Department of Biological Sciences
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Item Dual Role for Zn2+ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease(Elsevier, 2007-11) Ghosh, SoumitraWe describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.Item Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme(American Society for Microbiology, 2008-09) Ghosh, SoumitraGlutamate racemase (MurI) provides d-glutamate, a key building block in the peptidoglycan of the bacterial cell wall. Besides having a crucial role in cell wall biosynthesis, MurI proteins from some bacteria have been shown to act as an inhibitor of DNA gyrase. Mycobacterium tuberculosis and Mycobacterium smegmatis MurI exhibit these dual characteristics. Here, we show that the two activities of M. tuberculosis MurI are unlinked and independent of each other. The racemization function of MurI is not essential for its gyrase-inhibitory property. MurI–DNA gyrase interaction influences gyrase activity but has no effect on the racemization activity of MurI. Overexpression of MurI in vivo provides resistance to the action of ciprofloxacin, suggesting the importance of the interaction in gyrase modulation. We propose that the moonlighting activity of MurI has evolved more recently than its racemase function, to play a transient yet important role in gyrase modulation.Item Expression and functional analysis of rice genes involved in reproductive development and stress response(World Scientific, 2007) Sharma, RitaThe rice genome sequenced and annotated by the IRGSP has identified 37,544 protein-coding genes. In an effort to identify genes encoding transcription factors and signal transduction components, more than 7,000 genes belonging to 87 classes have been used to prepare a local database. Detailed analysis of genes for plant hormone response, CDPKs, C2H2 zinc-finger, and SET domain proteins unraveled interesting evolutionary aspects in relation to genes and the rice genome. A 51k microarray, SAGE analysis, and real-time polymerase chain reaction revealed differential expression of target genes during reproductive development and stress conditions. Several genes specific to reproductive floral organs and seed development have been identified. A large number of SAGE tags are observed from intergenic regions and antisense strands reflecting the unexplored transcription potential of the rice genome. Analysis of rice gene promoter activities has been undertaken in transgenic tobacco/Arabidopsis to demarcate regions conferring anther-/pollen-specific expression. OSISAP1, a gene coding for a stress-associated zinc-finger protein, and its promoter have been functionally validated in transgenic tobacco and rice. Genes for proteins interacting with OSISAP1 have also been found to be stress-inducible. Investigations on functional analysis of stress-responsive genes are in progress.Item Plasmodium falciparum: genetic diversity of C-terminal region of MSP-1 in isolates from Indian sub-continent(Elsiever, 2005-08) Saxena, Vishal; Das, AshisMalaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that merozoite surface protein-1 (MSP-1) is an effective target eliciting a protective immune response. The MSP-142 region comprising two EGF-like domains is involved in generating protective immune response in humans and other experimental animals. Searching for point mutations in this region is essential in view of vaccine development. We have investigated the sequence variations in Plasmodium falciparum MSP-1 carboxy terminal region in field isolates from different regions in India. Our study reveals the presence of eight variant types of MSP-119 in the Indian sub-continent, which comprise of E-TSR-L, Q-TSR-L, E-TSG-L, Q-KNG-L, Q-KNG-F, E-KNG-L, E-KNG-F, and E-KYG-F. The last named allele is a novel variant being reported for the first time.Item A Handbook of Genetic Engineering(Kalyani Publishers, 2007) Saxena, VishalItem Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes(Elsiever, 2001) Bhagavatula, VaniExposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (∼65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide 14C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSIItem Characterization of high temperature induced stress impairments in thylakoids of rice seedlings(NISCAIR, 2001-08) Bhagavatula, VaniExposure o f isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40°C) for 24 hr in dark on rice seedlings to characterize the extent of damage by inl vivo heat stress on photofunctions of photosystem II (PSII). Chi a fluorescence transient analysis in the intactrice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited > 50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thy lakoid membranes from heat exposed seed lings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by ~40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40°C heat exposure for 24 hr, the PSII reaction centers were impaired by inl vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII .Item A MATHEMATICAL MODEL FOR THE EFFECT OF TOXICANT ON THE IMMUNE SYSTEM(World Scientific, 2007) Dubey, Uma S.; Dubey, BalramIn this paper, a nonlinear mathematical model is proposed and analyzed to study the effect of environmental toxicant on the immune response of the body. Criteria for local stability, instability and global stability are obtained. It is shown that the immune response of the body decreases as the concentration of environmental toxicant increases, and certain criteria are obtained under which it settles down at its equilibrium level. In the absence of toxicant, an oscillatory behavior of immune system and pathogenic growth is observed. However, in the presence of toxicant, oscillatory behavior is not observed. These studies show that the toxicant may have a grave effect on our body's defense mechanism.Item MODELING THE INTERACTION BETWEEN AVASCULAR CANCEROUS CELLS AND ACQUIRED IMMUNE RESPONSE(World Scientific, 2008) Dubey, Uma S.; Dubey, BalramThis paper deals with the interaction between dispersed cancer cells and the major populations of the immune system, namely, the T helper cells, T Cytotoxic cells, B cells, and antibodies produced. The system is described by a set of five ordinary differential equations. Both local and global stability of the system has been investigated. It has been observed that under appropriate conditions this interaction is capable of controlling the growth of these cancer cells. The analytical findings are supported by numerical and computational analytical methods.Item Activated pericyte attenuates endothelial functions: nitric oxide – cGMP rescues activated pericyte-associated endothelial dysfunctions(CSP, 2007-11-23) Majumder, SyamantakHepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte–endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO–cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependent