department of Biochemstry, School of life sciences,

University of Hyderabad, Hyderabad, AP 500 046(India).

venkibiotech@gmail.com

The unicellular green alga Chlamydomonas reinhardtii has been explored as a eukaryotic model organism for decades. In C. reinhardtii the number of light harvesting complex (LHC) I polypeptides are currently uncertain. Till now, 9 LHCI’s are identified by proteomic analysis. In view of the facts that the C. reinhardtii LHCs is unique and the structural integrity of LHCI of PSI-LHCI supercomplexes are not known, it is difficult to isolate individual LHCI proteins to develop antibodies by direct method. We designed a series of peptide tags of LHCs by peptide finder, Abie pro 3.0 and ReMUS tools. Twenty five peptides were generated by these tools, where hydrophilicity varies from -1.03 to -1.69, length of peptides is from 12 to 17 amino acid residues. Among the generated peptides only few are taken for the peptide synthesis. All these peptides were screened based on their hydrophilic percentage. The peptides were chemically synthesized with C-terminal cysteine residue to facilitate cross-linking to carrier molecules. Chemically synthesized peptides were purified by HPLC and purity of the peptides was analyzed by MALDI-TOF. To raise the polyclonal antibodies, the peptides were conjugated to the carrier protein keyhole limpet hemocyanin (KLH) through the cysteine of the peptide by using standard glutaraldehyde method. Developing the polyclonal antibodies in Rabbit, against the LHCs are in progress.

PB 7

Photometabolism of sulfonated aromatic compounds by

Marichromatium indicum JA 120

¹Shobha, E., ²Sasikala, Ch. and ¹Ramana, Ch.V

¹Department of Plant Sciences, School of Life Sciences, University of Hyderabad,

P.O.Central University, Hyderabad 500 046, INDIA.

²Bacterial Discovery Laboratory, Centre for Environment, IST, JNT University

Kukatpally, Hyderabad 500 072, INDIA.

E-mail: shobhahcu@yahoo.com

Sulfonated aromatic compounds constitute a class of compounds in which sulfone functional group (SO₃H) is covalently attached to the benzene ring by a carbon sulfur bond. Sulfonated aromatic compounds are widely used in domestic, industrial and pharmaceutical processes thus a nearly ubiquitous pollution problem. A purple sulfur bacterium Marichromatium indicum JA120 is capable of utilizing sulfonated aromatic compounds viz, Benzenesulfonicacid, p-toluenesulfonicacid, 4-aminobenzenesulfonicacid, 5-sulfosalicylicacid, as a source of sulfur. Utilization of p-toluenesulfonic acid was light dependent and its catabolism proceeds through four distinct pathways viz, 1.Desulfurization, 2.Oxidation, 3.Reduction, and 4.Conjugation.

PB 8

Stages of in vitro tuber development in Dactylorhiza hatagirea (D. Don) Soó: A critically rare medicinal herb

ANJULI AGARWAL, D KHOKHAR, C PATHAK & GN GUPTA

GBPUA&T Agricultural Research Station, Majhera

Garampani (Nainital) 263 135 India

E-mail: anjulit@rediffmail.com

Key Words: Dactylorhiza hatagirea, Salampanja, Protocorm, in vitro

Dactylorhiza hatagirea (D. Don.) Soó (syn: Orchis latifolia L. family Orchidaceae.) commonly known as Salampanja is a critically rare medicinal herb. D.hatagirea is a terrestrial orchid in alpine meadows and has medicinally important paw shaped tuber. This specie has become endangered due to over-exploitation of tuber for its medicinal value. In nature, plant regeneration takes place through seeds but the seeds are minute and without endosperm, therefore, need mycorrhizal association for germination. For conservation and propagation of endangered orchids in vitro methods have proven to be useful. Protocol for in vitro seed germination and protocorm formation has already been standardized and reported earlier. Culture medium for protocorm multiplication and plantlet formation was supplemented with higher concentration of plant growth regulators as compared to protocorm development medium. Under in vitro condition protocorm gave rise to a plant which formed the tuber afterwards. Tuber developed in vitro is just like the naturally formed tuber. Present work describes the various stages of tuber development. As per the reports, most of the Dactylorhiza species are either diploid or autotetraploid, chromosome number ranging from 20 to 40. Investigation done by our group revealed that D.hatagirea specie has only 18 chromosomes. Ploidy manipulation using in vitro approaches shall be helpful to increase the size of tuber, henceforth, the available biomass. Cultures are treated with colchicine, screening for ploidy manipulation is in progress.