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International Symposium on Teaching,Research and Exploration in Biochemistry: 50 years of Journey Dated 6- 8 January 2006

Symposium Abstracts                                                                                                                                     Poster Abstracts

Investigating the association of a cryptic bacteria in

Arachis hypogea

Senjuti Sinharoy, Sandip Samaddar, Ayan Raichaudhuri, Manas Kanti Maity Susanta Roy Chowdhury and Maitrayee DasGupta. Department of Biochemistry, University of Calcutta.

A suspension cell culture was initiated from callus cells originating from Arachis hypogea cotyledon explants in 1997. These cells turned out to be auxin autotrophic in nature. Even after almost 342 passages performed to date auxin independence remain unchanged for these cells. The molecular basis of auxin autotrophy remains unknown. Through ribotyping, we have identified in these cells an unculturable bacteria belonging to the gamma proteobacteria division and Pseudomonas sub division indicating that the cultured cells harbor a cryptic bacterium. The same bacteria was identified in an Arachis plant in seed tissues and in specialized structures called nodules where nitrogen is fixed by symbiotic association with rhizobia. Both these tissues are obligatorily dependent on the rhizosphere for their maturation. Subcellular fractionation indicated these bacteria to be associated with the amyloplast fractions the significance of which is not understood. The association of the pseudomonas with Arachis may explain (i) the auxin autotrophy of the cell culture because they are known to contain auxin biosynthesis cassettes (ii) obligatory and unique relationship of this genus with the rhizosphere for its seed maturation.

Abhijit Bhattacharya, Sreya Chattopadhyay, Debaprasad Mandal, Lakshmishri Lahiry, Sankar Bhattacharya, Arindam Bhattacharyya, Arnab Goswami, Gaurisankar Sa and Tanya Das

Bose Institute, P1\12, C.I.T. Scheme VII-M Kolkata-700054

Over the years biological science has advanced with leaps and bounds but cancer still remains as a formidable foe. Modern science despite of providing potential anti-tumor drugs fails in of tumor cells without induction of concurrent toxic manifestations including immuno-suppression in the host. Prevention of cancer through dietary intervention is thus currently receiving considerable attention. In this study we have compared the efficacy of a popular dietary beverage, black tea, with a widely used cancer drug cisplatin. Interestingly, our study revealed that survival rate of Ehrlich’s Ascites carcinoma (EAC)-bearing mice upon cisplatin treatment was 66% of black tea-treated tumor-bearers and this popular beverage was more potent apoptogenic agent than cisplatin. Moreover, cisplatin further aggravated the tumor-induced immuno-suppression, hepatotoxicity and renal toxicity. interestingly, black tea ameliorated selective removal the tumor-induced immune suppression by inhibiting thymic and splenic lymphocytes apoptosis. The serum markers of hepatotoxicity and renal toxicity were brought back to the normal level in the black tea treated tumor-bearer. These results led us to compare the effect of black tea on cisplatin resistant EAC cell line, where cisplatin was unable to kill the tumor cell. Black tea showed considerable tumor cell killing. Showing its efficacy even on resistant tumor cell. To find out the mechanism of tumor killing level of tumor ROS was estimated. It was found that black tea considerably increases the tumor ROS and induces loss in MTP. All these results signify that although cisplatin has proven tumoricidal effects, it further aggravated immunosuppression, renal impairment and hepatic toxicity as already imposed by the developing tumor, thereby ultimately decreasing the survival rate of tumor bearers. Black tea, thus emerged as a better candidate since it not only successfully reduced tumor load but also ameliorated tumor induced immunosuppression as well as renal impairment and hepatic toxicity.

Biochemical & Molecular biological studies on Electron Dense Granules containing collagenolytic activity of E.histolytica

A. Mazumder, A. Debnath, S. Kumar, S. Ganguly and P. Das

Department of Microbiology, National Institute Of Cholera and Enteric Diseases, P-33 CIT Road Scheme XM, Kolkata-700 010, India

Key words : Microarray, Entamoeba histolytica, collagen

During the course of invasive intestinal amoebiasis, E. histolytica actually penetrates the mucosa and submucosa of host intestine. The actual mechanism by which parasite destroys the host tissue is still not clear. However role played by galactose binding lectins, certain adhesions, amoebapore forming proteins, toxins and proteolytic enzymes viz. cysteine proteinase, and metalloproteinase (collagenase) secreted by the parasite have been suggested to exert considerable influence in this regard.

Collagen being one of the major component of the basement membrane of human intestine and where E. histolytica first come in contact with host tissue and do vital activities, so collagenolytic activity was thought to be important factor in pathogenesis of amoebiasis. Later on in vitro studies it was demonstrated that pathogenic strains if inoculated with human collagen type I and Ca⁺², they secreted collagenolytic enzyme and probably related to lysis of host tissue. This collagenolytic activity was seen in special molecules called Electron Dense Granules (EDG). The latter was found first in plasma membrane and with the time released into the extracellular milieu. It is demonstrated that EDGs have 21 times higher collagenase activity than the whole trophozoites (0.76 ± 0.006 units of collagenase / mg of protein). Antibodies to EDGs, both polyclonal and monoclonal showed its use in differentiation of pathogenic to non-pathogenic strains of E.histolytica. Despite some information on the activity or functions of collagenase enzyme in E. histolytica is available nothing is known about the gene expression by human collagen type I and Ca⁺² interaction with E. histolytica.

To identify cellular mechanisms of trophozoite activation, the global variation in gene expression during collagen interaction with E. histolytica, a shotgun DNA micro array was constructed by use of 9600 random inserts from an E. histolytica genomic DNA library. Through differential hybridization, key differences between gene expression in collagen-activated trophozoites and that in nonactivated trophozoites were identified. Fourteen differentially regulated clones were reproducibly identified and selected for sequencing. Among the genes identified were those coding for (1) components of a signaling cascade that had been previously hypothesized to transmit responses to cell attachment, (2) adapter proteins for vesicle formation, and (3) proteins that are implicated in cytoskeletal reorganization and locomotion. Two known virulence-factor genes—those for cysteine proteinases and amoebapore—also were up regulated in response to collagen stimulation. These results provide important new clues about how a pathogen orchestrates responses to the host environment as well as a new tool for the analysis of other aspects of Entamoeba species infection and pathogenicity.

Co-aggregation of proteins regulating excretion of glycosidases

Suman Khowala, Sudeshna Chowdhury, Samudra P. Banik, Shakuntala Ghorai, Swagata Pal

Applied Biochemistry, Indian Institute of Chemical Biology, Kolkata-700032, India

 Co-aggregation of proteins is responsible for impairing their normal function and is frequently found to be associated with some diseased state in human beings. However reports of involvement of co-aggregation in modulating the secretion of proteins are not available. Sucrase is a secretory protein of the filamentous fungus, Termitomyces clypeatus, which seems to present an altogether unique perspective in these studies. Sucrase is a constitutive enzyme of the fungus, which breaks down sucrose to free glucose and fructose residues. The enzyme is very well known for its usage in commercial field. The enzyme is found to be intrinsically associated with cellobiase, another secreted enzyme of commercial interest belonging to the fungal glycohydrolase family. The studies here show this co-aggregation to be indispensable for the activity and stability of the latter, though to different extents in both intra- and extra cellular fractions. Separation of sucrase from the co-aggregate resulted in drastic fall of the activity and stability of the enzyme as confirmed by detail kinetic and biophysical studies of the glycosidases. Studies to track the association of these two enzymes en route secretion were done and sucrase was detected in vacuolar fraction of the fungus along with cellobiase. It was concluded that sucrase had a significant role in regulating protein excretion of cellobiase via co-aggregation under cellular signaling in the fungus. Results also include studies regarding the expression of sucrase (and cellobiase) in different growth media and role of glycosylation in secretion of the enzymes.

Studies on the structure-function relationship of the repressor of temperate mycobacteriophage L1

Tridib Ganguly, Partho Chattoraj, Malabika Das, Subrata Sau

Dept. of Biochemistry, Bose Institute, Kolkata.

Abstract

The wild-type repressor (CI) of mycobacteriophage L1 overexpressed in E. coli binds to its cognate operator DNA specifically. Further studies show that factors like time, temperature, salt, and pH greatly affect the binding of L1 repressor to its cognate operator. In order to achieve optimum operator binding of L1 repressor in tris buffer, the minimum requirements of time, temperature, salt, and pH have been estimated to be 1 min, 32° C, NaCl (50 mM), and 7.9, respectively. Interestingly Na⁺ but not NH₄⁺, K⁺, Li⁺, Cl^-, citrate^-, acetate^-, and H₂PO₄^- was found to augment significantly the binding activity of CI protein. An in frame deletion mutant of L1 repressor carrying no HTH motif (at its N-terminal end) was found not to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens etc. Taking together the results suggest that N-terminal end of L1 repressor carries a HTH motif. To determine the role of the C-terminal end of the L1 repressor, a variant of CI (CIts391) harboring a temperature sensitive point mutation has been cloned and characterized. Comparison of operator-binding activities between CIts391 and CI at 32° to 42° C shows that while 40 - 95 % operator-binding activity is retained at 35° to 42° C in CI, more than 75 % operator-binding activity was lost in CIts391 at 35° to 38° C. The CI repressor was also found to inhibit the growth of a clear plaque mutant of L1 phage more strongly than that of the CIts391 repressor at both 32° and 42° C. The half-life of CIts391- operator complex was found about 8 times less than that of CI - operator complex at 32° C. All these data suggest that point mutation at the C-terminal half of CI also plays a crucial role in binding the L1 repressor to the cognate operator DNA. Further analysis of the putative secondary structures of four mycobacteriophage repressors including L1 repressor reveals that two common regions encompassing more than 90% of primary sequence are present in all the repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions

Differential regulation Of Gastrin by IL1b promoter polymorphism

Meenakshi Chakravorty¹, Dipanjana Datta¹ De, Abhijit Choudhury², and Susanta Roychoudhury¹

¹Human Genetics and Genomics Division Indian Institute of Chemical Biology Kolkata 700 032, India.

²Departments of Medicine and Gastroenterology Institute of Postgraduate Medicine and Experimental Research.Kolkata-700 020, India.

  Gastric acid secretion has a major role to play in gastric disease outcome. This acid secretion depends on varied number of factors. Infection by Helicobacter pylori & host genetic factors play an important role in modulating gastric acid secretion. In our previous study (Human Mutation, in press.) we have reported association of IL1beta –511T/T and –31C/C genotype with Helicobacter pylori induced duodenal ulcer. Quantitative analysis of mRNA showed that individuals with these genotype secreted lower amount of IL1beta. IL1 beta is known to be an inhibitor of gastric acid secretion & Gastrin is a major physiological regulator of gastric acid secretion. Since our earlier findings suggest the role of the IL1B promoter polymorphisms upon its differential transcriptional regulation, we further elucidated the role of this differential expression of IL1B upon gastrin expression. Effect of IL1b on gastrin expression was established by incubation of AGS cells transfected with the gastrin promoter-luciferase plasmid with variable concentrations of IL1B. Cotransfection experiments were also carried out using the gastrin promoter luciferase construct and the IL1B cDNA plasmid in AGS cells. There was a gradual decrease in expression of luciferase on increasing amount of IL1beta. This proves that gastrin promoter is negatively regulated by the proinflammatory cytokine IL1B. To analyze the effect of the IL1B promoter polymorphism upon gastrin expression, cotransfection experiments of polymorphic IL1B constructs with gastrin promoter was also performed. It was found that differential promoter activity of IL1beta regulated the gastrin promoter differentially.

RELATION BETWEEN PROMOTER METHYLATION OF hMLH1 AND hMSH2 GENES AND MICROSATELLITE INSTABILITY IN HNSCC TUMORS

Shiladitya Sengupta¹, Susmita Chakrabarti², Chinmay K. Panda³ and Susanta Roychoudhury¹

¹Human Genetics and Genomics Division, Indian Institute of Chemical Biology, Kolkata,

India.

²Metro Hospital, Cleveland, USA.

³Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata,

India.

Microsatellite instability (MSI) has been found to be a characteristic feature in a subset of head and neck squamous cell carcinoma (HNSCC). Unlike in colorectal tumors, the mechanism of MSI in HNSCC is uncertain. So attempt has been made to understand the molecular basis of MSI in HNSCC with respect to alterations in the MLH1 and MSH2 genes. Mechanism of inactivation of these two genes was investigated by exon scanning, promoter methylation assay and expression studies. Direct sequencing of the mutational hotspot regions of MLH1 and MSH2 genes revealed no mutation in both MSI⁺ and MSI^- tumors. Promoter methylation assay showed that 21% samples were methylation positive (Me⁺) for MLH1 gene only, 13% Me⁺ for MSH2 gene only and 16% were Me⁺ for both the genes. Interestingly, 25% samples were found to be methylated at the adjacent normal tissue. Presence of expression in methylation negative (Me^-) cases and vice versa have been confirmed both at the mRNA level by semiquantitative RTPCR and also at the protein level by immunohistochemistry. 29 % samples were Me⁺ MSI⁺, 22 % were Me⁺ MSI^-, 33 % were Me^- MSI⁺ and 16% samples were Me^- MSI^- However, considering Me⁺ cases only, 14% samples were adjacent normal tissue Me^- MSI⁺ , 28% were adjacent normal tissue Me^- MSI^-, 44% were adjacent normal tissue Me⁺ MSI⁺ and 14% samples were adjacent normal tissue Me^- MSI^-. Taking into consideration the tobacco habit, 62% addicted patients were Me⁺(54% of which were Me⁺ at the adjacent normal tissue also) and 35% unaddicted patients were Me⁺. Thus it can be summarized that MSI in HNSCC occurs due to two independent pathways; one due to the defective MMR pathway by exclusively promoter methylation as far as MLH1 and MSH2 are concerned and the other due to some other mechanisms. Tobacco habit of the patients may be responsible for promoter methylation, which can initiate early before the initiation of tumorigenesis and those cases are strongly associated with MSI, which is generated during tumor progression.

Effect of fatty acids of dietary oils on astrocyte structure and function

Anindita Joardar and Sumantra Das

Neurobiology Division, Indian Institute of Chemical Biology, Kolkata 700 0064, India

We have previously reported that the omega-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), plays a unique role in facilitating some of the vital functions of astrocytes in the developing brain (J. Lipid Res. 2005 in Press). In astrocyte culture, supplementation of DHA causes distinct differentiation of the cells having morphology comparable to those grown in normal serum containing medium. DHA also selectively increased the number of beta-adrenergic receptors (-ARs) thereby facilitating downstream events of the -AR pathways, viz. the induction of protein kinase A and glycogen turnover. DHA is synthesized in the mammalian brain from the dietary intake of its precursors in the form of oils. The choice of fats and oils vary considerably amongst population in India. So also does the FA composition of each source of oils. In view of our previous observations, such varied dietary intake of oils envisages a closer look at the role of these in the development of astroglial cells. Fatty acids were isolated from commonly used oils in diet like sunflower oil, soyabean oil, mustard oil, coconut oil, safflower oil and rapeseed oil and their compositions were determined by GLC. Immunofluorescence studies of astrocyte cultures supplemented with 100 ng of FAs, isolated from each oil, demonstrated variations in morphology. -AR binding was increased in case of astrocytes supplemented with FAs isolated from mustard and soyabean oil. In Indian style of cooking, oils are exposed to high temperatures whereby the cis double bonds of PUFA are converted to trans and peroxidised forms. Hence parallel studies were conducted with FAs from oils, which were heated for 72 hours. GLC analysis showed there is a decrease in total unsaturation levels while saturated fatty acid content increases. Overall, the effect of various commonly used oils on astrocyte structure and function has been reviewed.

Thyroid hormone prevents morphine-induced apoptosis in astrocytes: role of nitric oxide

Ishani Deb and Sumantra Das

Neurobiology Division, Indian Institute of Chemical Biology, Kolkata 700 0064, India

Chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. Using primary astrocyte cultures grown in thyroid hormone (TH) deficient conditions, we on the other hand, observed that morphine significantly decreased cell viability. Further studies showed that the loss of cell viability was due to apoptosis of the cells. The effect is attenuated by TH supplementation to the culture medium. The observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, antagonized the decline in cell viability. Although apoptosis in astrocytes during exposure to chronic morphine was not accompanied by changes in the level of nitric oxide (NO) in the media, 7NI, a specific inhibitor of nNOS, completely blocked loss of cell viability suggesting that morphine induced intracellular NO production, which instead of being released by the cells are taken up by alternative signaling pathways. Further studies suggest that NO acts through a cGMP independent pathway. Unlike neurons, astrocytes show a differential response to NO induced inhibition of mitochondrial respiration by upregulating glycolysis through a AMP kinase pathway. The involvement of similar mechanisms in morphine-induced apoptosis during TH deficiency has been investigated. Collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid homone in cultured medium.

Inhibition of Phosphate Transport across Erythrocyte Membrane by the Modification of Band 3 in

Visceral Leishmaniasis.

Sudipa Saha Roy¹, Gargi Sen², Tuli Biswas¹

¹Indian Institute of Chemical Biology

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

²Indian Institute for Cultivation of Sciences

Jadavpur,Kolkata700032

Visceral leishmaniasis (VL) or kala-azar is a life threatening protozoan disease, which is prevalent in many parts of the tropical world including India. Anemia associated with oxidative damages of red cells plays a paramount role in the pathogenesis of this disease. Our results revealed membrane destabilization in erythrocyte, which contributes, in the premature hemolysis and development of anemia in V.L. The present study describes the effect of structural modification of band 3 on phosphate transport in V.L. using ³¹ P NMR spectra. The result showed progressive decrease in the rate and extent of phosphate transport with advancement of the disease, which may be related to the marked degradation of the anion channel protein band 3 during the post infection period. Infection induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.

E. histolytica cysteine proteases cause apoptosis in HeLa in a c Jun N-Terminal kinase dependent manner

Sudeep Kumar, Sadhak Sengupta, Arindam Mazumder, Sandipan Ganguly and Pradeep Das *

Present investigation represents a novel insight into the extraintestinal pathogenesis in amoebiasis. Cysteine proteases of E. histolytica are known to degrade the tissue matrix components including collagen, laminin, and fibronectin. These tissue matrix components serve not only as structural moieties it has a lot to do in maintaining the celluar survival. A number of cell lines including Hela that show anchorage dependency on their survival and proliferation, tend to die of apoptosis on denial of attachment to their substratum. In this study we have investigated the apoptosis potential of the cystein proteases isolated from the E. histolytica excretory secretory products (ESP). HeLa monolayer was treated with ESP and the cells were examined for the characteristic morphological and biochemical changes associated with apoptosis. ESP treated Hela were showing phosphatydylserine externalization, DNA fragmentation characteristic of apoptosis, caspase 3 activation and PARP dgradation. Further when cystein protease inhibitor was applied all the above observation were reversed suggesting that cystein proteases have a potential role in ESP mediated apoptosis of HeLa. Further the role of stress activated protein kinase JNK (c Jun N Terminal Kinase) has been investigated and a marked increase in c Jun phosphorylation occurred in ESP treated cells, suggesting that JNK is activated by the ESP treatment, also the cystein protease inhibitor treatment along with the ESP treatment reversed the JNK activity. Moreover JNK inhibitor rescue cells from apoptosis. All the observations clearly suggest that cystein proteses of E. histolytica, cause apoptosis of HeLa and which is dependent on JNK activity.

Interactions of prolactin and cytokines in regulation of monocyte / macrophage functions in malignancy

Sonali Paul, Tanima Roy, Diptendu Ghosh, Utpala Chattopadhyay, Rathindranath Baral, and Ratna Biswas

Department of Immunoregulation and Immunodiagnostics

Chittaranjan National Cancer Institute

37 ,S. P. Mukherjee Road ,Kolkata-700 026

Immune system, central nervous system (CNS) and endocrine system form a regulatory network and abnormality in neuroimmune regulatory network is associated with various diseases. Pituitary prolactin (PRL) and growth hormone (GH) constitute clear examples of ability of immune system to produce classic neurohormones and express their relevant receptors.

The present work aimed to understand the role of PRL, in induction of polarized type I or type II immune response in healthy individuals and in cancer patients. The cytokines released and the mechanism involved in suppression of PRL regulated immune response was studied.

PRL induced release of IL-12 from peripheral blood monocytes (PBM) of healthy women but not from Ca-Br or Ca-oral patients. The hormone induced release of IFN- by the peripheral blood lymphocytes (PBL) of healthy women but failed to stimulate the PBL of Ca-Br patient. PRL suppressed the enhanced production of IL-10 by PBM of healthy women and Ca-Br patients. This difference in PRL mediated response could be correlated with altered expression of PRL - receptor (PRL-R) protein and PRL binding in monocytes of Ca-Br patients. Using murine model system it was observed IL-10 secreted by the tumor inhibits PRL mediated immune response by altering expression and function of PRL-R on the thymocytes. Our observation indicates a role of PRL and PRL-R in regulation of Th1 response and its alteration during malignancy.

 Infection by cag negative Helicobacter pylori strain in C57/BL6 mice upregulates matrix metalloproteinase-9 expression and activity

Parag Kundu¹*, Asish K. Mukhopadhyay²*, Rajashree Patra², Aditi Banerjee¹, Douglas E. Berg³, and Snehasikta Swarnakar^1#

*Authors contributed equally

¹Indian Institute of Chemical Biology, Kolkata-700032, India; ²National Institute of Cholera and Enteric Diseases, Kolkata 700010, India; ³Department of Molecular Microbiology, Washington University Medical School, St. Louis, USA; #snehasiktas@hotmail.com

Abstract

MMPs are a family of zinc-dependent endopeptidases that selectively degrade or modify most of the extracellular matrix (ECM) components of gastric mucosa including collagen, laminin, proteoglycan, elastin, fibronectin and hyaluronic acid. Helicobacter pylori infection upregulates matrix metalloproteinases (MMPs), especially MMP-9 in cultured cells. Several of H. pylori’s virulence-associated factors including alleles in the cag pathogenicity island (PAI) augment the risk for gastric ulcer as well as gastric adenocarcinoma. Our aim was to learn if MMP-9 expression and its secretion depended on H. pylori’s cag PAI during infection in mice. A new mouse-colonizing Indian H. pylori strain (AM1) lacking the cag PAI was isolated from an ulcer patient. Different groups of C57/BL6 mice were intragastrically inoculated separately with cag positive Sydney strain (SS)1 and strain AM1. Mice were sacrificed at appropriate times post infection, gastric tissues were examined histologically and bacterial colonization was scored by quantitative culture. Denudation of mucosal layer, elongation of gastric pits and infiltration of inflammatory cells were observed in the gastric tissues infected either with AM1 or SS1. Strains AM1 and SS1 each upregulated proMMP-9 expression and activity by ~6-fold along with ~2-fold increase in MMP-2 activity in mouse gastric tissues. These increases were achieved within one day post infection and further increased by ~25% after another nine days. Both the strains were equipotent in increasing proMMP-9 expression and activity in mice, at the level of secretion as well as synthesis. Presence of cag PAI was not found requisite for induction of proMMP-9 in mice infected with H. pylori. In addition, Western blot analysis revealed that TIMP-1 and IL-1β were directly involved in the upregulation of proMMP-9 and TIMP-1 expression followed an inverse correlation with proMMP-9. Furthermore, IL-1β induced in mice by bacterial infection possibly increased proMMP-9 expression at the level of secretion and synthesis.

A novel role of melatonin and other antioxidants to block indomethacin-induced gastric ulcer through upregulation of matrix metalloprotease-2 activity and expression

Krishnendu Ganguly, Parag Kundu, Aditi Banerjee, Snehasikta Swarnakar^#.

^#Department of Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata –700032. snehasiktas@hotmail.com

Abstract

Gastric mucosal damage is directly associated with extracellular matrix (ECM) degradation where matrix metalloproteinases (MMPs) play a crucial role. Remodeling of connective tissues and loss of tissue integrity due to the action of MMPs are reported in several inflammatory diseases including gastric ulcer. MMP expression and activity are highly regulated and subjected to several levels of control, including gene transcription, post-translational modification and inhibition by their endogenous inhibitors i.e. tissue inhibitor of metalloproteinases (TIMPs). MMP-2 (72 kd gelatinase) is unique among the MMPs because its expression is constitutive and its activation is associated with the balance between membrane type1-MMP (MT1-MMP) and TIMP-2. Indomethacin-induced gastric ulceration involves the generation of reactive oxygen species (ROS) and reduction of MMP-2 activity. Our aim was to learn the mechanism for modulation of MMP activity by ROS during ulceration and possible action of antioxidants especially melatonin during healing. Melatonin (N-acetyl-5-methoxytryptamine) blocked mucosal cell disruption as judged by histology and blocked ROS production by inhibiting protein oxidation and hydroxyl radical and nitrite anion generation. In addition, melatonin and other antioxidants (e.g. curcumin and omeprazole) offered gastroprotection by upregulation of suppressed MMP-2 expression and activity at the level of secretion and synthesis in vivo. Moreover, suppression of MMP-2 activity by H₂O₂ in a dose and time dependent manner in vitro is blocked by melatonin, omeprazole and curcumin. We observed that melatonin and other antioxidants reversed the suppression of MMP-2 expression by upregulation of MT1-MMP and downregulation of TIMP-2. Hence, we hypothesize that antioxidants might have a redox-dependent regulation on MMP-2 expression and activity during gastroprotection.

 Curcumin restores tumor-induced immunodepletion through cytokine-dependent and -independent pathways involving Jak-3/Stat-5 and NFk B.

Sankar Bhattachcaryya, Debaprasad Mandal, Gaurisankar Sen, Shuvomoy Banerjee, Juni Chakraborty, Pallab Ray, Tanya Das and Gaurisankar Sa.

Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India

 Patients suffering with advanced cancer often exhibits multi faceted defects in their immune system, which are likely to contribute to an increased susceptibility to infections and disease progression. The primary aim of this study was to monitor the effect of tumor burden on the host immune system and the potential role of curcumin (a known anti cancer agent) as an immune-restorer. Here, we report that tumor-induced immunosuppression involves depletion of both thymic CD4⁺CD8⁺ double positive, CD4⁺ or CD8⁺ effecter as well as loss of circulating CD4⁺/CD8⁺ cells as indicated by our flowcytometric data. We observed that tumor burden disrupts the Bcl2/Bax ratio in the thymocytes and peripheral blood lymphocytes through the downregulation of g _c chain mediated Jak3-STAT5 signaling pathway. We also observed down regulation of CD3ζ chain expression and associated Jak 3 level in the PBMC of tumor bearers. Administration of curcumin to tumor bearing animals resulted in restoration of progenitor and effecter thymocytes as well as circulating T lymphocyte. Curcumin restores normal Bcl2/Bax ratio (as indicated by the western blot studies) in both these cells via restoration of cytokine inducible Jak-3 signaling pathway and may be via restoration of CD3ζ chain expression in the PBMC of tumor bearer. We observed another very interesting aspect of cancer-induced immunosuppression and role of curcumin as an immune-restorer, we found out that curcumin can restore the oxidative stress-mediated inhibition of NFκB activity in thymus and could also dowregulate tumor-induced increase in TNF Receptor-1 expression. This result indicates that curcumin can restore immune system while reducing tumor load and thus, raise the possibility of inclusion of curcumin in successful therapeutic regimen against cancer.

Molecular understanding of the Epigallocatechin gallate induced apoptosis of Sarcoma-180 cells in vivo

 S. Manna, S. Mukherjee, S. Banerjee, S. Das, P. Mukherjee and C. K. Panda.

Chittaranjan National Cancer Institute, 37 S.P. Mukherjee Road, Kolkata-700026.

The chemopreventive role of Epigallocatechin Gallate (EGCG), a tea polyphenol, seems to be mediated via apoptosis. To understand the molecular pathway of the EGCG mediated apoptosis Sarcoma–180 was taken as a model. The mice was treated with EGCG either 12mg or 15mg/kg body weight intra-peritonially 7 days prior to the inoculation of Sarcoma-180 in the peritoneum cavity and continued for another 7 days before its sacrifice. The induction of apoptosis was found to be 2.5 fold as observed by flowcytometric analysis. The expression of the pro-apoptotic genes p53 and bax was slightly increased at the transcript level, whereas the expression of anti-apoptotic gene bcl-2 decreased by approximately 25% to 32%. But expression of another anti-apoptotic gene bcl-xl increased about 30%. However mdm2 expression remain unchanged along with the expression of other cell cycle regulatory genes c-myc, cyclin-D1, and p21. In the Western blot analysis, P53 level increased >2 fold where as C-MYC, BCL-2 level decreased about 33% to 60%. This indicates that EGCG induced apoptosis might be due to the increased stability of the P53 proteins followed by decreased stability of the BCL-2 and C-MYC proteins. In analysis of the expression of the major UsnRNAs (U1-U6) the expression of U1 and U6 was reduced nearly 29%-46% indicating their role in apoptosis

NITROSATIVE STRESS ON YEAST: EXPLORING THE INACTIVATION PATTERN OF GLYOXALASE- I AND GLUTATHIONE REDUCTASE.

RUPAM SAHOO, TANMAY DUTTA, ARINDAM BHATTACHARJEE, RAJIB SENGUPTA, SOUGATA SINHA RAY, SANJAY GHOSH.

Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata- 700019, West Bengal, India.

e-mail: rupam_s04@yahoo.com

ABSTRACT:

Nitrosative stress is emerging as a new field for research in the stress- response research arena. Changes in expressions of some stress responsive genes, activation or inactivation of some proteins were reported during nitrosative stress. As a consequence of nitrosative stress intracellular S-nitrosoglutathione (GSNO) accumulation is common in all cell types. In spite of the presence of a protective enzyme against GSNO (NADH dependent GSNO reductase), we observed in vivo inactivation of two important enzymes- glyoxalase- I and glyceraldehyde-3-phosphate dehydrogenase under GSNO stress in two budding yeasts. We explored the nature of yeast glyoxalase- I inactivation by GSNO. In our further study, we first report the presence of GSNO reductase activity in fission yeast and its inactivation by GSNO. Using S. pombe as model system, we observed the effect of nitrosative stress on cellular redox status and inactivation of glutathione reductase (GR) and glutathione peroxidase (GPx) in presence of various reactive nitrogen species in vivo. This is the first report of in vivo inactivation of GR by peroxynitrite using S. pombe cells. Supportive in vitro results were also obtained and formation of 3-nitrotyrosine was found to be responsible for GR inactivation.

A novel cytosine-DNA methyltransferase in Vibrio cholerae and its role in global regulation of gene expression.

Sanjib Banerjee, Kalidas Paul and Rukhsana Chowdhury,

Infectious Disease Division, Indian Institute of Chemical Biology, Kolkata

Cytosine-C5-DNA methyltransferases (m5C-Mtases) are a class of enzymes that catalyze the transfer of methyl groups from S-adenosyl methionine (AdoMet) to 5’ position of cytosine bases in the DNA. In prokaryotes, m5C-MTase are components of restriction- modification (R-M) systems involved in defending bacteria against potential foreign genomic parasites like transposons and phages. In eukaryotes however, C-5 MTases have a major role in the global epigenetic regulation of gene expression.

A m5C-MTase gene, designated vchm, was identified in majority of V. cholerae strains. Vcm recognizes and methylates the first 5’-C in the sequence 5’-RCCGGY-3’. Vchm is associated with neither a restriction nor a mismatch endonuclease and hence is an ‘orphan’ MTase.The gene was cloned and a deletion mutant was constructed. RNA fingerprinting analysis revealed a global effect of vchm in regulation of gene expression in V. cholerae. A set of genes have been identified that are down regulated and a separate set of genes appear to be up regulated in the V. cholerae vchm mutant strain. Expression of some representative genes have been studied further by

qRT-PCR, the methylation status of the genes and their promoters have been analyzed Phenotypic characteristics of the V. cholerae vchm mutant regarding growth, motility and virulence has been studied.

Nitrogen-fixation by cellulose producing Gluconacetobacter sp. RG3, first member in Acetobacteraceae family.

Debasree Dutta* and Ratan Gachhui*

* Life Science & Biotechnology, Jadavpur University, Kolkata 700 032, INDIA

ratangachhui@yahoo.com

Nitrogen fixation and cellulose synthesis are both highly energy demanding processes and thus performed by very few organisms. Individual members of the bacterial family Acetobacteraceae are known to fix nitrogen viz. Gluconacetobacter diazotrophicus and synthesize bacterial cellulose like Acetobacter xylinus. A novel nitrogen fixing bacteria, Gluconacetobacter sp. strain RG3, that could also produce cellulose was isolated from Kombucha tea, a consortium of bacteria and yeast. In an optimized media for bacterial cellulose production Gluconacetobacter sp. strain RG3 could synthesize cellulose almost twice as fast as reported for Acetobacter xylinus. It could also efficiently utilize acid hydrolyzed rice straw as sole source of carbon for the production of bacterial cellulose. Bacterial cellulose credits special consideration in the current global concern for the environment, as it is the world's most abundant natural, renewable and biodegradable polymer. Also the ability of Gluconacetobacter sp. strain RG3 to fix atmospheric nitrogen will encourage its application as biofertilizer. Ribotyping of the strain RG3 indicated a close relatedness with a well known cellulose producer Gluconacetobacter hansenii. It remains to be probed from where the strain RG3 acquired the nitrogenase genes. The presence of these two properties ensures the strain to be of immense potential which remains to be exploited for commercial purposes.

Regulation of impaired PKC signaling by chemokines in murine macrophages during visceral leishmaniasis.

 Ranadhir Dey#, Arup Sarkar#, Nivedita Majumder#, Suchandra Bhattacharyya (Majumdar)#, Kaushik Roychoudhury*, Sandip Bhattacharyya++, Syamal Roy * and Subrata Majumdar#

 #Dept. of Microbiology, Bose Institute, P1/12, C.I.T. SchemeVIIM, Kolkata-700 054, India, *Division of Immunology, Indian Institute of Chemical Biology, 4, R. S. Mullick Road, Kolkata, 700 032, India and ++ School of Medicine,Washington University, St.Louis, USA.

  BACKGROUND: The protein kinase C (PKC) family regulates macrophage function involved in host defence against infection. In case of L. donovani infection impairment of PKC mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established the CC-chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 hours. OBJECTIVE: In this study, the role of MIP-1alpha and MCP-1 in the regulation of impaired PKC activity was investigated in the early hours of infection. METHODS: Protein Kinase C activity was measured by the incorporation of -³²P ATP into histone IIIS, superoxide anion generation measured by cytochrome C-fMLP method, migration of cells by Chemotaxis Assay, ROS detection by fluorescence of oxidized DCFDA, western blotting to study protein expression and PKC mRNA quantified by RT-PCR and real time PCR. RESULTS: These chemokines restored the Ca²⁺-dependent PKC activity and inhibited the Ca²⁺ independent atypical PKC activity in L. donovani infected macrophages both under in vivo and in vitro condition. Pre-treatment of macrophages with chemokines induced the superoxide anion generation by activating NADPH oxidase components in infected cells. In vitro chemokines induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricts the parasite load in both liver and spleen. CONCLUSION: Collectively, these results indicate a novel regulatory role of CC-chemokines to control the intracellular growth and multiplication of the L. donovani and suggest the antileishmanial properties of CC-chemokines in the disease process.

Studies on proteins implicated in the lysis-lysogeny decision of phage lambda

Kaustav Bandyopadhyay, Sabyasachi Halder, Pabitra Parua, Dipak Dutta and Pradeep Parrack

Department of Biochemistry, Bose Institute

P 1/12, C.I.T. Scheme VIIM, Kolkata 700054

E-mail: pradeep@bic.boseinst.ernet.in

The temperate coliphage lambda has to decide between the lytic and the lysogenic pathways of development. This decision depends upon factors such as temperature, presence of nutrients and multiplicity of infection. Crucial to this decision is the viral protein CII, an unstable transcription activator. The virus senses the growth conditions of the bacterium through a host-phage crosstalk that serves to alter CII stability. When CII is stabilized, E. coli follows the lytic pathway; destabilization of CII leads to lysogeny. Chiefly from genetic studies, a number of proteins from lambda (CIII) as well as from E. coli (HflB, HflX, HflK, HflC and HflD) have been implicated in influencing the lysis-lysogeny decision, through their effects on CII. Apart from HflB, an ATP-dependent metalloprotease that processively degrades CII, none of the other proteins have been studied at the structural level.

With a view to understanding mechanistic details of the functions of these proteins (with respect to the lysis-lysogeny decision) at the molecular level, we have purified CIII, HflX, HflK, HflC and HflD, and have studied their various biochemical and biophysical properties. Interactions of several of these proteins with CII and with HflB, as well as with each other have been examined by GST/Ni-NTA pulldown and other methods. We find that (i) CIII is a homodimer containing an S-S bridge, (ii) the S-S bridge is dispensable for CIII activity, (iii) HflK interacts with CII, (iv) HflD interacts with CII and with HflB, (v) HflX does not interact with any of the other proteins or with CII, and (vi) HflX is an ATPase as well as a GTPase, whose cellular function remains unclear.

CALCIUM SIGNALS ASSOCIATED WITH 1,25D-REGULATED EXOCYTOSIS IN OSTEOBLASTS

Payal Biswas and Laura P. Zanello, Department of Biochemistry, University of California, Riverside, CA 92521

Osteoblasts are secretory cells. They produce a variety of bone matrix proteins. These are secreted by constitutive and regulated exocytosis under the influence of hormones involved in mineral metabolism, such as 1α, 25(OH)₂ vitamin D₃ (1,25D). 1,25D increases bone matrix production by acting at the cell nucleus. In addition, 1,25D stimulates secretion of bone materials acting at a membrane associated VDR (non-genomic mechanisms).

We found a rapid (within seconds) exocytotic response triggered by nanomolar concentrations of 1,25D. Single exocytosis events were monitored in real time in live osteosarcoma ROS 17/2.8 cells with confocal microscopy. In addition, 1-100 nM 1,25D promoted a significant (2-fold) increase in capacitance, a measure of real time exocytosis, when single primary osteoblasts were measured by patch clamp. GTP (0.1 mM) promoted a Pertussis toxin insensitive capacitance increase. We detected the expression of a Ga q protein in ROS 17/2.8 cells. This suggests that exocytosis may occur through a membrane-associated VDR coupled to a G protein.

We found an elevation of cytoplasmic Ca²⁺ concentration in single live ROS 17/2.8 cells, 10 sec after the addition of 5nM 1,25D. Cells were labeled with the Ca²⁺-sensitive dye Fluo 3-AM and a series of confocal microscope Ca²⁺ images were obtained. Nifedipine (2 m M) and DIDS (200 m M) significantly reduced 1,25D-promoted capacitance changes, suggesting that this is associated with Ca²⁺ and Cl^- channel activation.

We conclude that the Ca²⁺ signal is likely to be the transducer of the hormonal stimulus into an electrical signal leading to exocytosis.

CIGARETTE SMOKE-INDUCED APOPTOSIS: PREVENTION BY ASCORBIC ACID

 Palash Maity*#, Shuvojit Banerjee¶, Dhruba J. Chattopadhyay*¶, I. B. Chatterjee* and Alok K. Sil#

 * Dr. B. C. Guha Centre For Genetic Engineering And Biotechnology,

¶ Department of Biochemistry,

# Department of Microbiology.

University of Calcutta

35 B. C. Road, Kolkata 700019.

 ABSTRACT

 Cigarette smoking has been identified to be the cause of many life threatening diseases, including chronic obstructive pulmonary disease (COPD), cancer of lung and other organs and cardiovascular disease. One of the major manifestations of COPD is emphysema. Pulmonary emphysema is defined by enlargement of air-space, inflammation, excessive proteolysis and apoptosis. In-fact, apoptosis is considered to a major event. Cigarette smoke exposure to cultured lung epithelial cells (A549) causes significant increase in apoptotic death. Apoptosis was detected by TUNEL assay and confirmed by activation of caspase3, as well as increased Bax/Bcl-2 ratio. One of the major causes of cigarette smoke-induced apoptotic death is oxidative stress. Ascorbic acid as an well known antioxidant and we found that ascorbic acid prevented cigarette smoke-induced oxidative damage and apoptosis in cultured cells (A549). We also extended our observation in a guinea pig model to establish that ascorbic acid prevents cigarette smoke-induced apoptosis of lung alveolar cells.

Mannosylated and Arabinosylated Lipoarabinomannan Differ In Regulating IL-10 Mediated Signaling In Murine Peritoneal Macrophages

^*The Department of Microbiology, Bose Institute, P1/12 C.I.T. Scheme VIIM, Kolkata-700054, India. , ^†National Centre for Cell Science (NCCS), Pune, India

^‡The Department of Chemical Technology, University of Calcutta, India

^**Neurobiology Laboratory, National Institute of Health Sciences, North Carolina, USA

 BACKGROUND: Various species of Mycobacteria produce a major cell wall associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. OBJECTIVE: In this study, we explored the differential role of the anti-inflammatory cytokine Interleukin-10 (IL-10) in Mannose-capped LAM (Man-LAM from virulent Mycobacterium tuberculosis) and Arabinose-capped LAM (Ara-LAM from avirulent Mycobacterium smegmatis) treated macrophages. METHODS: All the cell signaling parameters like PI3K, Akt, Caspases and PARP were studied by Western Blotting, Nitrite generation estimated by Greiss Reaction, cytokine level estimated by sandwich ELISA, iNOS mRNA expression studied by RT-PCR and the fate of the cells studied by Flow Cytometric analysis for AnnexinV-FITC binding. RESULTS: IL-10 blockage in Man-LAM treated wild type (WT) cells, as well as IL-10 double knockout IL-10 ^-/- cells treated with Man-LAM showed distinct down-regulation of survival factors such as Protein kinase B (Akt), and a marked up-regulation of Nitrite generation and iNOS2 mRNA expression, but enhanced expression of apoptotic factors Caspase 9, Caspase 3 and PARP fragments. Exactly the opposite effects were observed in both IL-10 blocked and IL-10^-/- cells treated with Ara-LAM, which shows that IL-10 has functions similar to that of the pro-inflammatory cytokine TNF-a . CONCLUSION: Hence, our results suggest that the cytokine, IL-10, differentially regulates the fate of Man-LAM and Ara-LAM treated macrophages.

Liposomal flavonoid inhibits arsenite induced liver fibrosis

Ardhendu K. Mandal¹, Mukul K. Basu¹, Rohini N. Chakrabarti² and Nirmalendu Das¹

¹Biomembrane Division, Indian Institute of Chemical Biology,

4, Raja S.C. Mullick Road, Kolkata-700032, India,& ²Ashok Laboratory, 390B Jodhpur Park,Kolkata-700068, India

The theme of the study was to evaluate the efficacy of galactosylated liposomal flavonoidal antioxidant [Quercetin (QC)] in combating arsenic induced hepatic fibrogenesis. The rats of the hepatic damage group were injected (s.c) a single dose of sodium arsenite (100.06 m mol / kg b.wt in 0.5 ml of physiological saline). Hepatocytes and reticuloendothelial cells (Kupffer cells, endothelial cells and stellate cells) were separated. Mitochondria and plasma membrane were isolated from all those separated cells. Oxidative damage was monitored at different isolated subcellular parts of different hepatic cells. Liver fibrosis was also induced by the injection of sodium arsenite. Galactosylated liposomal QC injection prior to arsenite treatment checked fibrogenesis completely by protecting the liver from oxidative attack in cellular and subcellular levels.

Application of galactosylated liposomal Quercetin may be a potent therapeutic approach for arsenite induced fibrogenesis through a complete protection against oxidative attack in cellular and subcellular parts of rat liver.

Mannosylated liposomal Quercetin in combating age- related ischemia-reperfusion induced oxidative damage in rat brain.

Sibani Sarkar and Nirmalendu Das Biomembrane Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road,Kolkata – 700 032, W.B., India.

Toxic reactive oxygen species like O₂^-, H₂O₂ and OH^- which are generated due to the oxidative stress alter the activities of the enzymes involved in the defence against free radicals and substantially influence the aging process and age-dependant neuropathology. Unilamellar liposomes were used to deliver flavonoidal antioxidant Quercetin (QC) to brain. Antioxidant potential of QC loaded in mannosylated (QC 6.85 µmol/kg b.wt.) liposomes (50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Wister young (2 months old, b.wt. 160-180gm) and aged (20 months old, b.wt. 415-440 gm) rats. Animals were made ischemic for 30 min by bilateral clamping of the common carotid artery followed by a 30 min cerebral reperfusion by withdrawing the clamping. Diene level and (GSSG/GSH) ratio were found to be higher in normal aged, compared to normal young rat brain. Endogeneous antioxidant enzymes like superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione S transferase activities were lower in normal aged rat brain. Further reduction of these antioxidant enzymes was observed in aged rat brain by the induction of cerebral ischemia and reperfusion.. Mannosylated liposomally encapsulated QC treatment resulted in a most significant preservation of the activities of antioxidant enzymes and a marked inhibition of cellular edema formation in neuronal cells of young and old rats.

Extracellular Reduction of α-lipoic acid by Entamoeba histolytica trophozoites: existence of a transplasma membrane redox system

Nilay Nandi^b_, Tanmoy Bera^a, D. Sudhahar^a, Md. Ali Akbar^b, Abhik Sen^b, Sudeep Kumar^b and Pradeep Das^b*

Abstract

Oxidative stress and red blood cell membrane disorganization in diabetes mellitus and diabetes mellitus associated cardiovascular disease: A comparative study.

Sangeeta Adak and Dr. Maitree Bhattacharyya

Department of Biochemistry, University of Calcutta.

35, Ballygunj Circular Road Kolkata-700019, India.

ABSTRACT:

Macrovascular disease, especially cardiovascular disease accounts for most of the mortality with Type 2 Diabetes mellitus. This work attempts to explore the correlation between these two diseases regarding oxidative stress, thermal stability and oxygen releasing capacity of hemoglobin and finally stress induced erythrocyte membrane disorganization. Enhanced oxidative stress was revealed by elevated protein carbonyl content observed both in plasma and hemolysate of the blood samples in type 2 diabetes and diabetes associated cardiovascular diseased subjects. Altered levels of cytoprotective enzymes are evidenced by decreased catalase and glutathione peroxidase activity, increased glutathione reductase activity and unaltered superoxide dismutase activity and a decrease in the non-enzymatic antioxidant such as reduced glutathione content. Peroxidative activity of pathologic hemoglobin was noted to be much higher compared to healthy control indicating possible conformational modification of this tetrameric protein as a result of disease induced oxidative stress. This result is in good agreement with the observation of reduced thermal stability of hemoglobin in diseased subjects. Enhanced oxygen releasing capacity of tetrameric oxyhemoglobin has been monitored in pathologic red blood cells, maximum increment being noticed in diabetic cardiovascular diseased subjects. Significantly lower fluidity of erythrocyte membrane was observed in diabetes and diabetic cardiovascular patients compared to control. The experimental results elucidate that hyperglycemia induced oxidative stress is responsible to affect the conformation of hemoglobin molecule as well as the dynamic property of erythrocyte membrane. Oxidative stress and consequent structural modifications are more pronounced in case of diabetes associated cardiovascular disease.

Conservation and relative importance of residues across protein-protein interfaces

Mainak Guharoy and Pinak Chakrabarti*

Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta

700 054, India

* Corresponding author

E-mail: pinak@boseinst.ernet.in

A core region surrounded by a rim characterizes biological interfaces [1]. We ascertain the importance of the core by showing the sequence entropies of the residues comprising the core to be smaller than those in the rim. Such a distinction is not seen in the two-fold related, non-physiological interfaces formed in crystal lattices of monomeric proteins, thereby providing a procedure for characterizing the oligomeric state from crystal structures of protein molecules [2]. This method is better than those that rely on the comparison of the sequence entropies in the interface and the rest of the protein surface, especially in cases where the surface harbors additional binding sites [3]. To a good approximation there is a correlation between the accessible surface area lost due to complexation and G values obtained through alanine scanning mutagenesis [4] – 26-38 cal per Ų of the surface buried – for residues located in the core, a relationship that is not discernable for rim residues. If, however, a residue participates in hydrogen bonding across the interface, the extent of stabilization is 52 cal/mol per Ų of the nonpolar surface area buried by the residue. As opposed to an amino acid classification used earlier [3], an environment-based grouping of residues yields a better discrimination in the sequence entropy between the core and the rim.

References:

[1] Chakrabarti, P. & Janin, J. (2002). Dissecting protein-protein recognition sites.

Proteins, 47, 334-343.

[2] Guharoy, M. & Chakrabarti, P. (2005). Conservation and relative importance of residues across protein-protein interfaces. Proc. Natl. Acad. Sci. USA 102, 15447-15452.

[3] Elcock, A. H. & McCammon, J.A. (2001). Proc. Natl. Acad. Sci. USA 98, 2990-2994.

[4] Thorn, K. S. & Bogan, A.A. (2001). Bioinformatics 17, 284-285.

Apoptogenic effects of theaflavins on human breast cancer cells

Lakshmishri Lahiry*, Debaprasad Mandal, Arindam Bhattacharyya, Gaurisankar Sa and Tanya Das

Bose Institute, P1/12 CIT Scheme VIIM, Kolkata- 700 054, India.

Breast cancer is a leading cause of cancer related deaths in female all over world, which initially respond to the popular chemotherapy regimen, but long-term treatments are ineffective due to development of resistance. In an effort to avoid concurrent toxic manifestations during the regimen of cancer therapy, scientists have aimed to identify chemopreventive properties of naturally occurring non-toxic dietary phytochemicals and black tea is one such emerging candidate. Recent reports indicate that the principal mediators of chemopreventive effect of black tea are the polyphenolic components, theaflavins, although the mechanism of their efficacy remains largely unresolved. Here, we examined the molecular effects of these compounds on human breast cancer cells (MCF-7) and the underlying mechanisms. Our results showed that theaflavins induced MCF-7 cell apoptosis in a dose dependent manner with IC50 of 25 μg/ml. Probing further into the molecular signals, we observed up-regulation of pro-apoptotic proteins, p53 and Bax, and mitochondrial translocation of Bax resulting in mitochondrial transmembrane potential (MTP) loss, reactive oxygen species (ROS) generation and mitochondrial cytochrome c release in these cells, although MTP loss and apoptosis were independent of ROS. In caspase-3 deficient MCF-7 cells, theaflavins activated caspases-6, -7 and -9 in MTP-dependent manner, but not caspase-8. Importantly, this anti-tumor dose of theaflavins did not have any adverse effect on normal human mammary epithelial cells and peripheral blood mononuclear cells. Together, our findings suggest that theaflavins induce perturbations in mitochondria to bring about apoptosis. To conclude, because of rapid and potent apoptogenic response triggered by theaflavins, it can be envisaged that theaflavins could be of help in purging approaches to breast cancer treatment.

CYP1B1 gene is directly implicated in juvenile onset primary open angle glaucoma

Mahua Maulik

1
, Moulinath Acharya
1
, Suddhasil Mookherjee
1
, Ashima Bhattacharjee
1
, Arun K Bandyopadhyay
2
, Sanjay K.D. Thakur
2
, 
and Kunal Ray
1 

¹Human Genetics & Genomics Division, Indian Institute of Chemical Biology, Kolkata; ²Regional Institute of Ophthalmology, Medical College, Kolkata

PURPOSE: CYP1B1 has been implicated in primary congenital glaucoma. Recent studies suggest role of CYP1B1 in primary open-angle glaucoma (POAG) as a modifier locus. The purpose of the study was to further investigate the potential role of CYP1B1 using Indian POAG patients.

METHODS: Two hundred unrelated POAG patients and 100 unrelated controls were enrolled in this study. The coding sequence of CYP1B1 was amplified by PCR from genomic DNA followed direct DNA sequencing to identify the allelic variants.

RESULTS: Six mutations were identified in 9 patients and none of the controls examined. One novel mutation (R523T) was detected in homozygous condition while three reported (W57C, E229K, R368H) and two novel mutations (S515L and D530G) were found in heterozygous state. The homozygous mutation of a conserved residue, detected in a familial JOAG patient (lacking MYOC or OPTN mutations), cosegregated with the disease locus in autosomal recessive mode of transmission. All the novel mutations (R523T, S515L and D530G) were detected in a region of CYP1B1 that did not harbor any of the 34 point mutations implicated in PCG. In addition, six reported (p.R48G, p.A119S, p.V432L, p.D449D and p.N453S & 372-12C>T in intron 1) and four novel (p.V395V, p.P400P, p.V518A & c.2016C>G in 3’-UTR) SNPs were also observed in POAG patients and controls.

CONCLUSIONS: Our observation provides compelling evidence for primary role of CYP1B1 in juvenile onset POAG and emphasizes the importance of screening for mutation in the gene of JOAG patients that are determined not to harbor mutation in already characterized candidate genes and loci for POAG.

The study has been supported by CSIR grants (NMITLI & CMM 0016).

Molecular Pathogenesis of Parkinson’s disease: Identification of Mutations in Parkin Gene in Indian Patients

Arindam Biswas¹, Arnab Gupta², Tufan Naiya¹, Gautami Das¹, Shyamal K. Das ³

Kunal Ray² and Jharna Ray¹

¹S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India; ²Human Genetics & Genomics Division, Indian Institute of Chemical Biology, Kolkata, India, ³Bangur Institute of Neurology, Kolkata, India

Parkinson’s disease (PD) is one of the most common neurodegenerative disorder, affects at least 1% of the population over the age of 50. PD is characterized by some clinical features, which include unilateral onset of rest tremor, rigidity, bradykinesia, postural instability and mask faces. However, very little information is available regarding the molecular basis of PD among Indians. Since the largest number of mutations has been detected in the Parkin gene among all known PD loci, we aim to use Parkin as the candidate gene to assess its role in PD related pathogenesis in Indian patients. A total of 138 PD patients, with the mean age of onset being 47 ± 14 (age range, 5-77 yrs), and 50 controls were recruited for the study from eastern India. Parkin mutations were detected by amplification of exons of the gene along with the flanking splice junctions by polymerase chain reaction, single stranded conformation polymorphism and DNA sequencing. A total of 18 nucleotide variants including 9 novel changes were detected. These include five missense mutations (Gln34Arg, Arg42Cys, Arg42His, Tyr143Cys andArg334Cys) detected in 8 patients in heterozygous condition and a homozygous deletion encompassing exons 3 and 4 in two sibs affected with PD. Clinical features of the Parkin mutants were compared. Among eastern Indian PD patients, mutation in Parkin was identified in 7.24% cases.

This study is supported by a grant from CSIR, Govt. of India (to JR) and a CSIR project CMM-0116 (to KR).

A NOVEL STRESS- RESPONSIVE ORF IN Schizosaccharomyces pombe

Geetanjali Sundaram, Santanu Pal Chaudhuri, K.Sheelarani and Dhrubajyoti Chattopadhyay

Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, Department of Biochemistry, University of Calcutta, 35 Ballygunje Circular Road, Kolkata-700019,INDIA

  Our lab has been investigating the cellular responses towards Cigarette smoke using S.pombe as a model system. Differential display studies at the transcript level identified a 321 bp ORF to be highly upregulated upon exposure to Cigarette smoke. Subsequent cloning and sequencing of this ORF revealed it to be a sequence orphan coding for a 11.4kDa protein. We have confirmed the translation of this differentially expressed transcript by polysome profiling. Studies on the expression profile of this gene show that it’s expression is upregulated under most stress conditions (Oxidative stress, Nucleotide depletion, heat chock etc), osmotic shock being the only exception. Computational sequence analysis further revealed interesting features about this ORF, including a major potential for phosphorylation and glycosylation. We have also investigated the possibility of any growth advantage provided to the cells during stress conditions upon overexpression of this protein. An N-terminal EGFP tagged version of the protein when overexpressed in S.pombe localises in the cytoplasm. FACS analysis of cells overexpressing this protein indicates its probable involvement in cell-cycle checkpoint regulation too.

Novel proteins: Regulators of sperm flagellar motility dynamics

Mahitosh Mandal, Bijay S. Jaiswal, Kaushik Das, Sudipta Saha,

Sandhya R. Dungdung & Gopal C. Majumder

Indian Institute of Chemical Biology,

4, Raja S. C. Mullick Road,

Jadavpur, Kolkata - 700 032, India

E-mail: majumdergc@yahoo.com

Abstract:

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility, which is essential for fertility potential. This study reports for the first time purification to apparent homogeneity of two novel motility-promoting proteins: one from buffalo blood serum designated as FMSF (Forward motility stimulating factor) and another from goat epididymal plasma termed as MIP (motility initiating protein). FMSF is a 66kDa monomeric protein rich in aspartate, glutamate and leucine with isoelectric point of 3.7 where as MIP is a 125kDa dimeric protein made up of two subunits: 70 and 54kDa with an isoelectric point of 4.75. Both FMSF and MIP are heat-stable and showed high protein specificity and affinity for activating forward motility and of goat cauda epididymal spermatozoa. At saturating level (approx. 0.5 M level) the factors induced forward motility to nearly 60-70% of spermatozoa. They are Mg²⁺-dependent glycoproteins and the sugar parts of the proteins are essential for their biological activity. Both the proteins are acidic in nature and strongly immunogenic and their antibodies serve as potent inhibitors of sperm motility. Liver is its richest source of FMSF although it is also present in testis and epididymis whereas epididymal plasma is the richest source of MIP. A special property of MIP is that it is also capable of initiating forward motility in the immature caput-epididymal spermatozoa in vitro. Caprine epididymal plasma also possesses a motility-inhibiting protein that has been purified and characterized. The motility inhibitor is a 170kDa protein, which is a dimer of two subunits: 80kDa. The motility inhibitor is heat-labile and it is maximally active at pH 6.9-7.0. The inhibitor is strongly immunogenic and it is also localized on the sperm surface. As regulators of sperm motility these proteins have tremendous applied potential for contraception as well as for solving some of the problems of human infertility and animal breeding.

Dr.Gopal C. Majumder, PhD, FNASc, FAScT, CC
Emeritus Scientist; Ex-Scientist-G (Director Grade)
Off:Indian Institute of Chemical Biology, Calcutta 700032, INDIA.

Tel: 91-33-2473-3491/0492/6793. Fax: 91-33-473-0284

Res: Vasundhara Apartment, Flat-B 3
201C,Raipur Road, P.O.-Naktala,
Kolkata-700 047.West Bengal. INDIA.
Tel. 91-33-2421-7363 ,Mobile- 93397-84545 ( New )
E-mail:majumdergc@yahoo.com

Amelioration of tumor-induced thymic atrophy by black tea: Role of cytokine signaling

Debaprasad Mandal*, Arindam Bhattacharyya, Lakshmisri Lahiry, Gaurisankar Sa and Tanya Das

Bose Institute, P1/12 CIT Scheme VIIM, Kolkata- 700 054, India.

* debaprasad@boseinst.ernet.in.

Recent evidences show tumor to suppress host’s immune system by two principle approaches - immunocyte-depletion and/or disruption of functional attributes. Furthermore during the regimen of cancer chemotherapy, various popular drugs in use, exert concurrent toxic manifestations including immunosuppression thereby making cancer therapy extremely difficult. Scientists are, therefore, in search of any biological response modifier that will not only kill cancer cells but also protect the immune system of the host from tumor-insult. In this regard, tea has gained immense importance as a probable potential anti-tumor agent. Our flowcytometric studies revealed that with development of Ehrlich’s ascites carcinoma (EAC) in Swiss albino mice, there was a significant decrease in total CD4⁺ and CD8⁺ in the peripheral blood with increased apoptosis amongst lymphocyte subpopulation that reached its peak by the third week. Although thymus, which is the principal site for T-cell maturation, initially tried to produce more of these isotypes, progressing insult of the developing tumor resulted in severe thymic atrophy by the third week. Search for the underlying mechanisms revealed that developing EAC inhibited IL-7 signaling in thymocytes, which is essential for T cell development and survival, by down regulating IL-7R and its JAK-STAT pathway. Black tea (2.5%) prevented such thymic involution and preserved CD4⁺ and CD8⁺ in the peripheral blood by protecting this signaling cascade from tumor insult. Moreover, this beverage prevented the Th1 to Th2 shift, which was otherwise noted in untreated tumor-bearing animals. This improved the immunological environment thus augmented more IFNγ secreting CD8⁺ cells to fight against the developing tumor. Black tea therefore successfully protected the immune system of the tumor-bearer both in terms of number and function.

Abstract

Identification of candidate tumor suppressor genes loci in chromosome 4 associated with the development of uterine cervical carcinoma of Indian patients.

R. K. Singh, ^a S. Mitra ^a, D. Indra ^a, R. K. Mondal, ^a P. S. Basu,^a A. Roy^b, S. Roychowdhury,^c C. K. Panda ^a

^a Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, India

^b Calcutta Medical College and Hospital, 88 College Street, Kolkata 700073, India

^c Indian Institute of Chemical Biology, Kolkata 700032, India

Uterine cervical carcinoma (CA-CX) is one of the most common cancers among women worldwide. The estimated new cases of CA-CX per year are 500,000 of which 79% occurs in the developing countries. In eastern India (Kolkata) CA-CX constitutes about 18% of the total cancers occupying second highest position among females.

Cytogenetic studies have identified non-random chromosomal changes involving deletion, amplification, translocation etc. in several chromosomal regions. The deletions in chromosomal regions 2q, 3p, 4p/q, 5q, 6q, 11q, 13q, 18q etc. were seen at different stages of CA-CX. In a micro-cell hybrid system, normal chromosome 4 could suppress the tumorigenecity of CA-CX cell lines indicating the location of at least one tumor suppressor gene (TSG) in this chromosome. Thus to identify and characterize the candidate TSGs in chr.4 associated with the development of CA-CX, deletion mapping was done in chr.4 using 22 highly polymorphic microsatellite markers in 78 primary CA-CX samples. High frequency of deletion was detected in the 5 chromosomal regions i.e. D1 (4p16.3; 40%), D2 (4p15.2; 32 - 48%), D3 (4q22.3; 35%), D4 (4q34.2-34.3; 37-56%) and D5 (4q34.3-35.1; 39-50%). Among these 5 highly deleted regions, deletions in the D1 and D4 seems to be associated with the development of premalignant uterine cervical lesions due to their high frequency of deletions 32-34% in cervical intraepithelial neoplasia (CIN) samples. A candidate TSG SLIT2 has been located in the D2 region. Interestingly high frequency (80%: 20/25) of methylation has been seen in the promoter region of the SLIT2 gene in the CA-CX samples, indicating it as a probable candidate TSG.

Abstract

Molecular understanding of the Epigallocatechin gallate induced apoptosis of Sarcoma-180 cells in vivo

S. Manna, S. Mukherjee, S. Banerjee, S. Das, P. Mukherjee and C. K. Panda.

Chittaranjan National Cancer Institute, 37 S.P. Mukherjee Road, Kolkata-700026.

The chemopreventive role of Epigallocatechin Gallate (EGCG), a tea polyphenol, seems to be mediated via apoptosis. To understand the molecular pathway of the EGCG mediated apoptosis Sarcoma–180 was taken as a model. The mice was treated with EGCG either 12mg or 15mg/kg body weight intra-peritonially 7 days prior to the inoculation of Sarcoma-180 in the peritoneum cavity and continued for another 7 days before its sacrifice. The induction of apoptosis was found to be 2.5 fold as observed by flowcytometric analysis. The expression of the pro-apoptotic genes p53 and bax was slightly increased at the transcript level, whereas the expression of anti-apoptotic gene bcl-2 decreased by approximately 25% to 32%. But expression of another anti-apoptotic gene bcl-xl increased about 30%. However mdm2 expression remain unchanged along with the expression of other cell cycle regulatory genes c-myc, cyclin-D1, and p21. In the Western blot analysis, P53 level increased >2 fold where as C-MYC, BCL-2 level decreased about 33% to 60%. This indicates that EGCG induced apoptosis might be due to the increased stability of the P53 proteins followed by decreased stability of the BCL-2 and C-MYC proteins. In analysis of the expression of the major UsnRNAs (U1-U6) the expression of U1 and U6 was reduced nearly 29%-46% indicating their role in apoptosis.

Environmentally sustainable Development – Biochemistry of Biomethanogenesis

Archana Sharma& Jurimoni Shyam, Bitechnology Division, R.R.L.,Jorhat, Assam

Two global issues of concern today are production of energy and abetment of pollution. Production of energy in the form of biogas by biomethanogenesis of agricultural and various organic biodegradable wastes is considered promising as it provides energy, biofertilizers from renewable sources, helps in the conservation of fossil fuels and abates environmental pollution. Production of biogas from cow-dung is quite well established in India with 3.71 million family size and 3902 number community size biogas digesters against a potential of 12 million digesters based mostly on cattle manure as feed. As cattle manure is a limiting feed even in a country like India, researchers have considered other renewables in the form of abundantly available plant biomasses and agro – industrial wastes as feed for biomethanogenesis . Early attempts at making a slurry of these biomasses and agro- industrial wastes after powdering them and using it in the conventional biogas plants failed miserably. New digester design, new technology   are needed for this purpose. A novel digester and a technology was developed at R.R.L. Jorhat for production of biogas from a variety of plant biomasses and agro- industrial wastes without any extensive and costly pretreatment of the feed.

In the last 20 years considerable improvement have been made in biogas technology due to our understanding of the biochemistry and microbiology of the process. This has allowed the delineation the most sensitive steps in the process and development of strategies to enhance the operational stability of methane reactor. The results have been the development of novel reactors where the slow – growing organisms are retained in the reactors even at high volumetric loading. With these advances it is now believed that almost any type of wastes can be treated by biomethanation technology.           

Prevention of aggregation of tubulin by Deuterium Oxide.

AmlanDas, Pinaki Paul, Dr.Gopal Chakrabarti.

Dr.B.C.Guha Center for Genetic Engineering And Biotechnology,

University of Calcutta.

Deuterium Oxide has a stabilizing effect on the assembly of tubulin into microtubules in vitro. In aqueous buffer tubulin tends to aggregate ,accompanied with the loss in polymerization property .Aggregation of tubulin in H2O based buffers has been observed at both 4⁰C as well as 37⁰C.Stabilizing effects of D2O on tubulin are observed at both the temperatures .These results have been confirmed by several biophysical techniques such as fluorescence spectroscopy ,UV-Visible Spectophotometry and Circular Diachromism.Unfolding of tubulin in H2O and D2O buffers has been observed by DTNB kinetics which confirms that rate of tubulin unfolding is higher in H20 ,as compared to D20.The mechanism responsible for the ability of D2O to stabilize tubulin may involve the enhancement of hydrophobic interactions in the microtubule lattice, and substitution of deuterium bonds for hydrogen bonds.

DOES DIETARY PROTEIN-CARBOHYDRATE VARIATION AFFECT IMMUNE RESPONSE DURING AGEING ?

S. Pal, M. K. Poddar, M. Sen, A. Dalal and A. Mandal.

Department of Biochemistry, University of Calcutta, 35 B. C. Road, Kolkata, India

 Among the most important factors that are associated to regulate the ageing process and its associated changes in immune function, the well-characterized factor that can regulate ageing process involves the manipulation of dietary variables. In the present investigation, the effect of variation of protein and carbohydrate content (without caloric restriction) in the diet, on the alteration of immune response of male albino rats at the level of lymphocyte viability, proliferating activity, cytotoxicity, DNA fragmentation of blood, spleen & thymus and corticosterone status of plasma and adrenal gland were studied in relation to dietary supplementation and ageing. Rats maintained with normal protein (20%)-normal carbohydrate (68%) diet showed an age-induced (due to increase of age from 03 to 18 months) decrease in immune response. Supplementation of low protein (8%)-high carbohydrate (80%) diet (LPHC) for short-term period of 15 consecutive days (STP) decreased immuneresponse in young rats with a little immunopotentiation in aged rats. These effects were more pronounced following the supplementation of same diet (LPHC) for long-term period. High protein (50%)-low carbohydrate (38%) diet (HPLC) supplementation for STP showed a little and significant immunopotentiation in young rats with a decrease in immune response of aged rats. Similar, but more amplified effects were observed following supplementation of HPLC under LTP. These results, therefore suggest that the immune response status of the mammals depends on the (a) variation of the amount of protein and carbohydrate in the diet, (b) duration of its supplementation and (c) age of the mammals.

(Supported by Indian Council of Medical Research, New Delhi, India and University of Calcutta, India)