NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 1

DICLOFENAC AND CURCUMIN DOWN REGULATE THE TELOMERASE AND CYCLOOXYGENASE ACTIVITY IN CHEMOPREVENTION OF COLON CANCER. IL- 4 Chandan Rana and S.N.Sanyal Department of Biophysics, Panjab University, Chandigarh Email id: [email protected]

The universal presence of telomerase in tumor suggests that telomerase activity is required for cell immortality in vitro and in vivo, and targeting telomerase may represent a prevalent marker to specifically block tumor cell growth with minor effects on normal cells. The present study demonstrates the potential chemopreventive and anti-inflammatory effects of Diclofenac and Curcumin in combination with DMH induced early neoplasm of colon via telomerase inhibition along with the pro-inflammatory cytokines and induction of apoptosis. Diclofenac and Curcumin lowered the COX-2 activity and NF-κB level while the expression of pro-apoptotic proteins Bax and Bad was found higher and a lowered Bcl-2 activity stating that these agents promote apoptosis. Diclofenac and Curcumin were able to increase the Reactive oxygen species (ROS) load in colonocytes thereby lowering down the mitochondrial membrane potential to induce apoptosis. Both Diclofenac and Curcumin exert their anti- neoplastic role via modifying physicochemical properties of membranes. Higher telomerase activity was observed in DMH group and lowered with treatment regimen. Both drugs seem to function via inhibition of telomerase and preventing inflammatory responses raised by NF- κB to induce apoptosis. Further, chemopreventive effect is found to be enhanced using the combination regimen of the two drugs than individual one.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 59 PP- 2 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

HEPATOPROTECTIVE EFFECTS OF AQUEOUS Azadirachta indica LEAF EXTRACT IN DMBA-INDUCED HEPATOTOXICITY

Sanjay Bharati, Vandna Mohan, Ashwani Koul* Department of Biophysics, Panjab University, Chandigarh 160014 *[email protected] , *[email protected]

Environmental pollutants including DMBA have been reported to induce hepatotoxicity in various animal models. Plants and plant products have been found to modulate the effects of these hazardous chemicals. Azadirachta indica (A.indica) possesses a wide range of pharmacological properties including its hepatoprotective role. In the present study, the effects of Aqueous A. indica leaf extract on DMBA-induced hepatotoxicity were evaluated in the liver of male Balb/c mice. Male Balb/c mice were randomly divided into four groups Group I served as control. Group II mice were administered AAILE at a dose of 200µg/g body mass, thrice a week on alternate days. Group III mice received an intraperitoneal injection of DMBA (40µg/g body mass). Group IV mice received AAILE and DMBA as described for Group II and III. AAILE administration commenced 8 weeks prior to DMBA injection. The overall physiological status of the liver was assessed in terms of variety of liver injury markers and functional tests, employed widely in the detection of injury, assessment of injury type and severity. The functional status of liver as assessed in terms of hepatobiliary clearance rate of radioactive mebrofenin was severely impaired in the DMBA group while AAILE treatment restored the normal pattern of mebrofenin hepatobiliary clearance. Serum tissue injury markers, clastogenic assays further confirmed that AAILE pre-treatment to DMBA challenged mice resulted in the reduction of deleterious effects of DMBA. The hepatic xenobiotic biotransformation enzymes were modulated in such a way that increased detoxification of DMBA reactive metabolite is expected after AAILE treatment. Thus, it could be deduced from the present study that AAILE provided effective hepatoprotective against DMBA induced hepatotoxicity.

60 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 3

LYCOPENE EXHIBITS CHEMOPREVENTIVE ACTIVITY IN NDEA INDUCED HEPATIC CARCINOGENESIS: STUDIES ON ASSOCIATED ALTERED ULTRA- STRUCTURE AND APOPTOSIS IN LIVER IL- 4 Prachi Gupta, M.P.Bansal, Nisha Bhatia, Ashwani Koul* Department of Biophysics, Panjab University, Chandigarh 160014 *[email protected], *[email protected]

Chemoprevention has received growing considerations as a potential means to control the rising incidence of liver cancer, the second leading cause of cancer deaths. It seems appropriate to investigate the structural, biochemical and molecular changes coherently in the tissue during carcinogenesis to delineate and manipulate altered pathway using a compatible chemopreventive agent. N-nitrosodiethylamine (NDEA), a known environmental hepatic carcinogen, has been used as an initiator in several hepatic cancer models. ROS generated during NDEA metabolism causes genetic damage to DNA and other cellular macromolecules stimulate tumor initiation. Such initiated cells adapt to the altered responsiveness to their microenvironment and achieve a proliferative advantage relative to the surrounding normal cells. A new strategy to address the issue of liver cancer is the use of lycopene a potent antioxidant as a dietary chemopreventive agent. Female Balb/c mice were randomly divided in four groups i.e. Control, NDEA (200mg NDEA i.p. /kg body weight, cumulative dose), LycT (5mg lycopene orally/kg body weight thrice a week) and LycT + NDEA. Histopathological observations of liver tissue using hematoxylin and eosin staining gave the primitive idea about the extent of carcinogenesis and their staging. Scanning and transmission electron microscopy technique were used to demonstrate the various structural and ultra-structural alterations in the liver tissue. The disturbance in the microenvironment of hepatocytes/hepatic tissue was assayed by measuring the level of redox ratio (GSSG/GSH) and lipid peroxidation during NDEA and LycT + NDEA exposure. COMET and TUNEL assay were done to quantify the apoptotic activity. mRNA expression studies of pro and anti-apoptotic genes were carried out to investigate the genetic changes in the treated liver. Histopathology and structural studies revealed carcinogenic alteration in the liver tissue on NDEA exposure. Numerical densities of mitochondria, RER, lipid granules, along with deformed nuclear membrane and chromatin were significantly correlated to carcinogenic effect of NDEA. Rounded and smaller hepatocyte with respect to polyhedral normal cells signifies alteration in cellular integrity on NDEA treatment. Being an antioxidant lycopene reduces the LPO level and redox ratio in the tissue and hence causes marked delay in tumor initiation. LycT pre-treatment had shown delay in the carcinogenic effect of NDEA. Moreover, increased apoptotic index was found in LycT + NDEA group when compared to NDEA mice.These comparative findings provide a novel mechanistic insight into the growth- inhibitory effects of lycopene in cancer, hepatocarcinogenesis in particular.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 61 PP- 4 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

PTEN REGULATES APOPTOTIC CELL DEATH THROUGH PI3-K/Akt/ GSK3β SIGNALLING PATHWAY IN COLON CARCINOGENESIS

Manpreet kaur saini and Sankar Nath Sanyal Department of Biophysics, Panjab University, Chandigarh –160014, India

Phosphatidylinositol 3-kinase (PI3-K) and Akt (protein kinase B), are both essential signaling molecules that are upregulated in various cancers. Here, we examined the molecular mechanisms by which PI3-K and Akt expression are regulated by glycogen synthase kinase- 3β (GSK-3β) and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the early stages of experimental colon carcinogenesis. 1, 2-dimethylhydrazine (DMH) was utilized for the induction of colon cancer while piroxicam, a traditional non-steroidal anti- inflammatory drug and c-phycocyanin, a biliprotein from Spirulina platensis (cyanobacterium) as the chemopreventive agents. Western blotting and immunofluorescence results indicated that the expression of PI3-K and Akt was promoted in the DMH group while least apoptosis was detected in this group as analyzed by Hoechst 33342-propidium iodide co-staining. DMH group further detected lower GSK-3β and PTEN expression as compared to other groups. Piroxicam and c-phycocyanin treatment resulted significant apoptotic cell death while showing low PI3-K and Akt expression. Mitochondrial membrane potential

(ΔΨM) alterations (examined by JC-1 and rhodamine 123 labeling of colonocytes) and fluorescence intensity measurement of ROS level, were also analyzed showing the raised

ΔΨM while reduced ROS levels in DMH group, however piroxicam and c-phycocyanin

treatment resulted in falling of ΔΨM although both stimulated the ROS production as analyzed by flow cytometry. The present study thus identified that piroxicam, a traditional NSAID and c-phycocyanin, a newly discovered COX-2 selective inhibitor, constitute remarkable chemopreventive targets in mediating apoptosis in the DMH induced early rat colon carcinogenesis via regulating PI3-K/Akt/GSK-3β/PTEN signaling pathways. Further, a combination of the two drugs provides a better therapeutic option, than the monotherapy regimen.

Keywords: Apoptosis, PI3-K, GSK-3β, Akt, PTEN, ΔΨM

62 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 5

EXPLORING Wnt/β CATENIN PATHWAY IN COX-2 SELECTIVE INHIBITOR, ETORICOXIB INDUCED APOPTOSIS IN EXPERIMENTAL COLON CANCER.

Pinky Sharma*, R.K. Vyas* and S.N. Sanyal IL- 4 *Department of Physiology, Dr. S.N. Medical College, Jodhpur Department of Biophysics, Panjab University, Chandigarh-160014

Non steroidal anti inflammatory drugs (NSAIDs) along with their anti inflammatory properties, inhibits neoplastic cell proliferation by inducing apoptosis. COX-2 inhibition appears to be the principle target of NSAIDs as it’s over expression is reported in several cancers including colorectal cancer. Also it catalyzes the synthesis of prostaglandin (PGE2) which is a critical pro inflammatory molecule. The present study explores the Wnt/ β catenin molecular pathway and role of GSK-3β in NSAIDs induced chemopreventive effect in experimental rat colon cancer.1, 2 di methyl hydrazine (DMH) was used for inducing experimental colon cancer and Etoricoxib, a COX-2 selective inhibitor was used as chemopreventive agent. After 6 week (early stage) and 18 weeks (promotion stage) treatment with DMH, morphological analysis reveals marked occurrence of pre neoplastic lesion and neoplastic features such as MPLs, ACF, adenoma in colonic mucosa. Histologicaly well characterized hyperplasia and dysplasia was also observed. Simultaneous co-administration of etoricoxib with DMH resulted in significant reduction of these features, proving the chemopreventive efficacy of Etoricoxib at its anti inflammatory dose. Etoricoxib only group lacks any prominent carcinogenic feature. DNA fragmentation and TUNEL positivity was checked to study apoptosis in colonic tissues. DMH up regulated the expression of Wnt, β- catenin, PI3K, Akt, COX-2 and VEGF but reduced GSK-3β as seen by Immunofluorescence analysis. Co-administration of etoricoxib decreases Wnt, β- catenin, PI3K, Akt, COX-2 and VEGF and increased the level of GSK-3β. Gelatin zymography showed prominent MMP-9 activity in the DMH treated group, while activity was nearly absent in all other groups thus confirming the target of NSAID as Wnt/β catenin signaling pathway to exert following actions i.e. induction of apoptosis, inhibition of cell proliferation and decreasing angiogenesis.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 63 PP- 6 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

REGULATION OF INFLAMMATORY CYTOKINES BY TRANSCRIPTION FACTORS NF-κB AND Stat-3 IN INFLAMMATION ASSOCIATED CARCINOGENESIS

Shruti Setia and SN Sanyal Department of Biophysics, Panjab University, Chandigarh-160014, INDIA

Inflammation augumented tumorigenesis forms the basis of the present study where colitis associated colon cancer model is investigated for the role of transcription factors Stat-3 and NF-κB regulated inflammatory cytokines and cell cycle check points. Celecoxib, a non- steroidal anti-inflammatory drug (NSAID) is incorporated as a chemopreventive agent against experimentally induced colitis and colon carcinoma. Our study was divided into eight groups: control, DSS, DMH, celecoxib, DSS + DMH, DSS + celecoxib, DMH + celecoxib and DSS + DMH + celecoxib. The expression of NF-κB was found to be elevated in inflammatory and carcinogenic models leading to the increased secretion of pro- inflammatory cytokines IL-1β, IL-2, IL-4, TNF-α, IFN-γ, along with the increased expression of iNOS and COX-2. These cytokines were found to activate Stat3, which is a major controller of proliferation and cell survival. It regulates the expression of various cell cycle regulators, pro-apoptotic proteins like Bcl-2 apart from the maintenance of NF-κB level in tumorigenesis. The expressions of Jak3, Stat3, cyclin D1, cyclin E, cdk2, cdk4 and Bcl- 2 were elevated in DSS, DMH and their combination group. Further, the fluorescent staining of isolated colonocytes revealed suppressed apoptosis in these three groups. Membrane parameters studied using the fluorescent probes pyrene, 1,6-diphenyl-1,3,5-hexatriene and N- NBD-PE indicated increased membrane fluidity in these groups. Celecoxib significantly altered these modulations studied in all the parameters, thereby suggesting the critical role of transcription factors, cytokines and cell cycle check points in its chemopreventive action.

Keywords: Cell cycle check points, colitis, colon carcinoma, cytokines, NSAIDs, transcription factors

64 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 7

PHYTOMODULATORY EFFECT OF WHEATGRASS IN EXPERIMENTALLY INDUCED DIABETES

* K Rana, V Chadha, D K Dhawan IL- 4 Department of Biophysics, Panjab University, Chandigarh 160014 *[email protected]

The present study reports the phytoprotective effect of wheat grass in experimentally-induced diabetes mellitus. The female Sprauge Dawley rats were divided into 4 groups, normal control (group I), diabetic (group II), wheatgrass treated control (group III) and wheatgrass treated diabetic (Group IV). Diabetes was induced in Group II and Group IV animals. Wheatgrass was administered to the animals belonging to group III and Group IV. In order to ascertain the development of diabetes in the animals the urine glucose levels were assessed by the urine reagent strips. A weekly record of body weight change of the experimental animals was maintained throughout the study. The effect of wheat grass on the carbohydrate metabolism was assessed by various carbohydrate metabolising enzymes. Moreover, the modulation of antioxidant defense system and acylation of proteins was also assessed. By 3rd day of intraperitoneal injection of alloxan monohydrate, animals indicated > 500 mg/dl glucose concentration in the urine and thus confirmed the presence of diabetes. The alloxan treatment in Group II and Group IV animals resulted in significant weight loss. However, pre-treatment of wheat grass to Alloxan group (Group IV) showed improvement in body weight. The hexokinase/ glucose-6-phosphate ratio was altered in Diabetes (Group II) and an improvement was observed by wheat grass treatment. A similar modulation of antioxidant defense system and thus glycation of proteins was also observed upon wheat grass treatment. The results indicate wheat grass might prove to be an effective agent against complications associated with diabetes mellitus.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 65 PP- 8 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EVALUATION OF NEUROPROTECTIVE ROLE OF CARBENOXOLONE IN PROTEASOME INHIBITOR INDUCED PARKINSON’S DISEASE MODEL.

Ankita and Bimla Nehru Department of Biophysics, Panjab University, Chandigarh, India.

Parkinson’s disease (PD) is the most common movement disorder associated with degeneration of substantia nigra and decrease in the dopamine levels in mid brain region as well as accumulation of cytoplasmic protein aggregates. Ubiquitin proteasome system (UPS) protects the cells by degrading the mis-folded proteins and preventing them from aggregation. Recently it has been seen that impairment in UPS is implicated in PD. Present study was designed to establish an animal model of Parkinson’s disease by using a proteasome inhibitor MG-132 in rats and to evaluate the neuroprotective role of carbenoxolone in this model. MG-132 (100 μM, 0.01mg in 2µl saline) was injected unilaterally into SNpc in male Sprague Dawley rats (200-250g). Carbenoxolone was injected i.p at the dose of 20mg/kg bodyweight of the rats, every day for 12 days. Animals were monitored continuously for the body weight changes and behavioural changes were observed by the bar test, actophotometer and rota-rod test. After 12 days animals were sacrificed. MG- 132 reproduces the behavioral features of Parkinson's disease in rats. It destroys dopaminergic neurons in mid brain, causing deficiency of dopamine which leads to impaired motor functions. A 45% decline in dopamine and 65% decline in DOPAC levels were registered after MG-132 administration. Data showed impaired motor function, significant increase in catalepsy and decrease in locomotor activity in MG-132 treated rats w.r.t control. Co-treatment with Cbx significantly attenuated the extent of motor dysfunction and improvements in the level of dopamine induced by proteasome inhibitor. Proteasome inhibitor resulted in increased oxidative stress and results into neuronal loss in mid brain region of rat brain. Co-treatment attenuates most of these changes. Increased oxidative stress also causes the induction of the apoptosis. Increased expression of caspase 3 and 9 in MG- 132 treated group, results in the induction of apoptosis. Cbx co-treatment effectively prevents the apoptosis induction. Histopathological analysis also indicated the loss of neurons in the SNpc in MG-132 administered rats w.r.t control. A better representation was observed in conjunctive group that received MG-132 and Cbx. Thus, present study provides evidence that Cbx is a potential neuroprotector for PD.

66 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 9

MODULATION OF MITOCHONDRIAL FUNCTIONS BY THE HEAT SHOCK INDUCER CARBENOXOLONE IN THE PARKINSON’S DISEASE

Poonam Thakur and Bimla Nehru IL- 4 Dept of Biophysics, Panjab University, Chandigarh, India [email protected]

Parkinson’s disease (PD) pathogenesis involves several factors like mitochondrial dysfunctions, oxidative stress, protein aggregation etc. Neurons try to employ several defense systems to combat these stresses like up-regulation of anti-oxidants mechanisms and induction of heat shock proteins (HSPs). Carbenoxolone (Cbx) is a novel HSP inducer, which has been shown to be neuroprotective in various CNS disorders like ischemia, epilepsy etc. In the present study we explored the neuroprotective mechanism of Cbx in a rotenone based model of PD. For this male SD rats were divided into 4 groups. One group was given rotenone at the dose of 2 mg/kg body weight for 5 week while another group was administrated Cbx (20 mg/kg body weight) along with rotenone. Cbx treatment was started 1 week prior to rotenone treatment and was continued till the end of the study. Vehicle treated control group and Cbx per se group were also run simultaneously. Our previous studies have shown that Cbx can prevent the decline in dopamine and TH levels as well as the motor functions. Presently, it was found that Cbx administration leads to the improvement in the mitochondrial GSH pool as well as Mn-SOD activity. Cbx co-treatment was also able to increase the levels of ATP which were significantly lowered in the rotenone treated animals. Due to the improvements in the mitochondrial anti-oxidant defenses, Cbx was able to prevent the decline in the activity of complex-I and complex-IV which are the most sensitive targets of rotenone exposure. Subsequently, improvements in the levels of apoptotic markers like caspase-9 and caspase-3 were also seen with Cbx co-treatment. All these changes were strongly associated with a significant up-regulation of the levels of Hsp- 70 and Hsp-90 following Cbx administration. Thus, Cbx can exert neuroprotective effects in PD by improving the mitochondrial functions and preventing apoptosis. These effects seem to be mediated by its HSP inducing and anti- oxidative properties.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 67 PP- 10 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SPECTROSCOPIC STUDIES OF METALLOTHIONEIN BINDING TO ARSENIC AND ZINC

Roobee Garla, B. P. Mohanty, Renuka Ganger, M. Sudarshan, M. P. Bansal and M. L. Garg Department of Biophysics, Panjab University, Chandigarh-160014 Email: [email protected]

Metallothionein (MT) is a family of ubiquitous low molecular weight (6000-7000 Da) proteins with an unusually high concentration of cysteine (30%). MT binds to a number of heavy metals and plays a crucial role in essential metals homeostasis (Zinc and Copper) and protection against heavy metal toxicity ( Cadmium, Mercury); but the exact mechanism is still not known. Arsenic, a known human carcinogen, has high affinity for biological thiol groups. To observe the role of MT in case of Arsenic toxicity in vivo, there is need for efficient and reliable isolation and characterization protocols for the analysis of MT. The stoichiometric analysis of metal induced MT will throw light regarding the metal and MT interactions in in-vivo conditions which will expand our understanding regarding the role of MT in metal homeostasis and toxicity. To achieve this, PIXE and ESI-MS analysis of rabbit MT-1 (procured from Bo Tai Biotech Co., Ltd., China), apo rabbit MT-1, As substituted rabbit MT-1(with various As concentration) was conducted. The PIXE analysis of rabbit MT shows the presence of copper in addition to Zinc. With increase in arsenic concentration, the

amount of arsenic bound to rabbit MT also increases. Ions of Zn5MT1, Zn6MT1 and Zn7MT1 were observed in the ESI-MS spectra of rabbit MT1. Zn induced MT was also isolated using Gel filtration and ion exchange chromatography from rat liver in our laboratory. The PIXE analysis of isolated MT from rat liver again shows the presence of copper in addition to Zinc and the sulphur to zinc ratio is high (3.05) even in

comparison to that of fully metallated MT (Zn7MT for which S:Zn is 1.468). This may be inferred as the presence of a mixture of partially metallated MT, apo-MT of fully metallated MT. Similar results were observed with ESI-MS for isolated rat liver MT which again shows

the presence of partially metallated ions of MT, Zn5MT1 and Zn1MT. To the best of our knowledge these are the first results on isolated MT where presence of Cu along with Zn has been reported. The induction of Zn-Cu MT in rat liver on Zn supplementation may be related to the role of MT in Zn and Cu homeostasis. Isolation and characterization of arsenic induced rat MT is future goal.

68 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 11

INVESTIGATION ON THE INTERACTION OF ZINC AND ARSENIC WITH REDUCED GSH THROUGH NMR AND ESI- MS SPECTROSCOPY

B. P. Mohanty, Rajbinder Kaur Virk, Jaspreet Kaur, Roobee Garla, IL- 4 Narinder Kaur, M. P. Bansal and M. L. Garg Email:[email protected]

Toxic metals are known to replace zinc from different enzymes thus affecting normal cellular processes. Arsenic (As) is one such environmental toxicant commonly found in soil, water and air and is classified as a human carcinogen. Reduced glutathione (GSH) is present in high concentration in most living cells from microorganisms to humans. Its biological significance is mainly related to the free sulphydryl moiety of the cysteine residue of GSH, confers to its unique redox and nucleophilic properties and plays a crucial role in Zinc (Zn) homeostasis. In case of arsenic toxicity, GSH plays an important role in reducing As(V) to As(III) and expelling As(III) from intracellular environment by forming As-GSH complex. This suggests that in presence of As, glutathione redox balance would be affected which may alter Zn homeostasis. At physiological pH GSH has 8 dense binding sites. The present study was carried out to identify the binding sites of GSH for Zn and As and the stoichiometry of the metal-GSH complex at physiological pH. To achieve this, the interaction of Zn and As with GSH was studied by solution NMR and ESI-MS spectroscopy. Different pulse sequences for 1H and 13C nuclei had been employed to extract necessary stoichiometric information on the complexation of these two metals with GSH. Through reaction studies of Zn with GSH, the binding of Zn with the sulfhydral, terminal amino and the three CO groups of GSH were observed. The binding of dimeric or multimeric form of GSH with Zn could not be confirmed. Zn-GSH complexation could not be observed with ESI-MS. NMR spectra of GSH with different concentration of As show the binding of it only with the thiol group of Cys. Moreover, the broad doublet structure of Cys β protons imply that more than one thiol group are binding with As. This fact was confirmed by ESI-MS where As(GS)3 complex was observed. These results suggest that Zn ions have affinity for multiple sites of GSH, due to which the valency of a Zn ion get satisfied by simultaneous coordination with these sites. However, As ions have been observed to have affinity only for the sulfahydral site. This may be the reason that only As forms polymeric GSH complexes not the Zn. The determination of three-dimensional structure of Zn/As-GSH complex using NOE based NMR is our future goal.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 69 PP- 12 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

GABAPENTIN A GABA ANALOUGE NEUROMODULATES THE CHANGES IN BEHAVIORAL AND NEUROTRANSMITTER SYSTEM INDUCED BY TRIMETHYLTIN INTOXICATION IN THE RATS.

Sukhwinder Kaur* and Bimla Nehru* *Department of Biophysics, Panjab University, Chandigarh. INDIA Email: [email protected] and [email protected]

Ingestion ofTrimethyltin (TMT) overt neurological and behavioral changes in rodents, which includes aggression, hyperirritabilty, tremors, spontaneous seizures. These symptoms may be related to their interaction with activators of the endogenous excitatory neurotransmitter system. Gabapentin a novel anticonvulsant has possible effect on glutamate and GABA metabolism in brain as they may relate to its anticonvulsant mechanism of action. The study was an attempt to explore the neuromodulatory effect of Gabapentin against impaired behavioral and neurotransmitter system induced by Trimethyltin exposure in the rats. Male SD rats, were administered single systemic injection of TMT (8.5 mg/kg b.w) after administration of TMT; rats were treated with gabapentin i.p (25mg/kg b.w) for 21 days. Cognitive deficits were studied by aggression scores, seizure score, active avoidance, passive avoidance and water maze at (3, 7, 14, 21 days) of treatment. At 21 day their hippocampal tissues were processed in order to access changes in neurotransmitters (GABA, serotonin, acetylcholine esterase). The co-administered of TMT with Gabapentin showed marked improvement in seizure, aggressive behavior which in turn reflected by the levels of serotonin and GABA. The deterioration of memory caused by TMT exposure was significant improvement in animals administered gabapentin along with TMT. These alterations in learning and memory were correlated well with modified acetylcholine esterase content with gabapentin coadministration. Thus, gabapentin ameliorated brain neurotransmitters which were impaired during trimethyltin exposure and offered protection against cognitive deficits. Keywords: Gabapentin, Trimethyltin, Cognition and Neurotransmitters

70 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 13

NEUROMODULATORY EFFECTS OF APOCYANIN IN LIPOPOLYSACCHARIDE INDUCED PARKINSON’S DISEASE MODEL

Neha Sharma* and Bimla Nehru IL- 4 Dept of Biophysics, Panjab University, Chandigarh-160014

Recent studies have revealed an essential role for neuroinflammation that is initiated by microglial and infiltrated peripheral immune cells and their toxic products (cytokines.chemokines etc) in pathogenesis of PD. Lipopolysaccharide, a bacterial endotoxin is the most extensively utilized glial activator for the induction of inflammatory dnergic neurodegeneration therefore, LPS at a dose of (5ug/5ul PBS) injected stereotaxically into the Substantia Nigra of rat brain was utilised for the establishment of PD model. This in turn leads to microglial activation and hence increased level of pro-inflammtory cytokines and activation of NADPH oxidase complex leading to excessive superoxide anion production which further resulted in neurotransmitter Dopamine loss and behavioural impairment whereas Apocyanin, ameliorated LPS induced alterations in neurochemical (Dopamine and its metabolites DOPAC and HVA), behavioural (Actophotometer, Rotarod , Bar test) - biochemical oxidative stress markers (GSH, O2 , SOD, Catalase, LPO) produced due to microglial activation following LPS exposure as assessed from decreased levels of proinflammatiory cytokines (TNF-α,IL-1β) using RT-PCR and decreased superoxide anion production from NADPH oxidase activation, increase in GSH, SOD and decreased lipid peroxidation following apocyanin treatment. Apocyanin is derived from the rhizome of Picrorhiza kurroa, has been used as a herbal medicine for treatment of a number of inflammatory disease. Results suggest that apocyanin due to its antioxidant as well as anti- inflammatory properties can prove to be beneficial for curing PD.

Key words: parkinson’s disease, NADPH oxidase, tumor necrosis factor, superoxide anion

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 71 PP- 14 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECTS OF ZINC AND ARSENIC ON MT PROTEIN AND ITS mRNA EXPRESSIONIN RAT LIVER

Renuka Ganger, Roobee Garla, M. L. Garg and M.P.Bansal Department of Biophysics, Panjab University, Chandigarh-160014

Arsenic (As) is a ubiquitous environmental toxic metalloid that is becoming a major public health concern worldwide. Metallothionein (MT), is a low molecular weight, metal binding protein which protects against metal intoxication. There are various dietary factors such as selenium, vitamin-A, iron, and zinc that are known to play a critical role in modulating arsenic toxicity. This study therefore was undertaken to determine whether the combination of zinc and arsenic has a modulatory effect on mRNA and MT protein expression. The rats were divided into four groups. Group 1 as control, group 2 received subcutaneous (sc)

injection of ZnSO4 (153µmol /kg) for successive two days; group 3 received NaAsO2

(75µmol/kg) sc for one day; group 4 receive ZnSO4 (153µmol /kg) along with NaAsO2 st (75µmol/kg) on 1 day, followed by ZnSO4 (153µmol /kg) on second day and were sacrificed after 18hrs of last injection. In the present study hepatic rat MT-1 was isolated from zinc treated group using gel filtration column and ion exchange chromatography which was further used as an immunogen for antibody generation. Rabbit model was used to raise polyclonal antibodies (pAbs) against rat MT-1. MT-pAbs were characterized by SDS-PAGE electrophoresis and western blot assay in different treatment groups. Western blot showed multiple bands of MT-1 at 44, 24 and 14 kDa due to the aggregation of MT-1. The MT expression was enhanced in Zn treated group but not in arsenic treated group however in combined group MT expression was more as compared to zinc treated group. The mRNA expression studies revealed no change in MT-1 mRNA expression. In conclusion, zinc and combined treatment of Zn+As has effect on MT induction at translational level but not at transcriptional level. No effect was observed in As treatment at both levels. In addition combination of Zn+As has more MT protein expression than zinc treatment. This may be inferred that As has synergistic effect along with zinc that might be increasing the MT protein expression.

72 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 15

EVALUATION OF NANOSESAMOL AS A CHEMOPREVENTIVE AGENT FOR SKIN CARCINOGENESIS IN MICE MODEL

1 *,1 1 2 Rishi Bhardwaj , Tranum Kaur , SN Sanyal , Kim Vaiphei , IL- 4 Vandita Kakkar3 and Indu Pal Kaur3 1Department of Biophysics, Panjab University, Chandigarh, India; 2Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 3University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India; *Email: [email protected]; [email protected]

Sesamol is a natural antioxidant and has been found to show anti-mutagenic and chemopreventive effect. Solid lipid nanoparticles (SLN’s) are at the forefront of the rapidly developing field of anti-cancer nanotechnology with several potential applications. In the present study, sesamol encapsulated in solid lipid nanoparticles were evaluated and compared with its free form in 7,12-dimethylbenz[α]anthracene (DMBA) induced skin cancer mice model for their improved and effective chemopreventive potential as topical delivery system. Chemopreventive potential of nanosesamol in comparison to the free sesamol was assessed in terms of histopathological examination, biochemical estimations (lipid peroxidation, and antioxidant enzyme estimations), body weight, tumor incidence, and expression pattern of cell death and cell proliferation markers assessed by immunoflourscence and western blot analysis. In our study, animals were divided into six groups. Group 2, Group 4 and Group 6 were subcutaneously injected with DMBA in 0.2ml of olive oil at dose of 30mg/kg body weight once a week for three weeks consecutively. Group 1, Group 3 and Group 5 were only given 0.2ml of olive oil subcutaneously. Group 1 served as control group which received blank gel whereas Group 3 and Group5 (per se groups) were topically applied with only encapsulated sesamol and free sesamol respectively. DMBA skin cancer induced mice were treated with free sesamol and sesamol-SLNs (4 mg/kg body weight) from one week before DMBA injection till the end of experiment (18 weeks). Results revealed lipid peroxidation levels in sesamol treated groups to be significantly lower in comparison to tumor bearing mice (Group 2). Sesamol treatment exerted chemopreventive effect by significantly increasing the antioxidant levels, thereby, decreasing the development and promotion of tumors. Immunofluorescence studies of bcl-2 and bax protein expression suggested the modulatory effect of sesamol. Overall, nanosesamol treatment gave better results in comparison to free sesamol and demonstrates anti-carcinogenic effect of sesamol. Therefore, sesamol may be regarded as the valuable nutraceutical because of its richness in antioxidants along with its excellent chemopreventive potential especially when delivered topically in its encapsulated form in solid lipid nanoparticles.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 73 PP- 16 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EXPLORING THE PROAPOPTOTIC & ANTI-ANGIOGENIC ROLE OF NOVEL TYROSINE KINASE RECEPTOR INHIBITOR, IMATINIB, IN EXPERIMENTAL COLORECTAL CANCER

Dilpreet Kaur and SN Sanyal Department of Biophysics, Panjab University, Chandigarh-160014 Email: [email protected]

Imatinib acts primarily by the inhibition of tyrosine kinase activity, responsible for excessive proliferation in tumorigenesis. The present study explores the chemopreventive role of Imatinib in 1,2 dimethylhydrazine (DMH) - induced colon carcinogenesis in the rat model. The animals were divided into four groups: Group 1 served as control and recieved the vehicle of the drugs. Group 2 recieved a weekly injection of 30mg/kg body weight of freshly prepared DMH in 1mM EDTA saline. Group 3 recieved DMH along with daily oral administration of Imatinib (30mg/kg body weight) and Group 4 recieved imatinib daily orally. Gross morphological analysis revealed the occurence of raised mucosal lesions called MPL or multiple plaque lesions, which were maximium in the DMH group and their number was regressed with the co-administration of imatinib. Further, aberrant crypts foci (ACFs), the regions of abnormal cell growth, were found to be decreased in imatinib co- administration groups. Histological analysis was also preformed and abnormal histo- architecture like hyperplasia and dysplasia were evident in the carcinogenic group, which were found to be reduced with imatinib co-administration. Cyclooxygenase enzyme (COX), which catalyzes the conversion of arachidonic acid to prostaglandins, will also be explored and its ubiquitious isoform COX-1 inducible isoform, COX-2 will be studied. Also, the signal transduction pathways for angiogenesis and apoptosis will be explored which way reveal that the tyrosine kinase inhibitor, Imatinib, could be a potential target for the chemoprevention of colorectal cancer.

Keywords : Colorectal cancer, COX, DMH, Imatinib.

74 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 17

EFFECT OF CHAPERONES AND METAL IONS ON AGGREGATION OF GAMMA GLOBULIN

Moupriya Nag and Soumen Basak IL- 4 Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064

Gamma globulin (γG), particularly in an aggregated form, combines with rheumatoid factor (RF) to form immune complex and activates the blood complement (C) system thereby releasing a variety of phlogistic mediators. We have studied the effect of metal ions and molecular chaperones on the conformational properties and aggregation kinetics of γG using absorption, fluorescence and circular dichroism (CD) spectroscopy. Room temperature incubation of γG (at concentrations as low as 5-10 µM and at neutral pH) leads to formation of insoluble precipitates, usually through large-scale aggregation of fibrils. Changes in CD spectra in presence of metal ions (Zn++, Al+++) showed that the latter induce conformational changes in γG, with the altered structures acting as possible precursors of γG-aggregates that formed faster upon incubation with the metals. The kinetics of fibril formation, measured by thioflavin T (ThT) assay, yielded sigmoidal curves that are indicative of a nucleation dependent polymerization process with a lag time of around 20 hrs in absence of metal ions. The time course of aggregation was also studied at higher temperatures (50-70 ºC) by monitoring the increase of apparent OD of a solution of γG and expectedly showed that the kinetics (always cooperative) became faster with increasing temperature. Both the lag time and rate constant of aggregation varied linearly with the logarithm of protein concentration, indicating that aggregation followed a first-order kinetics. The aggregation process was inhibited by the presence of αs-casein, which acts as a chaperone, with the degree of inhibition being proportional to the concentration of αs-casein.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 75 PP- 18 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

NSAID INDUCED MEMBRANE FUSION: EFFECT OF METAL CO-ORDINATION OF THE DRUGS.

Anupa Majumdar, Sreeja Chakraborty and Munna Sarkar. Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata-700064.

Membrane fusion is an extremely fundamental membrane mediated process. Although the fusion process in vivo or in vitro is almost always mediated by proteins and peptides, small drug molecules also have the ability to induce membrane fusion. Certain Non-Steroidal Anti- inflammatory drugs (NSAIDs) like Meloxicam and Piroxicam, have already been established by our group, to be fusogenic agents effective at very low drug: lipid ratios of around 0.03. It is already known that some copper (II) coordination complexes of NSAIDs exhibit enhanced anti-inflammatory and anti-cancer effects as compared to the bare parent drugs. A comparative study of the fusion kinetics for copper(II)-NSAID complexes and bare NSAID drugs induced membrane fusion was carried out in this work. FRET based membrane bilayer probe (NBD-PE/NRh-PE) dilution assay was monitored to study the first step of membrane fusion, lipid mixing. A dramatic increase in the rates of lipid mixing process was observed specially for Copper (II)-Meloxicam coordination complex as compared to bare meloxicam drug at the same effective oxicam drug concentration, probably hinting at a different fusogenic mechanism of the coordination complexes. Second derivative absorbance Spectrophotometry was used to determine the drug partition coefficients between lipidic bilayer and aqueous phase and a several fold increase in partition coefficients was observed for copper(II)-oxicam complexes as compared to the bare oxicam drugs. Comparable rates of content mixing obtained for both Copper (II) coordination complex at 15μM and bare oxicam drug at 30μM concentrations respectively were a clear indicator that the individual drug molecules participated in the fusion process in-spite of their connectivity. Transmission electron microscopy (TEM) images of the DMPC vesicles in the presence of Copper (II) complexes, revealed several fusion bodies consisting of more than two or three vesicles fused together which in a way could justify the several fold increase in partition coefficients in case of copper complexes. For the first time our study shows that the copper complexes are capable of anchoring two vesicles to initiate contact, the first step of the fusion process.

76 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 19

A FLUORESCENT PROBE AS A NEXT GENERATION MERCURY SENSOR: IN VITRO AND IN VIVO STUDIES

* Kallol Bera and Soumen Basak IL- 4 Chemical Sciences Division, Saha Institute of Nuclear Physics 1/AF Bidhannagar, Kolkata 700064

Mercury is a highly reactive toxic agent that damages the central nervous system, endocrine system, kidneys and other organs and causes many severe diseases upon ingestion in humans. Despite the progress of mercury detection in organic solvents, there remains a need for sensors with high sensitivity and selectivity that can detect mercury in water. We have developed a fluorescent probe that can detect and detoxify mercury in a single platform for the first time. The probe (RHDMSA), which can be prepared in one step from Rhodanine B hydrazide, responds rapidly to Hg2+ in aqueous solutions with 1:1 stoichiometry. Since the probe reacts with Hg2+ in an irreversible manner, it has advantages of both sensitivity and selectivity over existing reversible mercury probes in in vivo assays. The formation of water- soluble Hg2+-DMSA (meso-2, 3-dimercaptosuccinic acid) complex provides the opportunity for detoxification of Hg2+ by way of natural excretion from the system. In DMSO-water (1:9 v/v) solution, the fluorescence intensity of RHDMSA undergoes 20-fold increase and its emission maximum shifts from 583 to 587 nm upon addition of 1 equiv of Hg2+. By using Fluorescence Correlation Spectroscopy (FCS) we were able to detect mercury

levels as low as ~50 pM with high selectivity. The diffusion time (τD) of the probe increased from 32
sec to 38
sec as the concentration of Hg2+ was increased from 0 to 50 pM. The number of fluorescent particles within the FCS observation volume increased in linear fashion with increasing concentration of Hg2+, supporting the proposed 1:1 binding stoichiometry. The irreversibility of binding was verified by titration with excess DMSA at neutral pH. Fluorescence imaging assay was used to demonstrate the capability of RHDMSA to monitor the presence of mercury in mammalian cells. In vivo studies showed that the presence of mercury in HeLa cells could be detected by incubating the cells with Hg2+ (5 µM) and then with RHDMSA (5 µM). Confocal imaging experiments showed that the probe can penetrate HeLa cells with ease, thereby making in vivo Hg2+-detection a relatively straightforward procedure.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 77 PP- 20 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

MOLECULAR ANALYSIS OF ESBL AND MBL PRODUCERS AMONG DRUG RESISTANT UROPATHOGENS IN SOUTH MUMBAI

Mobashshera Tariq and Aruna K.* Department of Microbiology, Wilson College, Mumbai- 400 007, India. *Corresponding Author: Dr. Aruna K, Associate Professor, Head of Microbiology Department, Wilson College, Mumbai- 400 007, India. E-mail: [email protected]. Mobile no. 9867233673

β-lactamases are enzymes possessed by pathogens that cleave the β-lactam ring in the antibiotics thus rendering the organism resistant to that antibiotic. Extended spectrum β- lactamases (ESBLs) and Metallo β-lactamases (MBLs) are the two types of β-lactamases that have emerged as the major threat worldwide with limited treatment options. The present study was undertaken to study the occurrence of ESBL and MBL genes among uropathogens collected from various healthcare centers across south Mumbai. A total of 195 gram negative urine isolates were collected, identified and analyzed for ESBL production. Antibiotic sensitivity testing showed 50.25% (98/195) isolates to resist over 70% of the antibiotics used in the study. Phenotypic methods revealed the overall prevalence of ESBL and MBL producers to be 34.71 % (68/195) and 3.58% (7/195) respectively. Molecular analysis of ESBL and MBL producers showed presence of plasmids of approximately 50-60 kb in size. ESBL producers were further screened for presence of TEM, SHV and CTX-M genes by carrying out PCR. Isolates of E.coli (40/42), Pseudomonas aeruginosa (3/3), Proteus spp (6/7), Klebsiella pneumoniae (7/8), Enterobacter aerogenes (1/1) and Citrobacter diversus (6/7) showed presence of TEM gene making it the most common gene in this study carried out in south Mumbai. CTX-M gene existed in only 5 isolates (E.coli (2), K. pneumoniae, Proteus mirabilis and Citrobacter diversus). However, SHV gene was not detected in any of the isolates. Screening of MBL producers showed presence of NDM-1(New Delhi Metallo β-lactamase) gene in 4 out of 7 isolates. NDM-1 is a novel enzyme first reported in 2009 in New Delhi and has spread though out India since then. The current study also showed an alarming rise in the frequency of NDM-1 producers since majority of the MBL genes were of NDM-1 type. Restriction enzyme analysis of Plasmid DNA of β-lactamase producing uropathogen was also done using three restriction enzymes EcoR1, BamH1 and Hind111 to characterize the possible evolutionary relation between different ESBL and MBL producers and to determine the spread of different or same plasmid in the region of south Mumbai. This study will provide a fundamental base to the problems associated with ESBL and MBL producers among drug resistant uropathogens.

78 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 21

PURIFICATION AND ENZYMATIC PROPERTIES OF THREE LMW CYSTATINS

Vikash Kumar Yadav, Nirmal Chhikara, Kamaldeep Gill, Sharmistha Dey Sarman Singh, Savita Yadav IL- 4 Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, INDIA

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. Based on the molecule complexity, cystatins have been categorized into three families: Family 1 cystatins (stefins) are found mainly intracellularly and have molecular weights of 12 kDa, family 2 cystatins (S, SA, SN, C) are essentially found extracellularly and have a molecular weight of 14 kDa, and family 3 cystatins are the high molecular weight kininogens. Cysteine proteinase inhibitors (CPIs) are present in human semen in much higher concentrations than trypsin inhibitors, which are known to prevent fertilization process. The presence of cystatins in such a high concentration strongly indicates an efficient need of cysteine proteinase inhibition in reproductive tract. Therefore the aim of the study is to purify and characterize human seminal fluid (HSF) cystatins Three CPIs of low molecular weight (LMW) were isolated from HSF by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS–PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF- MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. The proteins were incubated at different temperatures, ranging from 20°C to 100°C, for 30 min. The inhibitory activity toward papain remained relatively stable up to 80°C. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, and pepsin and acts as a cross-class protease inhibitor in in-vitro studies.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 79 PP- 22 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURAL FEATURES OF TELOMERIC QUADRUPLEX DNA HAVING DIFFERENT TOPOLOGIES: STUDY BY MOLECULAR DYNAMICS SIMULATION

Angana Ray, Dhananjay Bhattacharyya Biophysics Division Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India

During the last decade, four-stranded DNA structures known as G-quadruplexes, or DNA tetraplexes, have emerged as a three-dimensional structure of special interest. In addition to their potential role in cell functioning, they play a very important role in telomere function. These G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer drug targets. Several structures from different synthetic constructs have been solved by X-ray crystallography and nuclear magnetic resonance spectroscopy. In order to understand the structure of ‘real’ telomeres, it is necessary to understand the structure of long human telomeric DNA sequences. We have therefore carried out detailed molecular dynamics simulation, with telomeric qDNA sequences having different topologies for 100ns until now. The human telomeric qDNA having parallel G-tetrad core containing potassium ions, has a high RMSD value, however, here the Guanine base positions remained largely constant along the trajectory showing high loop flexibility. The potassium ions initially present inside the core moves out into the aqueous medium and by the end of 100ns only 1 K+ ion resides inside the core. For the human telomeric qDNA having a similar parallel G-tetrad core containing no potassium ion, gives a comparable RMSD to that of the situation where potassium ions are present in the core. Here some sodium counterions from the solvent move inside the G-tetrad core after minimization. This is in accordance to the fact that presence of ions inside the G-tetrad core adds to the quartet stability. The Na+ and K+ ions involve in coordination with the O6 atom of Guanine base. The analysis of coordinate bonds formed inside the G-tetrad core, i.e., O6...K+...O6 and O6...Na+...O6 angles, indicates that the G- tetrad core may have a cubic or square antiprismatic geometry. The human telomere having mixed parallel and anti-parallel strand orientation and no potassium ions in it, has RMSD comparable to that of the parallel core structure but the RMSD value for Guanine base positions is significantly higher. This may indicate that the core type plays an important role in stability of the telomeric qDNA.

80 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 23

CRYSTAL STRUCTURE OF THE COMPLEX OF BOVINE LACTOPEROXIDASE WITH ASCORBIC ACID AT 2.35 A RESOLUTION

Yamini,S., Singh,R.P., Singh,A.K., Pandey,N., Kaur,P., Sharma,S., Singh,T.P. IL- 4 Dept. of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

Lactoperoxidase is a heme containing glycoprotein of molecular mass of 68 kDa. It catalyzes the conversion of inorganic ions such as Cl-, Br-, I- and SCN as well as a number of small aromatic compounds into the product which are toxic to a wide range of microorganisms. The reaction takes place in the presence hydrogen peroxide. The crystal structure of lactoperoxidase when complexed with ascorbic acid has been determined at 2.35 A resolution .The final Rcryst and Rfree factor were 0.226 and 0.250 respectively. The ascorbic acid has been found to bind lactoperoxidase in the substrate binding site on the distal heme side. Ascorbic acid forms hydrogen bonds with Arg-255, his-199 and conserved water molecule W1. The unbound protein has six conserved water molecules W1’, W2’, W3’, W4’, W5’ and W6’ in the substrate binding site .As a result of the binding of ascorbic acid four water molecules W2’, W3’, W4’, W5’ were removed from the binding site. The orientation of ascorbic acid in the substrate binding site is favorable for catalysis by lactoperoxidase. His- 109 plays an important role in proton- relay with the help of six linearly linked water molecules including W1-His109-W2-His226-W3-W4-W5 and W6. These results indicate a possible role of lactoperoxidase in the conversion of ascorbic acid into an antimicrobial product.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 81 PP- 24 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURAL AND FUNCTIONAL STUDIES OF Entamoeba histolytica SERINE ACETYLTRANSFERASES; INSIGHT INTO THE DIFFERENTIAL REGULATION OF ISOFORMS.

Sudhir Kumar, Mohit Mazumder and S. Gourinath School of Life Sciences, Jawaharlal Nehru University, New Delhi

Entamoeba histolytica has adapted to an anaerobic life style; still it consumes oxygen in small amounts and thus, produces toxic oxygen derivatives, similar to aerophilic organisms. It contains superoxide dismutase but lacks catalase, glutathione, and peroxidase. Therefore, cysteine becomes the major thiol for countering the oxygen free radicals. Cysteine is synthesized de novo in E. histolytica in a two step cysteine biosynthetic pathway. In first step L-serine is converted to O-acetylserine (OAS) by Serine acetyltransferase (SAT) and

consequently OAS is converted to cysteine by condensation with H2S catalyzed by O- acetylserine sulfhydrylase (OASS). EhSAT exists in three isoforms viz. EhSAT1, 2 and 3. EhSAT2 and EhSAT3 are about 73% and 48% identical with EhSAT1 respectively. Out of three isoforms, EhSAT1 has been reported to be feedback inhibited by cysteine almost completely while the other two isoforms are relatively insensitive to this inhibition. EhSAT1 was cloned, over-expressed and purified to homogeneity by affinity as well as gel permeation chromatography which showed EhSAT1 to be a trimer. EhSAT1 in native as well as serine/cysteine bound state was crystallized and structure was solved for all three states. EhSAT1 structure showed that due to its trimeric nature it could not form a decameric cysteine synthase complex. Careful observation of the sequences of both EhSAT1 and EhSAT3 revealed that all the active site residues except His208 are conserved, which was replaced with Ser208 in EhSAT3. Molecular modeling and dynamics studies show that His208 could be responsible for distinguishing between serine and cysteine. Kinetic studies on EhSAT1 and EhSAT3 mutants also reiterated the observations that His208 has effect over the binding of cysteine to active site. All these results corroborated the observation that E. histolytica has bypassed the complex regulatory mechanism to ensure the sufficient supply of cysteine to the infecting cell.

82 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 25

STRUCTURAL AND FUNCTIONAL STUDIES OF COACTOSIN FROM Entamoeba histolytica: CONTRARY TO THE OTHER ADF/COFILIN FAMILY PROTEIN

1 1 2 2 Nitesh Kumar , Somlata , Simanti Bhattacharya , Sankar Maiti IL- 4 And S. Gourinath1* 1School of Life Sciences, Jawaharlal Nehru University, New Delhi, 2Indian Institute of Science, Education and Research, Kolkata.

Entamoeba histolytica causes amebiasis that affect millions of people annually worldwide. A central contributor in E. histolytica pathogenesis is the highly effective actomyosin cytoskeleton, which allows fast morphological changes associated with amoebic motility and spatial reorganization of cellular components. The key players in this activity are the actin binding proteins (ABPs). Study of the actin cytoskeleton of the E. histolytica is interesting because it has been proposed that reorganization of the amoeba cytoskeleton is essential for several cellular functions involved in pathogenicity including the interaction between the amoeba and its target cell, phagocytosis of intestinal epithelial cells and human erythrocytes, cytolysis of target cells, cell migration, and escaping of the host immune response. We have been working on Coactosin which is a ADF/Cofilin family protein. The purified protein was crystallized at 289K by hanging drop vapour diffusion method using PEG1500 and diffracted X-rays to 1.8Å resolution. The SeMet derivative protein crystals diffracted to 1.5Å at selenium peak wavelength. The crystals belonged to P6 space group with unit cell parameters, a=b=76.59Å, c=54.649Å, α=β=90°, γ=120°. Actin depolymerisation kinetics shows that EhCoactosin, instead of depolymerising it stabilizes the actin filament. Crystal structure shows the protein to be rich in β-sheets and is similar to human coactosin like protein (CLP) with rmsd of 1.375Å. EhCoactosin does not behave like ADF/Cofilin family protein in vitro. It stabilizes actin filament even in presence of Xenopus cofilin. The mutation of the residue expected to interact with actin, Lys 75th did not alter its stabilizing property. Our study also associated this protein with phagocytic cup formation in E. histolytica.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 83 PP- 26 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CROWDING AGENT STABILIZES RIBONUCLEASE-A AT NEAR PHYSIOLOGICAL pH.

Moin Ishrat, Faizan Ahmad and Asimul Islam* Protein Research Lab., Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi. *Author to whom correspondence should be addressed. Phone: +91-11-9312812007. E-mail: [email protected]

The intracellular environment is highly crowded due to the presence of large amounts of soluble and insoluble biomolecules, including proteins, nucleic acids, ribosomes, and carbohydrates. The process of protein refolding in vitro has been studied extensively as a mean of understanding how proteins fold inside cells. These experiments are, mainly for practical reasons, commonly carried out in simple buffer system of 20-50 mM with low concentrations of protein (~ 1-2 mg/ml) in order to avoid aggregation during the refolding reactions. A major difference between these idealized conditions and those encountered within cells is that the intracellular environment is highly crowded (300-400 g/l) due to the presence of high concentrations of soluble and insoluble macromolecules in the cytoplasm. This has major thermodynamic and kinetic consequences on the properties of macromolecules present in the cell. These effects can be orders of magnitude different from those in the typical dilute solution used to study proteins in vitro. Biochemical equilibrium in a living cell may be quite different from those under idealized conditions. It is therefore surprising that the effects of macromolecular crowding on protein refolding have been mostly neglected with a few exceptions. In this study we have created an artificially crowded environment around ribonulease-A (RNase-A) through the use of dextran, an inert artificial molecular crowding agent. We have used near-UV absorbance to study the effect of macromolecular crowding on the stability of the protein at pH 6.0. We found that the high concentration of dextran stabilizes the protein. We heated the RNase-A at 287 nm in the presence of various concentrations of dextran. We

found that there was no change in Tm on addition of dextran from 0-150 g/l. However, there O O was a slight increase in Tm, 1.7 C and 2.6 C, in the presence of 200 and 250 g/l of dextran, O respectively. We found the maximum increase of 3.7 C of Tm at 300 g/l which happens to be in vivo concentration (300-400 g/l) of crowding agents in the cell. We propose that the high concentration of crowding agent plays an important role in the stability of proteins; however, further experiments are required to generalize the hypothesis.

84 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 27

OXIDATIVE STRESS MANAGEMENT USING GLUTATHIONE COATED ZnO- NPs IN GRAM (+) AND GRAM (-) BACTERIA

* Amit Kumar, Sakina Aamir, Z. A. Ansari, S. G. Ansari IL- 4 Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India 110025, *Email: [email protected]

Oxidative stress is seen as a cause of many disorders in living being though having a significant role in cell signalling. It constitutes a multiple source of free radicals which starts accumulating with age and during the early stage of the disease process and worsen over the course of disease. Therefore study and management of oxidative stress in cells is an ideal candidate for experimental investigations. Nanotechnology is seen to provide better solution to some previously known problems. In this regard the cell culture study on both gram (+) and gram (-) bacteria, E.coli and S.aureus respectively, using bare ZnO-NPs and glutathione coated ZnO-NPs has been performed and the dose– response relationship was characterized. The 5-10 nm spherical structured ZnO nanoparticles were synthesized via sol-gel process using zinc acetate dihydrate and zinc nitrate hexahydrate precursors with various amines respectively. These nanoparticles were characterized using XRD, UV- Vis, FTIR and FESEM. The bare ZnO-NPs show higer anti-bacterial activity by producing oxidative 4.0 stress against the gram (-) E.coli than the gram ZnO with acetate precursor and GSH )

(+) S. aureus. The anti-bacterial activity of e c

n 3.0 a

ZnO-NPs was more from acetate precursor than b control r

o GSH1mM s the nitrate precursor. However, the oxidative b GSH2mM a (

GSH3mM

y 2.0 t i

stress and thus anti-bacterial activity was s n e D significantly reduced when glutathione, an l

a 1.0 c i t

antioxidant, coated ZnO-NPs were used for p both precursors. The minimum inhibitory O 0.0 concentration (MIC) was high for the cell 0 4 8 12 16 20 24 Growth Time (h) walled gram (+) S. aureus while it was lower for the gram (-) E.coli. Keywords: Zinc oxide, Glutathione, Nanostructure, E.coli, S.aureus.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 85 PP- 28 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ENZYME BASED SENSOR FOR DETECTION OF BIOMOLECULES USING DOPED METAL OXIDES

Mazharul Haq, Arun Sharma, Nishat Akhtar, Z. A. Ansari, S. G. Ansari* Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India, *Email: [email protected]

Electrochemical enzyme sensors with high sensitivity, long term stability and low cost detection of specific biological binding events have extensively reduced sampling and testing time for biomolecule detection and high degree of reproducibility than conventional analytical techniques. In this paper biosensing of urea, glucose, cholesterol is presented using

nanostructured doped TiO2. Doped TiO2 was synthesized by hydrothermal method. Sensors were fabricated in the form of thick film using nanostructured powder and their electrochemical properties were measured as function of analyte concentration. Particular units of enzymes were immobilized on the films by physical method for soaking for few hours. In general, conductivity of film increases after enzyme immobilization for urea and glucose detection whereas it decreases for cholesterol. The immobilized films were found sensitive over a wide concentration of analyte. For some of the mateirals, different sensitivity regions are observed viz. (i) lower concentrations; (ii) linear region and a (iii) saturation region with varying sensitivity. Effect of nanostructure on the sensing performance revealed that higher surface area is better for sensor material. The sensor responses were reproducible, reliable, reversible and selective, with a response time of <10 S.

2.4

200 0.12 2.2 After immobilization )

6 - 180 0

1 0.10 ) x After Immobilization

) After Immobilization V I

V 2.0 d d d / / / 160 I I V d d ( d ( (

y 0.08

t y y i

t 1.8 t i v 140 i i v v t i i i t t s i i s s n 0.06 n e n

Before Immobilization e e 1.6 S 120 S S Before Immobilization Before immobilization 0.04 1.4 100

100 200 300 400 500 50 100 150 200 250 300 350 50 100 150 200 Urea Concentration(mM) Glucose Concentration (mg/dL) Cholesterol Concentration (mg/dl)

Urea sensing curves of Cu-TNT Glucose sensing curves of Sn-TiO2 Cholesterol sensing curves of Sn-TNT

86 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 29

STRUCTURAL AND FUNCTIONAL DETERMINANTS OF XIAP MEDIATED ACTIVATION OF PRO-APOPTOTIC SERINE PROTEASE HtrA2/Omi

Nitu Singh, Kakoli Bose IL- 4 Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi-Mumbai. [email protected]

HtrA2 (High temperature requirement A 2) is a mitochondrial serine protease which antagonizes inhibitor of apoptosis proteins (IAPs) thereby activating caspases. Although, HtrA2 was primarily identified as an IAP-binding proapoptotic protein, its other functions such as caspase-independent induction of apoptosis and serine protease activity are poorly characterized. Moreover, recently it has been observed that the IAPs are also substrates of HtrA2. Looking into its complex trimeric three-dimensional structure (N-terminal domain, a serine protease domain and a PDZ domain) and trying to account for its low protease activity and narrow substrate selectivity, researchers hypothesized a model which suggests intricate PDZ-protease coordination, rearrangement in their relative orientations and conformational changes at PDZ-protease interface as prerequisites for substrate cleavage by HtrA2. However, if this model is true, it might hold good for only a subset of interactions since IAPs bind the N-terminal domain. Therefore, in the present study our aim was to understand the structural determinants of N-terminal mediated activation of protease activity of HtrA2 and compare it with the current hypothesis so as to develop a model for its substrate-binding and cleavage. HtrA2 mutant, deficient for IAP binding, showed reduced proteolytic activity as compared to wild type; indicating N-terminal binding of IAPs is crucial for mediating its serine protease activity. Also, studies carried out using different domains of IAPs and HtrA2 variants further provided insights on this novel activation mechanism of serine protease HtrA2/Omi.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 87 PP- 30 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

BIOPHYSICAL CHARACTERISATION OF HAX1, A KEY PLAYER IN THE INTRINSIC PATHWAY OF APOPTOSIS

Raja Reddy Kuppili, Pruthvi Raj .B, Kakoli Bose* ACTREC, Kharghar, Navi Mumbai- 410210

HAX-1 is a ubiquitously expressing protein in murine and human tissues which appears to be predominantly localized to mitochondria. Reports suggesting its overexpression in several human cancers and its mutations in neurodegenerative diseases further emphasize its antagonistic role in apoptosis. Recent literature has reported HAX-1 as a binding partner cum substrate of HtrA2, a mitochondrial pro-apoptotic serine protease suggesting an alternate novel apoptotic pathway. The hypothesis is that this interaction leads to the activation of HtrA2 and subsequent degradation of HAX-1 showcasing an early event in apoptosis while HtrA2 is still localized in the mitochondria. The structural details, stoichiometry and mechanism of HtrA2-HAX-1 interaction and the event that subsequently follows in the mitochondria are still to be elucidated. To attain the objective of studying their interaction in vitro, recombinant HAX1 was expressed as a fusion protein and purified by affinity chromatography. Biophysical studies of HAX-1 helped us determine its secondary and tertiary structures. Apart from this, thermal stability and equilibrium unfolding studies were performed for the protein giving us insights into the unfolding pattern. For better structural clues of HAX1, a model was generated. Owing to its low sequence homology to known proteins threading was performed to generate the model. The experimental validation for this model was done by fluorescence quenching studies using the tryptophan positions predicted by the model as reference. This model was used for in silico interaction studies with HtrA2 to obtain leads for the interaction interface and the residues critical for this interaction. These biophysical studies along with the structural details of the interaction would help dissect the underlying mechanism of HtrA2- mediated HAX-1 cleavage and understand its implications in early stages of apoptosis.

88 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 31

ROLE OF STRUCTURAL PLASTICITY AND INTERDOMAIN CROSSTALK IN REGULATING HtrA2 FUNCTIONS

Lalith Kumar Chaganti, Kakoli Bose IL- 4 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, India. E mail: [email protected]

HtrA2 is a mitochondrial proapoptotic serine protease which antagonizes inhibitor of apoptosis proteins (IAPs) during apoptosis thereby activating caspases. However, it has been recently observed that HtrA2 is capable of inducing apoptosis in caspase-independent manner via its serine protease activity, the mechanism of which needs further characterization. High sequence similarity with its E. coli counterpart HtrA/DegP, suggests that mammalian HtrA2 might be involved in switching between protective and proteolytic responses toward mitochondrial stress. We hypothesize that the intricate oligomeric structure and crosstalk between serine protease and PDZ domains regulate protease activity and its specificity. HtrA2 forms a trimeric structure comprising multiple domains (N-terminal oligomerization, serine protease and C-terminal PDZ domains) which undergo huge conformational changes with temperature and peptide binding. Understanding the structural integrity and subtle conformational changes at PDZ-protease interface and at the active site is critical towards understanding its mechanism of action. Therefore, secondary and tertiary structural properties of various domains and monomeric variants of HtrA2 were studied by spectroscopic probes and their oligomeric properties were determined to elucidate their structure-function relationships. Protease activities of different variants and domains were analyzed to understand how the structural integrity modulates its protease activity and specificity. Overall, these studies suggest that intricate conformational changes and requirement of proper active site conformation do play a role in regulating HtrA2 functions.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 89 PP- 32 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECT OF STRUCTURAL VARIATION ON THE OPTICAL RESPONSE OF TWO DRUG-LIKE MOLECULES IN HOMOGENEOUS SOLVENTS AND BIOLOGICAL MACROMOLECULES

Sujay Ghosh, Amrit Krishna Mitra and Samita Basu Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064

Structure is the most important aspect for the design of a biologically important molecule. Even a very small structural alteration can change or altogether diminish its biological activity. On the contrary, the photophysical behavior alters only very slightly in homogeneous medium. In the current study we have selected two recently synthesized drug- like molecules KTHC-67 & KMTHC-67. The molecules differ only in the presence of a methyl group in KMTHC-67 attached to the nitrogen in the indole ring. Such a small change in structure is expected to cause small changes in the electronic distribution of the molecule as is seen in the optical response of the molecules in homogeneous solvent. Both the molecules are sensitive to the surrounding microenvironment, especially to the presence of H-bond. Such H-bond sensitivity is highly important in binding to biological macromolecules. We have studied the binding in presence of beta-cyclodextrin and also to a transport protein, Human Serum Albumin. Unlike our observation in homogeneous solvents, a significant change has been observed in the optical response of the molecules bound to beta-cyclodextrin and human serum albumin. The fluorescence emission spectra of the molecules undergo blue shift on addition of beta-cyclodextrin. Under the same circumstance, an isosbestic point has been found and also the fluorescence lifetime has been found to change. Fluorescence resonance energy transfer (FRET) has been observed from human serum albumin to the molecules to a good efficiency. The binding has been found to get affected with varying ionic strength of the solvent. Changes in fluorescence anisotropy, circular dichroism, fluorescence lifetime have been observed under same circumstances.

90 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 33

PROTEIN N-HOMOCYSTEINYLATION CAN BE DELAYED BY TREATMENT WITH PROLINE.

Tarun Kumar and Laishram R. Singh, IL- 4 Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi.

Homocysteine thiolactone is a toxic metabolite of homocysteine. It mediates the post- translational incorporation of homocystine into protein. It results in protein N- homocysteinylation by targeting the side chain amino group of lysine residue in protein, which is having very detrimental effects on protein structure and function. This may in turn lead to the pathophysiological effects in human body. It has been shown that N- homocysteinylation results in aggregate formation, enzyme inactivation, amyloid protofibril formation and multimer formation in proteins. Therefore, compounds that can inhibit these processes will be of great use to treat homocysteineuria. Here we have investigated the effect of a large set of osmolytic compounds (low molecular weight compounds accumulated by cells under stress conditions) in inhibiting N-homocysteinylation of horse cytochrome-c. Serendipitiously we discovered that the kinetics of N-Homocysteinylation could be delayed by treatment with proline. The study implicates that proline based drugs could be used for the treatment of homocystinuria.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 91 PP- 34 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

HOMOCYSTEINE THIOLACTONE INDUCES AMYLOID FORMATION IN ALPHA LACTALBUMIN

Gurumayum Suraj Sharma and Laishram R. Singh* Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110 007 *Correspondence: Laishram R. Singh (E-mail: [email protected])

It has been reported that elevated homocysteine levels in blood serum are an independent risk factors for cardiovascular diseases in human. The cytotoxic relevance of the compound is yet to be clearly understood. Besides lipids peroxidation, the compound is also known to cause protein–adducts formation (especially to the lysine residues) bringing about gross conformational changes in the protein structure and thus affecting its functionality and eliciting auto-immune response. Here in our present study, we discovered that homocysteine thiolactone induces amyloids formation in Alpha Lactalbumin. However, other protein having similar Lysine and Arginine residues are neither aggregated nor converted to amyloids. The results therefore indicate that homocysteine-induced amyloids are protein specific. Taken together, protein aggregation or amyloids formation induced by homocysteine might be the major factor for the cause of artherosclerotic plague.

92 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 35

EXPRESSION, PURIFICATION AND AGGREGATION STUDIES OF HUMAN ISLETAMYLOID POLYPEPTIDE

Saumya Prasad, Jaspal Garg, Vasudevan Seshadri IL- 4 and Rajaram Swaminathan Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India

Protein misfolding, aggregation, and amyloid deposits are the common hallmarks of amyloid diseases, such as Alzheimer's disease and Parkinson's disease. Type 2 diabetes mellitus is one such disease which is associated with the amyloidogenicity of human islet amyloid polypeptide (IAPP). It is a 37 amino acid long polypeptide which is co-secreted along with insulin by the β-cells of the pancreatic islets of Langerhans. IAPP is currently considered the most amyloidogenic peptide, but the molecular basis of its aggregation is still not understood completely. But the detailed characterization of the mechanism of amyloid formation requires large quantities of pure material. Chemical synthesis or recombinant expression and purification of IAPP constitute important steps toward the elucidation of the mechanism of amyloidogenicity. Therefore, this polypeptide along with two of its single amino acid variants were expressed as fusion proteins in E.coli BL21 CodonPlus cells. Along with wild type IAPP, we also decided the bacterial expression of two of its single amino acid variants. The mutants chosen were C7A-IAPP in which the Cysteine at 7th position is replaced by Alanine and Y37W-IAPP in which last Tyrosine is replaced by Tryptophan. We aim to label these peptides with potential fluorescent dyes to monitor the kinetics and aggregation mechanism of the peptide using various biophysical techniques.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 93 PP- 36 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

TAXANE DITERPENOIDS AS MICROTUBULE STABILIZING AGENTS

Umesh Yadava*, Hariom Gupta, Mihir Roychoudhury and Devesh Kumar$ Department of Physics, DDU Gorakhpur University, Gorakhpur-273009 India $Department of Applied Physics, Babasaheb Bheemrao Ambedakar University, Lucknow- 226025 (*Email: [email protected])

Microtubules are formed from the molecules of tubulin, whose dynamics is impartment of many functions in cell, the most dramatic of which is mitosis. Taxol is known to interact within a specific site on tubulin. Taxol is believed to block cell-cycle progression during mitosis by binding to and stabilizing microtubules. Along with the tremendous potential that taxol has shown as an anticancer drug, clinical problem exist with solubility, toxicity and development of drug resistance. The crystal structure of taxane diterpenoids namely, “10,13- deacetyl-abeo-baccatin-IV” (I) and “5-acetyl-2-deacetoxydecinnamoyl-taxinine-0.29hydrate” (II), 7,9-dideacetyltaxayuntin (III) and Taxawallin-K (IV) are very similar to the taxol molecule. These molecules are supposed to be the alternative of Taxol with less side effects. These molecules form cage like conformations which is found to be necessary for binding to tubulin. In the present work, the Molecular docking of these taxane diterpenoids have been carried out with the tubulin alpha-beta dimer (1TUB) and refined structure of microtubule (JFF) in order to assess the potential of tubulin binding of these cytotoxic agents. The Glide module of the Schrodinger suite has been used for molecular docking. Results reveal that molecules I - IV dock into the taxol binding site of tubulin with comparable docking energies to taxol. The docking energy, glide score and hydrogen bonding interactions show that compound I has better tendency of binding with bovine tubulin(1JFF), while II has better binding capability with 1TUB than other diterpenoids.

94 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 37

ROLE OF Ca2+ IN PREVENTING CROWDING-INDUCED AGGREGATION: AN INSIGHT TO NEURODEGENERATIVE DISEASES

Shruti Mittal and Laishram R. Singh* IL- 4 Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi,Delhi- 110 007, India *Correspondence: Laishram R. Singh (E-mail: [email protected])

Biological macromolecules perform their functions in a highly crowded environment (~400 g/l) that consists of a large number of soluble and insoluble macromolecules like proteins, nucleic acids, ribosomes and carbohydrates. It is known that highly crowded conditions induce defective protein folding and/or loss of enzyme activity, and potentiate protein aggregation/amyloid plaque formation relative to dilute solutions (e.g., Tris-HCl). Therefore, any agent that can alter the thermodynamic and structural consequences of the crowding- induced changes on macromolecule will be of good use for a large number of neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease etc). Calcium is a signalling molecule and involved in many biological processes. Here, we investigated the effect of calcium on the crowding-induced aggregation of Holo α-lactalbumin (HLA). We report here that calcium inhibits HLA from aggregation. The study indicates that manipulating calcium concentration inside the cell could serve as a good strategy to many neurodegenerative diseases.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 95 PP- 38 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

OSMOLYTE MIXTURES HAVE DIFFERENT EFFECTS FROM THAT OF SINGLE OSMOLYTES ON PROTEIN FOLDING AND FUNCTION

Marina Warepam and Laishram R. Singh* Dr.B.R.Ambedkar center for Biomedical Research, Delhi University,Delhi-110007 * Correspondence : Laishram R. Singh (E-mail : [email protected])

Osmolytes (small organic molecules) are observed to increase protein stability, correct protein folding defect under stressful situations that which would otherwise leads to defect in protein folding. Four decades of research have covered many landmark discoveries of the effect of each of the single osmolytes on protein folding and stability. In fact, cells under a specific stress conditions accumulate mixtures of osmolytes rather than a single osmolyte. To date, systematic investigation has not been done on the effect of mixture of osmolytes on proteins. Here, using model protein RNase-A, we investigated the effect of various osmolyte cocktails (binary) that are found in the cellular environments. We found out that osmolytes when in a mixture behave differently from that of the single osmolytes. Osmolyte mixtures are quite often synergistic in nature and poorly additive. Such finding lead us to speculate that mixture of different osmolytes may be clinically and proteotoxically important than those of individual osmolytes.

96 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 39

INTERACTION OF N, N-DIMETHYL PYRROLIDINIUM IODIDE IONIC LIQUID WITH BOVINE SERUM ALBUMIN IN AQUEOUS MEDIUM: A THERMODYNAMIC APPROACH IL- 4 Meena Kumari and Rajan Patel* *Corresponding author at: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central University), New Delhi-110025, India. E-mail address: [email protected]

The interaction of N, N-dimethyl pyrrolidinium ionic liquid (IL) with bovine serum albumin (BSA) was investigated by fluorescence spectroscopy. The binding mechanism was determined by applying Stern Volmer Equation. Thermodynamic parameters like entropy (ΔS), enthalpy (ΔH) and free energy (ΔG) change at different temperatures were calculated. Result shows that binding of IL to BSA was spontaneous and hydrophobic forces play a major role in complex formation.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 97 PP- 40 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECT OF DIFFERENT CONCENTRATIONS OF GEMINI SURFACTANT ON PROTEIN CONFORMATION

Muzaffar ul Hassan Mir, Jitendra Kumar Maurya and Rajan Patel* *Corresponding author at: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central University), New Delhi-110025, India. E-mail address: [email protected]

The interaction of the cationic Gemini surfactant with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra. The intensity vs wavelength graph shows the obvious dip in the fluorescence intensity of bsa and a simultaneous shift in the maximum

emission wavelength. The Stern–Volmer quenching constants KSV and the corresponding thermodynamic parameters ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The effect of Gemini surfactant on the conformation of BSA was evaluated by synchronous fluorescence spectroscopy. The results show that the conformation of BSA was changed dramatically in the presence of Gemini surfactant.

98 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 41

EFFECT OF 6 MEV ELECTRON IRRADIATION ON BSA PROTEIN

Sarika Hinge1, S.D.Dhole2,A.G.Banpurkar3,G.R.Kulkarni4 Department of Physics, University of Pune, Pune-411007 IL- 4 Email:[email protected] [email protected]

In the present work, the effect of 6MeV energy electrons on Bovine Serum Albumin protein has been investigated using UV-Visible, FTIR Spectroscopy and Surface Tension Measurements. 6 MeV energy electrons have been generated using the Microtron Accelerator and the respective electron fluence was varied in the range of 5 x1014 e-/cm2 to 10 x1014 e-/cm2.BSA protein samples having area around 4 cm2 were irradiated uniformly with 6MeV electrons. FTIR spectrum of BSA protein was compared before and after electron irradiation. The FTIR spectrum of non irradiated BSA protein shows the peaks at the wave numbers 1326cm- 1,1412.28cm-1,1557.7cm-11,1703.13 cm-1,2371.15 cm-1,2979.13 cm-1,3379.75 cm-1 and 3925.80 cm-1 & transmittance at 22.02, 22.30, 19.24, 10.91, 15.22, 20.36, 19.10 and 29.94 respectively. The FTIR spectrum of 6 MeV electron irradiated BSA protein at the fluence of 5 x1014e-/cm2 and 10 x1014e-/cm2 shows decrease in the percentage transmission. UV-visible absorption spectrum of non-irradiated BSA protein shows the peak at 289nm.Moreover, the UV absorption for BSA protein irradiated at the fluence of 5 x1014e-/cm2 and 10 x1014e-/cm2 show the peak at 239.25nm and the respective increase in the absorbance. Surface Tension of BSA protein solution was measured using Pendant drop method and shows decrease in the interfacial tension with increase in the fluence from to 5 x1014 e-/cm2 to 10 x1014 e-/cm2.It suggest that the electron irradiation causes conformational changes in the protein structure.

Keywords: FTIR, UV-visible, Surface Tension, fluence, Microtron

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 99 PP- 42 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

INVESTIGATION OF NANO- ARCHITECTURE OF BUTTERFLIES USING GONIOMETRIC SPECTROPHOTOBOARD

Ekata Ghate1, K.P.Adhi1, Gauri Kulkarni1 1Department of Physics, University of Pune, Pune -07, India Email: [email protected]

Iridescent feathers of peacock, shiny surface of sea shells and metallic color of some butterflies and beetles are some lustrous ‘biological surfaces ‘.Nano architecture of these biological surfaces is responsible for such brilliant colors and can be investigated by using electron microcopy and spectrophotometer. A spectrophotoboard has been designed for spectroscopic measurements of butterfly wings.The device include rotational stage, goniometric stage, visible range spectrophotometer (USB -4000 Ocean optics, USA) and spectrophotoboard with two fiber optic probe holders. Rotational stage helps in the present study for rotation of the wing sample from 0 degrees to 360 0 with a precession of 10. Goniometric stage is used in the present study for tilting the sample from 00 to 15 0 with a precession of 10 Goniometric optical fiber probe holders are so designed that angle of incidence can be varied from 00 degrees to 900 with a precession of 10 . Spectrophotometric study of Large Oakblue (Arhopala Amantes) butterfly has been carried out in this present study. Goniometric measurements of reflection spectra of butterfly wing reveal shift of wavelength when the wing is rotated or tilted. It has been observed that the maximum value of reflectance also changes when wing is rotated or tilted. Hues of different color are visible when butterfly wing is observed from different angles. Hues of the color and changes in peak value of reflectance are quantified by optical fiber spectrophotometer. Goniometric measurements of reflection spectra is a very important part of this study as measurement of reflection spectra keeping angle of incidence constant is measuring only a part of reflection spectrum reflected from butterfly wing. Keywords: Nano architecture, biological surfaces, reflection spectra, spectrophotoboard

100 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 43

UREA Vs GdmCl INDUCED DENATURATION OF YEAST iso-1-cytochrome c STUDIED BY ABSORPTION AND CD SPECTROSCOPY.

1 1 1 1 Md. Anzarul Haque , Shah Ubaid-ullah , Sobia Zaidi , Md. Imtiayaz Hassan , J. K. IL- 4 Batra2  Faizan Ahmad1 1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi – 110025, India. 2Immuno-chemistry Lab, National Institute of Immunology, New Delhi –1100 67, India E-mail: [email protected]

Cytochrome c is a member of an extended family of heme-proteins involved in electron transfer functions in the mitochondrial respiratory chain, bacterial photosynthesis and apoptosis. More than 270 sequences of cytochromes c have been reported. Multiple sequence alignment suggests that the yeast iso-1-cytochrome c has five extra residues at the N-terminal of the polypeptide chain. In order to investigate the role of these amino acid residues in the stability of the protein, we constructed variants of the protein deleting each amino acid residue. These variants,

-5/-1),
(-5/-2),
(-5/-3),
(-5/-4) and
(-5/-5) were expressed in BL21 and subsequently purified. Urea and GdmCl-induced denaturation of these variants and wild type protein in the native buffer were measured by monitoring changes in absorbance at 405 nm (

405) and CD at 222 nm ([
]222), 405 nm ([]405) and 416 nm 0 ([]416) at temperature 25  0.1 C. It was found to be reversible over the entire concentration range of the denaturant. Using a non-linear least-squares method, the entire data (y(D), [D]) o of each denaturant-induced transition curve were analyzed for GD , m and Cm using the relation (Santoro and Bolen 1988). Comparison of the free energies of denaturation for the all variants except (-5/4) indicate that mutations had a destabilizing effect. These observations will be explained in the light of the known 3-D structure of the wild type yeast iso -1- cytochrome c.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 101 PP- 44 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ELUCIDATION OF UNIQUE INSERTION-BASED MECHANISM OF COMPLEX LIPIDS SYNTHESIS IN Mycobacterium tuberculosis

Renuka RM1, Priyadarshan.K1, Ketan.D.patel1, Priyanka Verma2, Rajesh S. Gokhale2, 3 and Rajan Sankaranarayanan1 1CSIR-Centre for Cellular and Molecular Biology,Uppal Road, Hyderabad -500 007. 2National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067 3CSIR- Institute of Genomics and Integrative Biology, (CSIR), Mall Road, Delhi-110 007.

Insertions and deletions play an important role in evolution by modifying an existing function or creating new functions. The fatty acyl-AMP ligases (FAALs) of Mycobacterium tuberculosis (M.tb) present an interesting case of modification of function in a common acetyl CoA synthetase fold [1]. The structure of N-terminal domain of FAAL28 revealed the presence of a unique FAAL-specific insertion that allowed the diversion of common lipid pool in M.tb towards biosynthesis by polyketide synthases (PKS). The FAAL-specific insertion presents a conserved non-polar interface with the N-terminal domain, a feature absent in insertion-lacking counterpart’s fatty acyl CoA ligases (FACLs) [2]. However, the precise mechanism by which FAALs transfer activated lipids on 4’ PhosphoPantetheine arm (4’PP) of carrier proteins (CP) instead of Coenzyme-A is not known. We are currently carrying out work to decipher the full length structure of several FAALs like FAAL29, FAAL10 and FAAL32. The full length structure will shed light on mechanistic details how FAALs are able discriminate between the phosphopantetheine arm of CP and CoA. The non- polar interface of insertion was used for in silico identification of many FAAL-like proteins in many life forms including Drosophila, Zebra fish, Mouse and Humans. These FAAL-like proteins are being cloned and purified to ascertain their identity by structural and biochemical studies. Knockout studies on these FAAL-like proteins in multiple model systems like Mouse, Zebra fish and Drosophila are being pursued to characterize them functionally. Reference 1. Arora P. et al. Nature Chemical Biology 5, 166 - 173 (2009). 2. Aneesh Goyal. et al. J. Mol. Biol. 416, 221–238 (2012).

102 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 45

STUDY OF A BIOMARKER OF MUTATION AND CANCER: FORMATION OF 8- OXOGUANINE DUE TO REACTION BETWEEN GUANYL RADICAL AND CARBONATE RADICAL ANION IL- 4 Amarjeet Yadav* and P. C. Mishra Department of Physics, Banaras Hindu University, Varanasi – 221 005, India *E.mail: [email protected]

Mechanisms of reactions of four different tautomers of guanyl radical (G(-H1)·, G(-H2a)·, · · .- G(-H2b) and G(-H9) ) with carbonate radical anion (CO3 ) producing the mutagenic product 8-oxoguanine (8-oxoG) have been investigated theoretically using density functional theory and second order Møller-Plesset perturbation (MP2) theory. Geometries of reactant, intermediate and product complexes and those of transition states were optimized at the B3LYP/6-31G(d,p) level and single point energies were calculated at the B3LYP/AUG-cc- pVDZ, BHandHLYP/AUG-cc-pVDZ and MP2/AUG-cc-pVDZ levels of theory in gas phase. Solvent effect of aqueous media was treated by including eight specific water molecules in a hydration cell during optimization at the B3LYP/6-31G(d,p) level and single point energy calculations at other levels employing the polarizable continuum model. It is found that the specific water molecules catalyze proton transfer involving the different sites of the guanyl radical during the reaction. Possible roles of aeration, stirring and photoirradiation of reaction media which are employed to facilitate the reaction in experimental studies are explained. The carbonate radical anion is found to be a very effective reactive oxygen species.

Oxidation

PP-47Product Complex Reactant Complex

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 103 PP- 46 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

TEMPERATURE INDUCED MELTING OF DNA DOUBLE HELIX: AN INSIGHT FROM MOLECULAR DYNAMICS SIMULATION

Sangeeta Kundu1, Sanchita Mukherjee1 and Dhananjay Bhattacharyya2 1 Biophysics Divion Saha Institute of Nuclear Physics 1/AF Bidhannagar Kolkata -700064 2 Computational Science Division Saha Institute of Nuclear Physics 1/AF Bidhannagar Kolkata -700064

Structural polymorphism of DNA plays a crucial role in several biological processes such as replication, transcription, DNA repair, packaging, etc. For the above functions, DNA double helices need to unwind or melt locally. Therefore, understanding the pathway of unfolding is very essential to interpret these biological processes. Considering this we have carried out molecular dynamics (MD) simulations of a DNA double helix of d(CGCGCGCGAATTCGCGCGCG) sequence, where the nucleation might initiate in the A:T rich region. MD simulation of this particular sequence with water and charge neutralizing counter ions at different temperatures ranging from 300K to 500K has been performed by NAMD software using Charmm-27 force field. We would like to investigate the melting signature of this heteropolymeric DNA at different temperatures with an aim to understand the dynamic properties of the oligomer. Estimation of all the structural parameters for the oligomer at different temperatures have been explored that may facilitate to figure out the factors governing thermodynamic stability of this sequence. Analysis of the trajectory reveals that the terminal base pairs start unstacking first. The duration of stable terminal base pairs depends on the temperature of the simulation i.e. at elevated temperature the terminal basepairs melts early. We found the central AT rich region also undergoes melting after 15ns at 460K wheras the terminal base pairs start opening within 3ns. Such simulations of different oligomeric sequences would facilitate us to predict probable melting pathway in a double helical DNA.

104 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 47

INTERFACE PEPTIDE OF ALZEIHMER’S AMYLOID BETA: APPLICATION IN PURIFICATION.

a a b b a a Pokhriyal R , Das U , Ganguli M , Pasha S , Hariprasad G , Srinivasan A IL- 4 aDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi-110029, India bInstitute of Genomics and Integrated Biology, Mall Road, University of Delhi, North Campus, Delhi- 110007, India

A common problem observed during bacterial expression of eukaryotic proteins is that of protein precipitation, where expressed proteins comes in inclusion bodies. This has been resolved to some extent by the use of solubilizing agents. However, the high concentrations of these additives prevent the downstream procedures. If such solubilzing agents can be employed at very low concentrations the recombinant proteins can be used in function-based purification protocols. Sequence based protein-protein interactions can be used for such methods. However, in the event of protein aggregation and higher order structure formation by proteins, the interface peptide sequences are inhibitors. In this study, we have studied how a partial sequence of Alzeihmer’s amyloid beta peptide can inhibit the aggregation. This has been used to advantage for purification of proteins containing the above mentioned sequence.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 105 PP- 48 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

INHIBITION OF PROTEIN AGGREGATION: SUPRAMOLECULAR ASSEMBLIES OF ARGININE HOLD THE KEY.

Komal Ra, Das Ua, Hariprasad Ga, Ganguli Mb, Pasha Sb, Srinivasan Aa aDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi-110029, India bInstitute of Genomics and Integrated Biology, Mall Road, University of Delhi, North Campus, Delhi- 110007, India

The aggregation of unfolded proteins proceeds through interaction of exposed hydrophobic surfaces. Therefore mechanism to prevent their aggregation must explain prevention of such hydrophobic interactions. It is widely known that arginine can be used as an aggregation suppressor; however the mechanism for the same is vaguely understood. Through our methodology and subsequent observations, we propose a mechanism based on hydrophobic interactions of arginine. We have studied arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Using mass spectroscopic analyses, we have observed that arginine forms molecular clusters in the gas phase and the cluster composition is related to the solution conditions. Light scattering studies show that arginine exists as clusters in solution. Another significant observation is the modification of the reverse phase

chromatographic elution profile of Alzheimer’s amyloid beta 1-42 (Aβ1-42) peptide, in

presence of arginine. Modifications in the hydrodynamic volume of Aβ1-42 in the presence of arginine as measured by size exclusion chromatography indicate that arginine binds to

A
1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium and decreases

the aggregation of Aβ1-42 as observed by atomic force microscopy. On the basis of our experimental results we propose that molecular clusters of arginine in aqueous solutions show a hydrophobic surface by the alignment of its three methylene groups. These hydrophobic surfaces on the proteins interact with the hydrophobic surface presented by the arginine clusters. This causes masking of hydrophobic surface which inhibits protein-protein aggregation. The same mechanism is also responsible for the hydrotropic effect of arginine on various compounds.

106 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 49

BIOMARKERS OF OXIDATIVE STRESS AND THEIR ASSOCIATION WITH THE LEVEL OF TOLL LIKE RECEPTOR 9 IN MALIGNANT AND BENIGN BREAST DISEASES IL- 4 Kanchan Karki1, Deepti Pande1, Reena Negi1, Seema Khanna2, Ranjana S. Khanna3 and H D Khanna1 1Institute of Medical Sciences, Banaras Hindu University, Varanasi, India -221005, 2Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India -221005, 3Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, India -221005

Introduction Oxidative stress has been implicated in pathogenesis of breast diseases. The objective of the study is to evaluate the biomarkers of oxidative stress and its correlation with the serum concentration of Toll like receptor 9 (TLR 9) in breast diseases and controls. Material & Method 51 patients with breast carcinoma at different clinical stages, 20 patients with benign breast tumor, and 20 healthy subjects were recruited into the study. Levels of TLR 9, 8-hydroxy-2 deoxy Guanosine (8-OH-dG), Protein carbonyl (PC), and Malonaldyhide (MDA) along with total antioxidant status (TAS) level were measured in the serum of patients of breast diseases and controls. Receiver operating characteristic (ROC) analysis was done to study the diagnostic potential of TLR 9. Result & discussion Significantly increased levels of TLR 9, 8-OH-dG, PC, MDA and significantly decreased level of TAS were observed in breast carcinoma patients in comparison to the benign and normal control groups. TLR 9, 8-OH-dG, PC and MDA were positively correlated with disease staging but negatively correlated with total antioxidant status. As exhibited by ROC analysis TLR 9 is a good indicator of cancer presence. Conclusion Our results indicate that elevated levels of oxidative damage biomarkers are significantly associated with breast cancer. High accuracy of TLR 9 in indicating cancer presence can be used as discriminatory marker for efficient diagnosis.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 107 PP- 50 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IN SILICO STRUCTURE-FUNCTION ANALYSIS OF PATHOLOGICAL VARIATION IN THE FAS1 DOMAIN 4 OF TGFB1 GENE SEQUENCE

Umay Kulsum, Khurram Mushtaq, Divya Dube, Preeti Paliwal, T.P. Singh, Arundhati Sharma and Punit Kaur Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110 029, India

A progressive alteration of the cornea resulting in loss of transparency occurs in a set of hereditary diseases known as Corneal Dystrophies (CD). A number of these dystrophies have been linked to mutations in TGFBI (kerato-epithelin) also called as Transforming growth factor-beta-induced protein BIG-H3. However, the mechanism by which the mutations cause this disease is still unknown. To screen a cohort of corneal dystrophy patients from North India for mutations in the transforming growth factor beta induced (TGFBI) gene, the genomic DNA was isolated and screened for mutations by polymerase chain reaction (PCR) and direct DNA sequencing. The structural basis for the corneal dystrophies caused by mutations in the beta (ig)-h3 protein was investigated. Protein modeling studies on the wild type and TGFBI mutant structures were carried out to determine the effect of mutations in the overall geometry and folding of the protein. The stability and odds of misfolding in the mutant structures was also examined. Protein molecular dynamics (MD) simulations were performed to assess the structural changes in mutants. Out of total 44, 10 mutations from two region comprising residues 623 to 628 and 537 to 542 are present in the mid region (core) and alter the protein stability. Remaining 32 mutations are present in the peripheral regions and are more likely to interfere with the proper secretion of the protein or implicated in affecting the quaternary association of the protein with that of other proteins.

108 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 51

A SYSTEMATIC ANALYSIS OF PREDICTING NEW MOLECULAR TARGETS FOR RETT SYNDROME USING CYTOSCAPE.

Parul Sharma, Seema Singh, Supriya Dixit, T.P. Singh, Punit Kaur IL- 4 Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110 029, India

Rett syndrome is a neurodevelopmental disorder of the grey matter of the brain that exclusively affects females. Mutations in the methyl-CpG-binding protein 2 gene (MECP2) found on the X-chromosomes is the major cause of Rett Syndrome. Only few drugs with lower efficacy have been reported in the literature, also not many drug targets are known for the Rett syndrome. Moreover there exists a lack of complete knowledge of the gene Co- Expression network and pathogenesis for Rett Syndrome. System networks that are a central paradigm in biology help us identifying new drug targets which in turn can generate more in- depth understanding of the mechanism of diseases. We have implemented a computational platform that integrates gene-gene interactions, differentially expressed genome and literature mining data to result in comprehensive networks for drug-target identification. We used Cytoscape and its various plugins for prediction of the probable drug targets, to study the expression of genes in various biological processes and to identify highly interconnected clusters of genes. Five interconnected high scoring clusters of genes were found using AllegroMCODE which depicts the importance and functionality of a subnetwork in the network. The highest ranking subnetwork was found to have 6 genes including FXYD1, MECP2, CDKL5, FKBP5, GDI1 and NTNG1 which are known to cause Rett syndrome, and can play a major role in the pathogenesis for this disease. Analyzing the topology of the network we have detected the 5 potential drug targets by using Cytoscape. The probable drug targets were found using Cytohubba. The gene products which were found to be the potential drug targets for this disorder are HDAC1, MECP2, SIN3A, DNMT1 and RCOR1. In this work we presented an integrated approach to predict targets for Rett syndrome by exploring and integrating the information from the genomic space.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 109 PP- 52 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

INTERACTION OF HUMAN ACYL CARRIER PROTEIN DOMAIN WITH THIOESTERASE DOMAIN USING NMR SPECTROSCOPY

Pinakin K. Makwana, Ambrish Kumar and Monica Sundd National Institute of Immunology, New Delhi-110067. E-mail: [email protected]

Human fatty acid synthase (FAS), the multienzyme complex, which governs the synthesis of fatty acid chains, is an important target for anti-obesity and anti-cancer drugs. In the complex, the acyl carrier protein (ACP) is a central player that carries and protects the growing fatty acid chain. The ACP molecule is initially synthesized as apo-ACP (inactive) and is post-translationally modified to the holo-ACP (active) by the acyl carrier protein synthase which attaches a phosphopantetheine group to a serine side chain. Various acyl intermediates are formed during the biosynthetic process by the elongation of the acyl chain bound to the phosphopantetheine moiety of the holo-ACP. The acyl chain is released finally

by the thioesterase domain once a certain length has been achieved viz. C16, C18. The molecular mechanism by which the thioesterase domain interacts with the acyl intermediates, recognizes the length of the acyl chain and cleaves it while it is still tethered to the ACP molecule is still a mystery. Therefore, structural studies are underway to understand the mechanism of interaction of the human ACP domain with the thioesterase domain using NMR spectroscopy.

110 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 53

THERMODYNAMIC INVESTIGATION ON THE BINDING OF THE PLANT ALKALOIDSBERBERINE AND PALMATINE TO SERUM ALBUMINS

Asma Yasmeen Khan and Gopinatha Suresh Kumar IL- 4 Biophysical Chemistry Laboratory CSIR-Indian Institute of Chemical Biology Kolkata 700 032 Email:[email protected]

A study of structural and energetic aspects of drug-protein interactions provide a fundamental understanding of drug action at molecular level. The isoquinoline alkaloids, berberine and palmatine have potential role in medicinal chemistry due to their wide spectrum of demonstrated therapeutic utilities. The thermodynamics of the interaction of these two pharmaceutically important alkaloids with bovine and human serum albumin was investigated using calorimetric techniques, and the data was supplemented with fluorescence and circular dichroism studies. The thermodynamic results show that there is only one class of binding for both alkaloids on BSA and HSA. The equilibrium constant was of the order of 104 M-1 for both the alkaloids to serum albumins but the magnitude was slightly higher for HSA. Berberine showed higher affinity over palmatine to both proteins. The binding was enthalpy dominated and entropy favoured for both the alkaloids to BSA and HSA. Salt dependent studies suggested that electrostatic interaction had a significant role in the binding process that reduced at higher salt conditions. Temperature dependent calorimetric data yielded heat capacity values that revealed the involvement of different molecular forces in the complexation of the two alkaloids with BSA and HSA. 3D fluorescence, synchronous fluorescence and circular dichroism data suggested that the alkaloids changed the conformation of the proteins on binding to them by reducing their helicity. Destabilization of the protein conformation was also confirmed from differential scanning calorimetry studies. Overall, the results suggest berberine to be a better binder than palmatine to both serum proteins.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 111 PP- 54 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURAL STUDIES OF ACYL-COENZYME A BINDING PROTEINS (ACBP) OF Leishmania major

Richa Arya1, Suman Kundu1 and Monica 1Department of Biochemistry, University of Delhi South Campus, New Delhi-110021, 2National Institute of Immunology, New Delhi-110067. E-mail: [email protected], [email protected]

Leishmaniasis is a major public health concern with no effective vaccine available till date. Given the fact that the co-infection of HIV and Leishmania has rendered most of the commercially available drugs ineffective, it is necessary to understand essential biochemical pathways of this pathogen and identify new drug targets that can selectively inhibit the growth of the pathogen. Biochemical studies have shown that trypanosomatids synthesize ergosterol and other 24-methyl sterols that are indispensable for their growth and viability. These sterols are absent in the mammalian cells. Therefore, the sterol biosynthetic pathway from Leishmania could serve as an excellent target for drug design. Our focus is on a class of proteins, called acyl coenzyme-A binding proteins (ACBPs), that have the ability to bind acyl coenzyme-A. ACBP mainly functions as an intracellular acyl-CoA transporter and pool former, and is critical to lipid metabolism in cells. The ACBPs in Leishmania have not been characterized yet and thus presents immense scope for investigation. Our study involves the characterization of ACBPs (six in Leishmania major) in order to understand the expression, localization, structure-function relationship of these carrier proteins in Leishmania using biophysical techniques (Fluorescence, CD, NMR), biochemical assays as well as in vivo studies. Some recent results obtained will be presented and discussed.

112 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 55

THE Na-K-ATPase ACTIVITY IN RESPONSE TO EXOGENOUS ESTRADIOL ADMINISTRATION IN AGEING RAT BRAIN

ab a b b a Rashmi Jha , Priyanka Mishra , A.AMahdi , Shivani Pandey , Najma Z Baquer IL- 4 and Sudha M Cowsika aSchool of Life Sciences, Jawaharlal Nehru University, New Delhi-110067 bKing George’s Medical University, Lucknow, Uttar Pradesh-226021

The Na-K ATPase is an enzymatic activity responsible for the active transport of Na+ and K+ in most eukaryotic cells. Na-K-ATPase activity decreases with ageing in female rat brain synaptosomes. In the present study, we observed the in vitro effect of Tachykinin neuropeptide NKB, Amyloid Beta (25-35) and combined NKB and Aβ (25-35) on 17β estradiol (E2) treated synaptosomes isolated from ageing female rat brain synaptosomes of 3 months (young), 12 months (adult) and 24 months (old) age groups. An in vitro incubation of isolated synaptosomes with Aβ (25–35) showed toxic effects while NKB showed stimulating effect on the Na-K-ATPase activity and the combined NKB+Aβ (25–35) incubations showed a partial effect as compared to the Aβ (25–35) alone. It has been well documented that estradiol increases Na-K ATPase activity. To understand whether E2 affects the expression of Na-K-ATPase molecules, we examined the expression of Na-K-ATPase in ageing female rat brain synaptosomes. For this purpose, α1 and β2 Na-K ATPase monoclonal antibody was used to label Na-K ATPase molecules. The enzyme was quantified by SDS PAGE and western blot analysis in control and estradiol treated rat brain. We observed that the expression of α1 and β2 Na-K ATPase molecules increased and reversed to normal level in estradiol treated synaptosomes. These results confirmed that estradiol increased turnover of Na-K ATPase molecules in ageing rat brain. The present findings suggest a possible role of NKB with 17βestradiol (E2) in the age related changes in the brain.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 113 PP- 56 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ANTI-INFLAMMATORY AND ANTI-FUNGAL NOVEL PROTEIN FROM ALOE VERA: ISOLATION AND CHARACTERIZATION OF LOW MOLECULAR WEIGHT PROTEIN FROM GEL

Suresh Kumar, Kusum Singh, Kamaldeep Gill , Swagata Das, Mou Sinha, Sujata Sharma, Tej. P. Singh and Sharmistha Dey. Department of Biophysics, All India Institute of Medical sciences, New Delhi, India.

Aloe vera, also known as the Medicinal Aloe, is a species of succulent plant that probably originated in northern Africa with numerous medicinal properties. Since all succulents have self-repairing properties, Aloe vera also possesses the property of healing an injury or wound by simply making a water seal. Various polysaccharides with strong immunomodulatory and antitumour properties, phytosterols, lectins etc and invitro antibradykinin activity have been reported in the leaf gel of the Aloe vera. But till date no protein having anti-microbial (anti- fungal) property has been isolated from the Aloe vera gel. We have identified and purified a novel anti-fungal and anti-inflammatory protein with a molecular mass of 14 KDa from the Aloe vera leaf gel. The isolation procedure involved ion exchange chromatography using DEAE-cellulose and affinity chromatography using Affi-gel blue gel with 10 mM Tris-HCl buffer (pH 8.5)in both the steps. The unadsorbed protein fraction from DEAE-cellulose was loaded on Affi -gel blue gel column, from which the elution of adsorbed protein was done with the different concentration of sodium chloride ().1-1.0M. The purified protein of 14 kDa exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example Candida paraprilosis, Candida krusei and Candida albicans. In addition, the purified protein also showed anti-inflammatory properties against lipooygenase enzyme in the presence of substrate linoleic acid. The percentage inhibition was 75 %. This anti- inflammatory property was also verified by binding pure protein with the immobilized human protein Lipoxygenase and Cyclooxygenase-2 on sensor chip CM5 in the BIAcore.

114 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 57

CLONING, EXPRESSION AND PURIFICATION OF HUMAN TRUNCATED SIRT1

Rahul Kumar, Abhishek Gupta and Sharmistha Dey Department of Biophysics, All India Institute of Medical Sciences, IL- 4 New Delhi, India

Sirtuin or silence information regulator comprises an universal conserved family of NAD+ (Nicotinamide adenine dinucleotide) dependent deacetylases found in bacteria to human and play a role in a wide variety of important biological processes including development, heterochromatin formation, transcriptional silencing, DNA recombination and repair, genomic stability, apoptosis, axonal protection, fat metabolism, metabolic regulation and longevity. The anti ageing effects of human homologous of sirtuins are also seen by animal and human associated studies.SIRT1, the most studied of 7 sirtuin family members, is a key mediator of the pathways downstream of calorie restriction, dietary regimen that is known to delay the onset of ageing and reduce the incidence of age related diseases. The conserved part of amino acids residues 244-498 of full length human SIRT1 have the functional activity. To construct a functional form of SIRT1 by DNA recombinant technology, we have expressed and purified truncated protein of 420 (193 to 611) amino acids of MW 47 kDa in prokaryotic system, Ecoli BL21 (DE3) strain. The primers were designed based on the sequence of human SIRT1 mRNA sequence which was obtained from NCBI (NM_012238) of 4110 bp .The cloned gene includes an ORF of 1260 bp encoding 420 amino acids residues. The protein was characterized by mass spectroscopy, SDS PAGE and Western Blot was found to be of 51 kDa and pI value of 5.1. The specificity of protein was identified by using its specific antibody by western blotting and SPR. This functional protein has a potential to be used as a diagnostic tool and for the development of new therapeutic agent for age related disease.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 115 PP- 58 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

G-QUADRUPLEXES: INCREDIBLE TOPOLOGY

Anju Singh, Mahima Kaushik, Savita Joshi and Shrikant Kukreti Department of Chemistry, University of Delhi, Delhi-110007 [email protected]

Structural Multitude of nucleic acids serves basis for its multiple merits and varied applications. One of such structures are G-quadruplexes which are formed by intramolecular or intermolecular folding of the stretches of G-rich sequences. There are ample numbers of evidences supporting the involvement of these structural motifs in vital cellular processes such as transcription, translation, recombination etc. With the help of the knowledge of stability and strucutural polymorphism exihibited by G-quadruplexes in in vitro condition along with the widespread occurrence of putative G-quadruplex forming sequences in genome, it is easy to predict their in vivo existence and to understand their biological implications. To observe the general trends with respect to the influence of G-tract length and loops on structure and stability of G- quadruplex structures, we studied oligodeoxynucleotides with stack of G5 and with different number of T bases in loop. In the present study we have used Circular Dichroism, Native gel electrophoresis and UV-monitored thermal denaturation techniques in order to establish the

topology of putative quadruplex forming sequences - TG5T (1-4) G5T and its extended version

[(TG5T (1-4) G5)2T], under different ionic condition The major changes in topology were observed for the short G-stretches with respect to longer one. Surprisingly, the former sequences (with different no. of Ts in loop) was shown to forms parallel as well as antiparallel G-quadruplex structures in presence of Na+, whereas in K+ ion they form only parallel species of varied molecularity. The extended version with single T in loop, also behaved differently in presence of different cations. Gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. This report envisages the possibility of the formation of various forms of G-quadruplex structures with varied and unusual topologies. References: 1. M. Kaushik, A. Bansal, S. Saxena, S. Kukreti, Possibility of an Antiparallel (Tetramer) Quadruplex Exhibited by the Double Repeat of the Human Telomere. Biochemistry,(2007)46: 7119 – 7131.

2. Marko Trajkovski,† Mateus Webba da Silva,‡ and Janez Plavec*,†,§, Unique Structural

∥

Features of Interconverting Monomeric and Dimeric G-Quadruplexes Adopted by a Sequence from the Intron of the N-myc Gene, J. Am. Chem. Soc. 2012, 134, 4132−4141.

116 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 59

TRIPLEX FORMATION AT C-ALLELE OF A COMMON PROMOTER VARIANT - 765 G > C (rs20417) OF COX-2 GENE: A POSSIBLE CAUSE FOR SIGNIFICANTLY REDUCED PROMOTER ACTIVITY IL- 4 Swati Mahendru, Kapil Roy, Mohan Kumar and Shrikant Kukreti Department of Chemistry, University of Delhi, Delhi-11000788 [email protected]

Complexity of highly evolved organism is not reflected in the number of genes but in the higher percentage of non-coding part of genome. The human genome would be nearly hollow if one were to remove the non-coding sequences from the genome. Time and again it is being increasing demonstrated that the sequences that were termed JUNK could actually be “Just Unexplored Noncoding Knowledge”. This non-coding region harbour a large number of regulatory sequences like promoters, enhancers, transcription factor binding sites, silencers etc. that direct the expresion of a particular gene. Single nucleotide polymorphisms (SNPs) have been hailed as the most common polymorphism and believed to be responsible for individual’s response to environment and risk of disease. COX-2 (cyclooxygenase-2), the inducible isoform of COX-1, plays a crucial role in oncogenesis. Many risk factors are accused in the causation of diseases such as cancers of the stomach, oesophagus and prostate, and asthma, heart attacks and stroke especially a common variant -765G>C of COX2 gene. In the present work, we used a combination of UV-spectroscopy, Gel electrophoresis and Circular Dichroism to study the stability of triplex formed between a 57-mer duplex having -765 G>C polymorphic site and a triplex forming oligonucleotide which exist in the 3`-flanking region of COX-2 gene. The biophysical and biochemical attempts suggest that triplex formd in case of C-allele is much stronger than that in G-allele. Such triplex formation could be responsible for significantly reduced activity of COX-2 promoter and can be further used for gene silencing via anti-gene stategy. References: 1. He J.; Zhang Q; Ren Z; Li Y; Li X; Zhou W; Zhang H; Meng W; Yan J; He W. Cyclooxygenase-2 -765 G/C polymorphisms and susceptibility to hepatitis B-related liver cancer in Han Chinese population. Mol Biol Rep (2012) 39:4163–4168. 2. Rizzo M.T. Cyclooxygenase-2 in oncogenesis. Clinica Chimica Acta (2011) 412: 671–687. PP-61

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 117 PP- 60 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

BINDING PREFERENCES OF HEMAGGLUTININ PROTEIN FROM INDIAN STRAINS OF AVIAN INFLUENZA A/ H5N1 TO AVIAN/HUMAN RECEPTORS USING DOCKING AND MOLECULAR DYNAMICS SIMULATION

Abhisek Behera, Pratip Shil, Sarah Cherian Bioinformatics Group, National Institute of Virology, Pune 411001

Influenza A viruses are pathogens that cause significant morbidity and mortality in humans and a variety of animal species. Among the Avian Influenza virus subtypes, highly pathogenic avian influenza (HPAI) A H5N1 viruses continue to pose a serious threat to global public health especially as they may cross the species barrier and adapt to human hosts. Of the two glycoproteins, hemagglutinin (HA) and Neuraminidase (NA) on the viral envelope, the HA protein is responsible for viral binding to host receptors and enabling entry into the host cell through endocytosis. The HA binds to receptors containing glycans with terminal sialic acids and their precise linkage determines the species preference. Binding preference of HA to sialosaccharides is an important area for understanding the amino acid mutations that could be the cause for avian-to-human transmission. India experienced the first outbreak of HPAI H5N1 in poultry during January-April 2006 in Maharashtra and Gujarat while between 2008-2009 wide spread infection of influenza A/H5N1 was reported in north-eastern states. The present work aims at understanding the binding preference of HA of Indian strains of Influenza A/H5N1 to sialic acids connected to galactose in α2-3 linkages (avian receptors) and to α2-6 linkages (human receptors). The 3D structure for HA protein of Influenza A / H5N1 Indian strains of 2006 and 2009 and a strain from West Bengal of 2008 possessing a S221P mutation in the receptor binding domain were predicted using the homology modeling protocol. Molecular docking of both Sialic acid α2-3 / α2-6 Galactose to the HA protein was carried out using GOLD package. Protein- ligand interactions and binding stability were evaluated in terms of GOLD fitness function and ligand binding energy. Stability of protein-ligand complexes in the presence of water were also tested using molecular dynamics protocol as implemented in the YASARA package. Analysis revealed that the vital difference between avian and human receptors were their binding that possessed TRANS and CIS conformations respectively to the HA protein. The Influenza strain possessing the S221P mutation indicated increased binding to α2-6 linked sialic acids when compared to the binding of strains that did not possess the HA-221 mutation. However further studies are required to fully understand the importance of mutations at residue position 221 in converting the HAs of H5N1 viruses known to recognize the avian receptor to ones that would enhance recognition to the human receptor.

118 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 61

MOLECULAR ELECTROSTATIC POTENTIAL (MEPS) CALCULATIONS OF 5 2 5 2 BASE PAIRMODELS OF ΤM S U34:G3 AND ΤM S U34:A3 OVER RM1 PREDICTED MOSTSTABLE STRUCTURE OF 5-TAURINOMETHYL-2-THIOURIDINE 5 2 (ΤM S U). IL- 4

Asmita D. Kamble1, Bajarang V. Kumbhar1, Susmit B. Sambhare1 And Kailas D. Sonawane1 Structural Bioinformatics Unit, Department of Biochemistry, Shivaji University,1Kolhapur-416 004, (M.S.) India.

Modified nucleosides are present in the anticodon loop of Transfer ribonucleic acids (tRNAs). Transfer RNAs are essential molecules required in protein biosynthesis process. Posttranscriptional modifications present at the wobble position of the tRNA anticodon participate precisely in decoding of the genetic code. tRNA contains various modified nucleosides instead of four standard bases. Such modified nucleosides occur more frequently at the (34th) ‘wobble’ position and at anticodon 3’- adjacent (37th) position of anticodon loop in tRNA of various species. The hypermodified nucleoside 5-taurinomethyluridine (τm5U) and its derivative 5-taurinomethyl-2-thiouridine (τm5s2U) present at the 34th ‘wobble’ position in the anticodon loop of mammalian mitochondrial tRNA. The modified nucleoside τm5s2U present in anticodon loop of tRNALys, tRNAGln and tRNAGlu. Lack of such taurine- containing uridines in mutant mitochondrial tRNAs causes MERRF. Hence, Molecular electrostatic potential (MEPs) calculations and single point energy calculation studies of base 5 2 5 2 pair models of τm s U34:G3 and τm s U34:A3 have been performed over RM1 predicted most stable structure of 5- taurinomethyl-2-thiouridine (τm5s2U). The single point energy calculations have also been made over the three different proposed deprotonated models I, II 5 2 Lys 5 2 and III of τm s U34:G3. The results show that modified tRNA containing τm s U modification at 34th position participate in recognition of correct codons AAG than AAA.

5 2 5 2 Keywords: tRNA, Modified nucleosides, τm s U34:G3, τm s U34:A3, RM1 and MEPs.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 119 PP- 62 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IN VITRO VIRAL GENES KNOCK-DOWN BY PNA USING GOLDNANOPARTICLES- AN ANTIVIRAL STRATEGY FOR A RNA VIRUS

V.G. Joshi, A.P. Sahoo, A.K. Singh, V.D. Dighe, A.K. Tiwari and Satish Kumar CIF Bio-Engg., Division of Veterinary Biotechnology, I.V.R.I., Izatnagar 243122 (UP)

Designing a rational delivery mechanism for effective antiviral strategy is gaining importance in post genomics era. Use of nanomaterials for improved delivery of biomolecules for antigene interventions is at prime focus especially use of gold-nonmaterial’s based delivery designs in different formats. Peptide-nucleic acids (PNAs) are DNA mimetics and charged neutral molecules thus have high binding affinity towards complementary gene targets. PNAs relating to poly-purine/poly-pyrimdine stretches in gene/DNA can form a stable duplex and triplex. Despite PNAs high clamping abilities to target gene its use as antigene molecule met with limited success primarily due to poor cellular uptake. PNA can be delivered in to cell either by covalent conjugation with peptide or with non-covalent peptide-PNA interactions. Earlier design however robust, have drawback as peptide may interfere with PNA activity and in later lower delivery efficiency is major concern. In present study we demonstrate a simple GNP based cellular delivery of PNA for having both cytoplasmic and nuclear deliveries as observed by confocal-microscopy. We evaluated antiviral efficiency of PNA delivered trough this mechanisms in vitro virus culture systems using Newcastle disease virus as a model for RNA virus. We have prepared PNAs targeting F-gene of Newcastle Disease Virus (NDV) and evaluated them successfully using GNPs based delivery. NDV is fetal disease of poultry affecting all species of birds. Outbreak of NDV causes heavy economical losses as mortality rate of disease is 80% and above. We observed 6 fold reduction of viral HA titer and 75% reduction in F gene expression in presence of PNA as antiviral intervention in comparison with virus control. This study provides an alternative to make use of GNPs as possible carrier for PNAs without affecting their antigene activity, similar to non-covalent peptide based delivery used by us. The modification of GNP-PNA by incorporating molecular handles can provides better strategy for drug homing in specific tissues.

120 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 63

UNDERSTANDING EFFECT OF TRANSPORTERS ON RICE METABOLISM

Rahul Shaw and Sudip Kundu Department of Biophysics, Molecular Biology & Bio-informatics, IL- 4 University of Calcutta

An eukaryotic cell has different compartments which are specific to different biological activities. The total cellular metabolism of a cell is also compartmentalized. The inter- compartment transporters within a cell are responsible to transport the products of one compartment to other. The main aim of this work is to formulate a model to understand the utility of different transporters and apply this in a real case. Here, we have taken a partially compartmentalized genome scale metabolic model of rice (developed in our group). Depending on the gene-expression, the transporters available to transport the metabolites from one compartment to other would change. We studied the effect of transporter's capacity on the overall metabolism. We find that depending on the effectiveness of transporters, the photon demand for a rice leaf's biochemical machinery to synthesize the necessary biomass from inorganic nutrients, changes upto two fold. We also observe, some mitochondrial and chloroplastid reactions are associated with this change.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 121 PP- 64 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SEQUENCE AND 3D STRUCTURE BASED ANALYSIS OF TNT METABOLIZING PROTEINS IN Arabidopsis thaliana

Amrita Bhattacherjee, Rahul Shubhra Mandal, Santasabuj Das, Sudip Kundu Department of Biophysics, Molecular biology and Bioinformatics, University of Calcutta Biomedical Informatics Centre, National Institute of Cholera and Enteric Diseases

In spite of its very recent introduction to natural environment, many plants (along with various bacterial taxa) have been found to possess the ability to metabolize TNT either completely or at least to a less toxic form. The metabolism of TNT has been widely studied in Arabidopsis and has been found to begin with its conjugation to an glucose molecule by the enzyme UDP Glucosyl transferase(UDPGT). Although the details of the pathways are still being elucidated in different plants the conjugation step is assumed to be common to all of them. Interestingly, while Arabidopsis genomes contain more than 100 UDPGTs extensive experimental analysis suggests the role of only 5 of these UDPGTs in the TNT metabolic process. In addition to reviewing genome wide expression and gene architecture of these UDPGTs the present work attempts to identify sequence and structural signatures that discriminate the TNT metabolizing UDPGTs from others Arabidopsis UDPGTs. Homology modeling for the structural prediction for both TNT degrading and non degrading is done along with docking for the probable binding site of TNT in formerly mentioned proteins. The difference of interaction with the TNT molecule also be presented.

122 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 65

VIRUS STRUCTURE REMODELING FOR DRUG DELIVERY

Saumya Bajaj and Manidipa Banerjee Kusuma School of Biological Sciences, IIT Delhi IL- 4

The capsids of non-enveloped viruses are, in effect, stable protein shells which incorporate the properties of safe packaging, penetration within cellular membranes and swift disassembly. As such, they are ideal for delivering therapeutic biomolecules to specific cellular locations. We are attempting to develop a non-enveloped insect virus, Flock House Virus (FHV) into a nano-container for targeted delivery of chemotherapeutic drugs to tumor cells. The advantages of using FHV as a delivery vehicle are numerous - it is non-infectious to humans, genetically tractable and undergoes programmed disassembly in animal cells. Utilizing baculovirus-mediated expression in insect cell line Sf21, we have engineered modified versions of FHV, which have tumor-homing peptides (peptides that bind to growth factor receptors over-expressed in tumor cells) engineered onto the capsid outer surface. These particles are being characterized for proper structural assembly, stability and cell penetration ability. In parallel, we have expressed and purified FHV capsid protein from bacterial expression systems and are attempting in vitro assembly of the protein into virus- like particles. Our ultimate goal is to specifically target modified particles loaded with chemotherapeutic drugs like doxorubicin to tumor vasculature. It is hoped that FHV-based nanoparticles can be eventually utilized to target anti-cancer therapeutics specifically to diseased cancerous tissue, sparing normal non-diseased cells, and reducing side effects of chemotherapy.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 123 PP- 66 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

INVESTIGATIONS OF CHRONIC LEAD TOXICITY ON BRAIN ENERGY METABOLISM USING 1H-[13C]-NMR SPECTROSCOPY.

K. S. Varadarajan, Anup Nirmal Chugani, Puneet Bagga and Anant Bahadur Patel Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India 500007

Lead is a toxic heavy metal, shown to inhibit the Ca2+ dependent release of acetylcholine, dopamine and amino acid neurotransmitters and increases their basal level even at low levels of exposure (Needleman 2004). Neurotoxic actions of lead include apoptosis, excitotoxicity, neurotransmitters storage and release, and damage to the astroglia and oligodendroglia (Lidsky et al 2003). These findings suggest that exposure to lead will alter the level of neurotransmitters and cerebral metabolism involving glutamate and GABA pathways. In this study, we have investigated the effect of chronic exposure of lead on the regional cerebral glutametergic, and GABAergic neurotransmitter functions in mouse model by using 1H- [13C]-NMR spectroscopy. C57BL6 mice (2 month age) were divided into two groups: Control (n=6) and Lead (n=6). Lead group was exposed to lead acetate (500 ppm/day for 60 days) added in drinking water while the control mice received sodium acetate. For metabolic study, overnight fasted mice were intravenously administered with 13C labeled glucose for 10 min (Tiwari and Patel

2012). At the end of the infusion, brain was then frozen in liquid N2 and metabolites were extracted from frozen tissue obtained from different brain regions. Concentration and 13C labeling of metabolites were measured using 1H-[13C]-NMR spectroscopy (de Graaf et al 2003). Level of neurometabolites remains unperturbed with lead exposure. 13C Labeling of glutamate-C4, glutamine-C4 and GABA-C2 is reduced in cerebral cortex with lead exposure. However, other regions exhibit reduction in the labeling of glutamate-C4 and glutamine-C4 only. These findings suggest that chronic exposure to lead reduces the excitatory activity in all the brain regions while inhibitory function is perturbed only in the cortical region. References: Needlemam H (2004) Annu Rev Med 55:209; Lidsky et al (2003) Brain 126:5; Tiwari et al (2012) J Alzheimer's Dis 102:5588; de Graaf et al (2003) Magn Reson Med 49:37. Acknowledgment: This study is supported by funding from CSIR-CCMB

124 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 67

INVESTIGATIONS OF THE GLUTAMATERGIC AND GABAERGIC FLUXES DURING NORMAL AGING IN MICE

Pandichelvam Veeraiah, Mohammad Shameem, Vivek Tiwari and IL- 4 Anant Bahadur Patel NMR Microimaging and Spectroscopy, Centre for Cellular and Molecular Biology, Council for Scientific and Industrial research, Uppal Road, Hyderabad, India

Normal aging is associated with a decline in brain function, including cognitive, memory and sensory processes1. Although change in mitochondrial function have been implicated in age- related neurodegenerative diseases and in the loss of cognitive function with healthy aging 2, its significance at the cellular level is not fully understood with normal aging. As glutamatergic and GABAergic neurotransmitter energetic is supported by oxidative glucose metabolism, healthy aging will lead to impairment in these pathways. In this study we have investigated TCA cycle and neurotransmitter cycle fluxes associated with glutamatergic and GABAergic neurons in cerebral cortex of young adult and aged mice. Two groups of C57BL6 mice, young adult (6 month) and aged (24 month), have been used for the study. Mice were administered with 13C labeled glucose via tail vein for different period of time. Concentration and 13C labeling of cortical metabolites were measured in tissue extracts3 by 1H-[13C]-NMR spectroscopy4. Metabolic fluxes were estimated by fitting a metabolic model to the 13C turnover of amino acids5. Level of cortical glutamate, aspartate and taurine was found to be reduced significantly (P≤0.03) while the level of choline (P = 0.04) was elevated in aged mice as compared to young adult. The total (glutamate+GABA) TCA cycle and the neurotransmitter cycle rates were found to be lower in aged mice compared to young adult. The decrease in metabolic rates in aged mice was solely accounted by reduction in the activity of glutamatergic neurotransmitter and GABAergic function seems to be unchanged with normal aging. Our finding indicates that normal aging attenuates the excitatory function in cerebral cortex which may explain the loss in cognitive function during aging. References: 1. Hedden and Gabrieli JD (2004) Nat Rev Neurosci 5:87; 2. Reddy PH (2007) Antioxid Redox Signal 9:1647; 3. Patel et al (2001) Brain Res 919:207; 4. De Graaf et al (2003) Magn Reson Med 49:37; 5. Patel et al (2005)Proc Natl Acad Sci USA 102:5588.

Acknowledgement: This study is supported by funding from Department of Biotechnology and CCMB.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 125 PP- 68 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SENSITIVITY OF Cu(II)-NSAIDs TO SEQUENCE SPECIFIC DIFFERENCES IN DNA BACKBONE

Sreeja Chakraborty and Munna Sarkar Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata E-mail address: [email protected]

Drugs belonging to the Non-Steroidal Anti-Inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing it with a bioactive metal like copper show an enhancement in their anti-cancer effects, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that a possible molecular mechanism behind the anticancer effects of Cu(II)-NSAIDs is direct binding to the DNA backbone by the copper complexes of NSAIDs namely meloxicam and piroxicam. Base sequence dictates the nature of binding of a drug with the DNA backbone altering the strength and the mode of binding. Preferential binding to a specific base sequence guides its function in vivo [53]. A detailed elucidation of the strength of binding and possible binding mode is important in predicting the binding sites of the drugs on the genome sequence. In this work,wWe have identified the differences in the interaction of Cu(II)-piroxicam and Cu(II)-meloxicam with hompolymeric polydA-polydT and alternating polydA-dT as well as with homopolymeric polydG-polydC and alternating polydG-dC. Differential response of the two Cu(II)-NSAIDs to the subtle variation in backbone structures of polydA-polydT and polydA-dT, was expressed in the difference in their binding parameters. Both the complexes show similar binding modes with polydA-dT which is intercalative, but for polydA-polydT, the results point to a mixed mode of binding. Even for the mixed mode, there is a significant difference in the binding of Cu(II)-piroxicam and Cu(II)-meloxicam to polydA-polydT. However, for the GC rich sequences, Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC to a weak binder of polydG-polydC, while Cu(II)-piroxicam changes from a strong binder of polydG-polydC to a weak one of polydG-dC, point to the sensitivity of these complexes to changes in the backbone structures/hydration. Hence, Cu(II)-NSAID complexes are highly sensitive to differences in backbone sequences.

126 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 69

NEED TO ASSESS NUTRITIONAL STATUS OF ADOLESCENT GIRLS FOR FUTURISTICS TASKS

*a Navneeta Desale IL- 4 *a Department of Zoology, S.P.H.Mahila Mahavidyalaya Malegaon-camp (Nashik), 423105, (M.S.) India Email: [email protected]

Adolescent constitute over 20.4% of the population in India. This period needs special attention because of adolescence faces different stages of development, different circumstances, different needs & diverse problems. Unfortunately assessment of nutritional status of adolescent girls has been the least explored area of research particularly in a rural India. In this work we have collected data of 176 adolescent -college girls of age group between 17 to 19.Information of weight, height, BMI, nature of diet were assessed. Hemoglobin concentration was measured. The data was collected and analyzed using WHO standard guidelines.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 127 PP- 70 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SIMULATION OF DICTYOSTELIUM CULMINATION

Saikat Dutta Chowdhury and Ansuman Lahiri Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta 92 APC Road, Kolkata 700009, India

The cellular slime mould (Dictyostelium discoideum) is considered to be an excellent system for studying morphogenesis since growth and development are separated in its life cycle. Because of its amenability to experimental studies, a lot of detailed information is available about the differentiated states of its cells, the types of cell-cell interaction and relative movement, the signalling molecules and their distribution. On the basis of these data, comprehensive models have been developed using cellular automata technique, foremost among which is the Glazier-Graner-Hogeweg (GGH) model.

A novel gene, trishanku (triA), has been identified in Dictyostelium discoideum and it has been shown to be expressed strongly in prespore cells. Disruption of triA expression has a pleiotropic effect: it destabilizes cell fate, reduces aggregate size, the stalk becomes thicker, the spore:stalk ratio becomes lower and the spore mass is subterminal. It was established that the upper cup does not develop correctly in a triA- mutant. A likely reason for the inability of the upper cup to lift the spore mass is defective intercellular adhesion. A previous model of the phenomenon of culmination was developed on the basis of cAMP signalling and differential adhesion, combined with cell differentiation and slime production. However, it did not consider the role of the upper cup in culmination. To study the effect of adhesion between upper cup and spore mass, we have carried out a simulation of the culmination phenomenon, where we considered a model with six cell types and their relative adhesion properties. We found that even such a simple system generates a culmination-like process. We are currently refining the model to incorporate more realistic features.

128 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 71

COMPARATIVE STUDY OF MODIFIED TFO

Rakesh Kumar Tiwari* and Rajendra Prasad Ojha Department of Physics, DDU Gorakhpur University, IL- 4 Gorakhpur, U.P.India. 273009 *[email protected]

A comparative study of triple helix formation by natural oligonucleotides and PNA, as a third strand, is presented here. The parameters of PNA are prepared using G-6-31(d) basic set. The RESP charges are calculated and used for the MD simulation. The initial structure of duplex is generated by LEAP and NAB under AMBER11 and Na ions are placed near to phosphate to neutralize the system. In this study we have taken two sets of purine rich mixed sequence, in which first and second strand is common and the third strand is parallel to the first strand of natural oligonucleotides and made of natural oligonucleotides and PNA. Common method is applied to both the sets of complexes i.e. the ions are randomized with water before starting the equilibration of 1 ns dynamics and after this, system is simulated for 20 ns dynamics. DNA parameters are calculated using PTRAJ by using ff10 force-field under AMBER11 software. The free energy calculations show that PNA can form stable complex with the mixed sequence of DNA and structure is stabilized during dynamics with “O” type of hydrogen bindings in base triplets. The MD results support a dynamically stable model of the modified DNA triplex over the entire length of the trajectory in both cases. We observed that the bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding in mixed sequences but they form hydrogen bonds with the bases of both the strand of DNA duplex. RMSD results and atomic fluctuations have been calculated and discussed which suggest that PNA strand has potential to use as drug.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 129 PP- 72 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EVALUATION OF EXPRESSION LEVELS OF LOX IN BREAST CANCER AND DESIGN OF PEPTIDE INHIBITORS AGAINST THE SAME

Abhay Kumar Singh1, Ratnakar Singh2, Rajinder Parsad3, Rahul Kumar1, Amrendra Pratap Singh1, Vishal Sahu1 and Sharmistha Dey1* Department of Biophysics1, Biochemistry2 and Surgery3, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India - 110 029.

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors such as epidermal growth factor (EGF) with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid which can be further metabolized by cyclooxygenase and lipoxygenase pathways to produce prostaglandins. The high concentration of prostaglandins have been associated with chronic inflammatory diseases and several type of human cancers. This is due to the over expression of inflammatory enzymes like COX, LOX etc. This work involves to quantitate the serum LOX-12 of breast cancer patients, its effect after chemotherapy to establish serum LOX-12 as a prognostic marker and the inhibition of LOX-12 by structure based designed peptide inhibitors. The evaluation of LOX-12 was done by real time Surface Plasmon Resonance (SPR) technology in BIAcore 2000 system. A significant increase in LOX-12 levels was observed in breast cancer patients (Mean±SD=40.54ng/ml±13.61) as compared to healthy controls (13.42ng/ml± 2.47) (p<0.0001). Serum LOX-12 levels were significantly higher in patients with lymph node involvement. More than 75% patients showed significantly reduced LOX-12 levels after chemotherapy(p<0.0001). The expression levels of LOX-12 increases according to the stage and can evolve as a potential marker in breast cancer. This study was followed by the design of specific peptide inhibitors based on the structure of active site of LOX. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (KD) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.3961028 M and 8.661028 M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.4561027 M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC50) of 75 mM and 400 mM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 mM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p>0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

130 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 73

MULTIMODAL IMAGING OF MOTILE GLIOMA CELLS UNDER TREATMENT WITH EXTRACELLULAR NUCLEOTIDES.

1 2 W. Klopocka , P. Pomorski ; IL- 4 1Nencki Inst Exp Biol, 2NanoFun Laboratory of Multimode Microscopy of Cell Motility and Adhesion, Nencki Inst Exp Biol, Warszawa, Poland

While microscopy is crucial for development of live sciences from decades, the molecular biology revolution put this technique into the shade. From twenty years, however the real microscopy revolution take place. The aim of this revolution is to use biochemical and molecular biology techniques inside the living cell. Our scientific interest focuses on the process especially suited for use of the microscopy: cell motility. To address requirements of the real time subcellular studies of motile cell, we have decided to construct multimodal system allowing us to make parallel imaging using both fluorescence and transmitted light methods. The system is organized around three main microscopy methods: long term DIC Nomarski cell observation, IRM (Internal Reflection Microscopy) and fast multiwavelength fluoresce microscopy. The fluorescence microscopy mode is optimized for fast Fura2 measurements yielding up to 30 frame-pairs per second in full camera resolution. This configuration allows overlay of calcium concentration maps on Nomarski contrast motility tracks and IRM adhesion measurements resulting in almost complete motility analysis. Presented results show the influence of P2Y2 receptor induction by UTP and resulting calcium signal on motility of Glioma C6 cells and it substratum adhesion visualization. The signal parameterization showed previously (Wypych D, Pomorski P (2010). Signaling Goes Local - Subcellular Visualization of Calcium Signal Formation in Glioma C6 Cells. Mol Biol Cell 22, 82 (abstract 171)) as well as adhesion pattern is overlaid onto DIC images. While in the control, motile cells calcium signal is strongly polarized, our results show clearly how signal polarization disappears after cell actomyoisin contractility inhibition by Y-27632, a selective ROCK kinase inhibitor.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 131 PP- 74 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURES OF THE COMPLEXES OF LACTOPEROXIDASE WITH ISONIAZID AND PYRAZINAMIDE REVEAL THE MODE OF ACTIONS OF PRODRUGS

Amit Kumar Singh, Nisha Pandey, Rashmi Prabha Singh, Mau Sinha, Nagendra Singh, Sujata Sharma and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi – 110029, India

Tuberculosis is a fatal infectious disease which is caused by Mycobacterium tuberculosis. The alarming rate at which the incidence of bacterial resistance to known antibiotics has been rising is a serious cause of concern. At present, the two well known antituberculosis drugs, isoniazid and pyrazinamide are used in the front-line multi-drug chemotherapy but they suffer from bacterial resistance. Both these compounds are prodrugs which are converted to active forms by enzymes catalase peroxidase and pyrazinamidase respectively. However, their mode of bindings with these enzymes are not known because crystal structures of these enzymes with isoniazid and pyrazinamide are not known. However, our structural studies of lactoperoxidase and its complexes with isoniazid and pyrazinamide have revealed the mode of binding of these compounds to lactoperoxidase. These studies also provided insights into the mechanism of their conversion into active forms. The modes of binding and mechanisms of action of catalase peroxidase and pyrazinamidase with isoniazid and pyrazinamide respectively appear to be very similar to those of lactoperoxidase. Also there is a clear evidence that INH is converted into active form in a similar way by enzymes lactoperoxidase and catalase peroxidase. In case of pyrazinamide the nature of active forms produced by lactoperoxidase and pyrazinamidase may differ but their effects are similar. These results suggest that lactoperoxidase may be a very useful enzyme against tuberculosis.

132 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 75

CRYSTAL STRUCTURE OF MAMMARY SIGNALING GLYCOPROTEIN (SPX-40) IN COMPLEX WITH 2-METHYL-2,4-PENTANEDIOL AT 1.6Ǻ RESOLUTION REVEALS TRYPTOPHAN AS A THREE-WAY SWITCH IN REGULATING THE FUNCTION OF THIS PROTEIN IL- 4

Anshul Chaudhary, Pradeep Sharma, Mau Sinha, Sujata Sharma, Punit Kaur andT. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi

The growth and development of mammary gland is regulated by a complex set of factors that begin with mammary gland development and lactogenesis and conclude with mammary gland involution. Mammary gland involution is marked by large scale tissue remodeling and a massive loss of epithelial cells due to programmed cell death called apoptosis. During this process, the increased production of a 40 kDa secretory glycoprotein SPX-40 (X denoting the species) suggests an important regulatory role for this protein. A similar protein called breast- regression protein (BRP-39) with a sequence identity of 69% to SPS-40 has been discovered in certain types of breast-cancer cells. SPS-40 also shows sequence identities of 53% with chitinases. However, it lacks chitinase-like activity due to replacement of an essential Glu residue by Leu in the active site of the protein. The lack of hydrolytic activity with a residual capability to bind saccharides at the carbohydrate-binding groove implicates a binding role for SPX-40. We have determined the crystal structure of SPS-40 isolated from sheep mammary secretions in complex with 2-Methyl-2,4-pentanediol (MPD) at 1.6Ǻ. The overall structure revealed the presence of two MPD molecules in the sugar-binding groove of the protein. The binding of MPD molecules in the TIM-barrel core results in considerable conformational perturbations in various residues like Gly77, Trp78, Phe80, Glu269 and Ile272, the most significant being Trp78. Trp78 occupies the core of the sugar-binding groove. Binding of hexasaccharide, N-acetylglucosamine (GlcNAc6) to SPS-40 alters the conformation of Trp78 from the native conformation (1 = -65.5, 2,1 = -78.8, 2,2 = 97.5) to the stacked conformation (1 = -170.0, 2,1 = -114.3, 2,2 = 61.6). The very tightly bound MPD molecules at subsites -2 and -1 induced a rarely observed conformation of Trp78 (1 = 55.9, 2,1 = 90.2, 2,2 = -88.9). With these three distinct conformations of Trp78, the protein SPS-40 exists in three states: (i) a resting native state with pinched conformation, (ii) a saccharide-bound state with a stacked conformation and (iii) an inhibited state as observed in the complex with MPD with obstructive conformation.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 133 PP- 76 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CRYSTAL STRUCTURE OF THE COMPLEX OF LACTOPEROXIDASE WITH FOUR INORGANIC SUBSTRATES, SCN-, I-, Br- AND Cl-

Harsh V. Sirohi, Amit K. Singh, Nisha Pandey, Mau Sinha, Punit Kaur, Sujata Sharma, and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi -110029

Lactoperoxidase (LPO) is a member of the family of mammalian heme peroxidases. It catalyzes the oxidation of halides and pseudohalides in presence of hydrogen peroxide. LPO has been co-crystallized with inorganic substrates, SCN, I, Br and Cl. The structure determination of the complex of LPO with the above four substrates showed that all of them occupied distinct positions in the substrate binding site on the distal heme side. The bound substrate ions were separated from each other by one or more water molecules. The heme iron is coordinated to His-351 N2 on the proximal side while it is coordinated to conserved water molecule W-1 on the distal heme side. W-1 is hydrogen bonded to Br ion which is followed by Cl ion with a hydrogen bonded water molecule W-5 between them. Next to Cl ion is a hydrogen bonded water molecule W-7 which in turn is hydrogen bonded to W-8 and N atom of SCN. W-8 is hydrogen bonded to W-9 which is hydrogen bonded to I. SCN ion also interacts directly with Asn-230 and through water molecules with Ser-235 and Phe- 254. Therefore, according to the locations of four substrate anions, the order of preference for binding to lactoperoxidase is observed as Br > Cl > SCN > I. The positions of anions are further defined in terms of subsites where Br is located in subsite 1, Cl in subsite 2, SCN in subsite 3 and I in subsite 4.

134 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 77

CRYSTAL STRUCTURE OF C-TERMINAL LOBE OF LACTOFERRIN IN COMPLEX WITH PATHOGEN ASSOCIATED MOLECULAR PATTERNS REVEAL THE STRUCTURAL BASIS OF ITS ANTIMICROBIAL EFFECT IL- 4 Mau Sinha, Prakash Shukla, Lovely Gautam, Sujata Sharma,Punit Kaur and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

Lactoferrin is a multifunctional bilobal protein found in milk and exocrine secretions. Lactoferrin is one of the key elements that exerts defense activity against a broad-spectrum like bacteria, fungi, protozoa and viruses. It exerts its antimicrobial action by sequestration of iron. Lactoferrin can also directly bind to some components like pathogen associated molecular patterns (PAMPs) of the bacterial cell wall. The well known pathogen-associated molecular patterns include lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. Both LPS and LTA are glycol-lipids that are composed of an amphipathic lipid component and a hydrophilic polysaccharide core. Several reports suggest that lactoferrin binds to LPS and LTA and decreases the toxicity induced by these PAMPs. Proteinase K divides this protein into two equal halves. N-lobe is generally degraded on exposure to microbial environment. So we investigated the role of C- lobe of lactoferrin in its antimicrobial action. The purified samples of iron-saturated C-lobe were co-crystallized with LPS and LTA. The structures of LPS and LTA were obtained at 2.0Å and 2.1Å respectively. This site of binding of PAMPs is located on the other side of the iron binding cleft and separated by the longest -strand of the molecule. The glycol portion of the PAMPs make hydrogen bonds with several residues like Thr 430, Glu659, Thr660, Leu661 whereas the acyl chains have aligned in a way to maximize hydrophobic interactions with the residues of the protein. This study gives an idea of the structural basis of binding of C-lobe of lactoferrin with PAMPs and hence can explain the mode of anti-microbial activity.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 135 PP- 78 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURE OF BOVINE CARBONYL LACTOPEROXIDASE AT 2.0Å RESOLUTION AND INFRARED SPECTRA AS A FUNCTION OF pH

Naseer Iqbal, Amit K. Singh., Shavait Yamini, Mau Sinha, Punit Kaur, Sujata Sharma and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I− into antimicrobials and small aromatic organics after being itself oxidized by H2O2. LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO-LPO at 2.0Å resolution and infrared (IR) spectra of the iron-bound CO stretch from pH 3 to 8.8 at 1 cm−1 resolution. The observed Fe-C-O bond angle of 132 is more acute than the electronically related Fe(III), CN-LPO with a Fe-C-N angle of 161. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III)moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN-LPO. The IR spectra of CO-LPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ≈ 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH 7; both band widths at half- height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.

136 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 79

CRYSTAL STRUCTURES OF A TYPE-1 RIBOSOME INACTIVATING PROTEIN FROM Momordica balsamina IN THE BOUND AND UNBOUND STATES

Nilisha Rastogi, Gajraj Singh Kushwaha, Nisha Pandey, IL- 4 Mau Sinha, S. Baskar Singh, Punit Kaur, Sujata Sharma and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We have determined six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2´-deoxyadenosine triphosphate (2
-dATP) (F). These were determined at 1.67 Å, 1.60 Å, 2.20 Å, 1.70 Å, 2.07 Å and 1.90 Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2
-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i)
1 =
66°and
2 = 165° in structures (A) and (B); (ii)
1 =
95° and
2 = 70° in structures (C), (D) and (E); and (iii)
1 =
163° and
2 = 87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 137 PP- 80 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURAL INSIGHTS INTO THE DUAL STRATEGY OF RECOGNITION BY PEPTIDOGLYCAN RECOGNITION PROTEIN, PGRP-S: STRUCTURE OF THE TERNARY COMPLEX OF PGRP-S WITH LIPOPOLYSACCHARIDE AND STEARIC ACID

Pradeep Sharma, Divya Dube, Mau Sinha, Punit Kaur, Sujata Sharma and T. P. Singh* Department of Biophysics, All India Institute of Medical Sciences, New Delhi

Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A-B and C-D. The A-B and C-D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A-B and C-D contacts. Two ligand binding sites, one at C-D contact and another at A-B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both Gram-positive and Gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C-D contact (Site-1) while SA binds to it at the A-B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS forms 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2Å) while SA forms 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN- due to LPS and SA decreased considerably upon the addition of CPGRP-S.

138 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 81

CRYSTAL STRUCTURE OF THE COMPLEX OF GOAT LACTOPEROXIDASE WITH AN ANTI-THYROID DRUG METHIMAZOLE

Rashmi Prabha Singh, Nagendra Singh, Amit Kumar Singh, Mau Sinha, Punit Kaur, IL- 4 Sujata Sharma and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi – 110029

Lactoperoxidase (LPO) belongs to the family of four mammalian heme peroxidases including myeloperoxidase (MPO), thyroid peroxidase (TPO) and eosinophil peroxidase (EPO). The methimazole (MMI) is an anti-thyroid drug and acts by inhibiting the function of TPO. Since the overall structure of distal heme binding site in TPO and LPO are likely to be similar, it was of interest to understand the mode of binding of MMI to LPO and also correlate if LPO could be playing any role in the state of hyperthyroidism. LPO was purified from the mammary secretion of goat (GLPO). It was crystallized with anti-thyroid drug MMI. The crystal of the complex belonged to space group P21 with cell dimension of a = 80.3Å, b = 93.0Å, c = 81.5Å an  = 89.9. It had two molecules in the asymmetric unit and formed a dimer. This was the first noncovalent dimer observed so far in the family of mammalian heme peroxidases. MMI binds to GLPO at the distal heme binding site. It replaces the catalytically important water molecule W1 and forms a coordinate bond with heme iron at a distance of 2.68Å. The binding of MMI resembles with that of amitrole and inhibits the function of LPO. It might be inhibiting the function of TPO in a similar fashion.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 139 PP- 82 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CRYSTAL STRUCTURE DETERMINATION OF RIBOSOME INACTIVATING PROTEIN IN COMPLEX WITH CYTIDINE

Sadanand Pandey, Shavait Yamini, Gajraj S. Kushwaha, Mau Sinha, Punit Kaur, Sujata Sharma and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi – 110029

Ribosome Inactivating Proteins (RIPs) inhibit protein synthesis in eukaryotic cells by catalytically damaging their ribosomes. RIPs hydrolyze N-glycosidic bond between adenine and ribose in highly conserved sarcin / ricin loop of the large subunit of ribosomes. Recently we have also shown that type1 RIP from Momordica balsamina (Mb RIP-1) recognizes mRNA cap structures that affect several cellular processing steps including mRNA splicing, translation initiation, mRNA stability and nuclear transport. Here we report the crystal structure of MbRIP-1 with cytidine. The structure of the complex of MbRIP-1 with cytidine revealed that cytidine is placed in the substrate binding site in such a way that it inhibits the catalytic action of MbRIP-1. It occupies the position close to Glu85 with which it forms two H-bonds. It also makes other H-bonds at this position with Ile71,Gly109,Arg163 and six water molecules. The binding of cytidine is further stabilized by more than 70 Van der Waal contacts. The strong binding of cytidine with MbRIP-1 suggests that pyrimidine bases at specific site in RNAs may be involved in neutralizing the lethal effect of MbRIP-1.

140 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 83

CRYSTAL STRUCTURE OF THE COMPLEXES OF DIHYDRODIPICOLINATE SYNTHASE FROM Acinetobacter baumannii WITH OXALIC ACID AND OXAMIC ACID IL- 4 Sanket Kaushik, Avinash Singh, Mukesh Kumar, Mau Sinha, Punit Kaur,Sujata Sharma and T.P. Singh Department of Biophysics, All India Institute of medical sciences, New Delhi 110029

The enzyme dihydrodipicolinate synthase (DHDPS) catalyses the first unique reaction of the (S)-lysine biosynthesis: an aldol condensation between (S) aspartate β- semialdehyde and pyruvate.The catalytic triad involves residue from two monomers. The reaction is critical for the survival of bacteria. The enzyme DHDPS from Acinetobacter baumannii was cloned and expressed. It was purified with His-tag and further purified to homogeneity using size exclusion chromatography. In order to study mode of inhibition, two compounds oxalic acid and oxamic acid were used to make its complexes. The complexes of DHDPS with oxalic acid and oxamic acid were crystallized. The crystals belonged to monoclinic space group P21, with unit cell dimensions a = 52.3 Å, b = 122.4 Å, c = 51.3 Å. The structures were refined to less then 20% values of their Rcryst and Rfree factors .There were more than 93% residues in the most favored region of the Ramachandran map. The final model consists of 4390 protein atoms and 602 water oxygen atoms. The structure of the subunit consists of an N-terminal

(/)8- barrel followed by a C-terminal  helix domain. Three amino acids from the catalytic triad: Tyr 133, Thr 44 and Tyr 107, whereby Tyr 107 is contributed by the adjacent monomer across the tight dimer interface. The buried surface at the dimmer interface was of approximately 850 Å. A comprehensive understanding of the structure and function of this important bacterial enzyme is essential for the design of potent inhibitors of the (S) lysine biosynthesis pathway.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 141 PP- 84 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CRYSTAL STRUCTURE OF A POTENT ANTIMICROBIAL LACTOFERRIN NONAPEPTIDE IN COMPLEX WITH SERINE PROTEASE PROTEINASE K

Satyaprakash Yadav, Prakash Shukla, Mau Sinha, Sujata Sharma, Punit Kaur and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

Proteinase K or endopeptidase K is a serine protease of the subtilisin family consisting of 279 amino acids. Lactoferrin contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. Earlier works with serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves. Proteinase K cleaves lactoferrin, an iron binding glycoprotein (molecular weight of 80 kDa) into two equal N-terminal and C- terminal halves each containing an iron binding site. The lobes were further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product did not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. It has been observed that peptides which are predominantly cationic and hydrophobic in nature show potent antimicrobial activity. In this study we have synthesized a lactoferrin nonapeptide having the same sequence as that of the residues 387 to 396 of C terminal lobe of lactoferrin. The structure of proteinase K in complex with the peptide has been determined using X-ray crystallographic method and refined to 1.3 Å resolution with Rcryst and Rfree factors of 0.202 and 0.249 respectively. The peptide is placed at the top of the active site. The recognition site residues are accessible to the solvent on one side, whereas the other ends of these strands terminate at the catalytic triad of Asp39 – His69 – Ser224. It is well known from earlier crystallographic studies on proteinase K that the substrate recognition site is formed by two peptide chains Asn99 - Tyr104 and Ser132 - Gly136. The inhibitor fills the recognition site completely. The peptide inhibitor is held by several intermolecular hydrogen bonds including active site residue His69 as well as Gly102, Gln103, Tyr104, Ser105, Leu133, Gly134, Ser221 and Thr223. Inhibitor binding does not cause any major conformational reorientation of the active site residues. The peptide also exhibited antimicrobial properties against Escherichia coli, BL-21 strains.

142 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 85

STRUCTURE OF PEPTIDYL-tRNA HYDROLASE FROM Acinetobacter Baumannii at 1.7 Ȧ RESOLUTION

Sujata Sharma, Sanket Kaushik, Shavait Yamini, Sanjit Kumar,Mau Sinha, Punit Kaur IL- 4 and T.P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi – 110029

Peptidyl tRNA molecules are produced in cell due to their premature dissociation from the ribosome during protein synthesis. In order to reuse the amino acids and tRNA in the ongoing protein synthesis process the peptide and tRNA components have to be separated. This process of recycling is done by peptidyl-tRNA hydrolase (Pth).The sequence and structure of bacterial Pth are different from that of human protein. Thus bacterial Pth forms an attractive target for the design of antibacterial agents. For this purpose we have cloned, expressed and determined the structure of Pth from Acinetobacter baumannii (AbPth) at 1.78

Ȧ. Purified protein was crystallized in space P212121 with cell dimensions of a= 34.5 Ȧ, b= 59.2 Ȧ and c= 109.2 Ȧ. The polypeptide chain of AbPth adopts a compact α/β globular fold with a twisted β- sheet comprising of seven β strands, β1(residues, 5-10) , β2 (residues,41- 43), β3(residues, 48-52), β4(residues, 58-64), β5(residues,89-96) ,β6 (residues, 103-108) and β7 (residues,130-136).The centrally located β-sheet is surrounded by six α-helices, α1(residues, 24-36), α2(residues,72-82) α3(residues, 116-125), α4 (residues 146-151), α5 (residues,156-179) and α6 (residues,181-188) .The segment Gly 138 - Val 150 is flexible as it lacks significant interactions with the rest of the protein. Other flexible loops Gly 109-gly117 and Asp 95- Asp 100 show variable placements with respect to each other. The hydrogen bonded interactions between Asn 116, His 22 and Asp 95 indicate a strereochemically favorable arrangement of these residues for the catalytic action. One of the most striking features of the peptidyl –tRNA hydrolase structure is the conformation of the loop Gly 138- Val 150 which is variable. In order to accommodate substrates of different sizes, this loop moves back and forth. The design of inhibitors of Pth requires to stabilize the conformation of this flexible loop with respect to the loop Leu 108-Leu 118 on the opposite side of the cleft Leu 108.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 143 PP- 86 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECT OF INTENSE, ULTRASHORT LASER PULSES ON DNA PLASMIDS IN THEIR NATIVE STATE

1,2Harish Bharambe, 1Jacinta S. D’Souza, 3Jayashree A. Dharmadhikari, 3DeepakMathur and 3Aditya K. Dharmadhikari 1UM-DAE Center for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai - 400098; 2National Institute of Pharmaceutical Education and Research (NIPER), EPIP campus, Industrial area, Jandaha Road, Hazipur (Vaishali) – 844102; 3Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai400005

Ultrashort laser pulses are beginning to be applied to study biological materials and associated phenomena such as cellular physiology, its functioning, understanding DNA damage and repair mechanisms, as well as for therapeutic purposes. Detailed understanding of mechanisms that govern interactions of biological matter with ultrashort laser pulses are important for gaining proper insights and for optimizing parameters of use in biomedical applications.When such laser pulses pass through an aqueous medium, a pool of low energy electrons (LEE) are generated which can induce single strand breaks (SSB) and double strand breaks in DNA (DSB). This damage may be readily visualized when a DNA sample is analyzed on an agarose gel post laser exposure: indications that we have utilized recently [1] are observations of the conversion of supercoiled form of a plasmid DNA into relaxed (major) and linear forms (minor). Details of the mechanism by which such conformational changes occur remain elusive, although recent work [1] has indicated that single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media by low-energy electrons and OH-radicals that are formed upon exposure to ultrashort laser pulses (820 nm wavelength, 45 fs, 1kHz repetition rate) at intensities of 1-12 TW cm−2. The strong laser field generates, in situ, electrons and radicals that induce transformation of supercoiled DNA into relaxed DNA, the extent of which has recently been quantified [1]. Introduction of electron and radical scavengers inhibits DNA damage; results indicate that OH radicals are four times more likely to cause DNA damage than electrons. In this work, we present new results on the scaling of DNA damage with femtosecond laser intensities for fixed concentration of DNA and exposure time.

1. J. S. D’Souza, J. A. Dharmadhikari, A. K. Dharmadhikari, B. J. Rao, and D. Mathur, Phys. Rev. Letters, 106, 118101 (2011).

144 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 87

BIOPHYSICAL CHARACTERIZATION OF A FLAGELLAR ASSOCIATED PROTEIN 174 FROM THE GREEN CHLOROPHYTE Chlamydomonas reinhardtii

1Venkatramanan G. Rao, 1Kalyana Venneti, 2Madhuri Chandra, 3G. Jithender Reddy, 4P. M. Dongre, 1,3R. V. Hosur IL- 4 and 1Jacinta S. D’Souza* 1UM-DAE Centre for Excellence in Basic Sciences, Santacruz (E), Mumbai-400098, India; 2BITS-Pilani, Hyderabad, India; Department of Chemical Sciences, Tata Institute of Fundamental research, Homi Bhabha Raod, Colaba, Mumbai-400005, India; 4Department of Biophysics, University of Mumbai, Santacruz (E), Mumabi-400098, India *Author for correspondence: [email protected]

Flagellum, a sensory and motility organelle is conserved in most ciliated eukaryotes. Its ease to culture under laboratory conditions and the standardized molecular toolkit have proven Chlamydomonas reinhardtii as a model organism to study this organelle. Moreover, the organelle shares considerable homology with the Human ciliary proteome. These features have helped study the flagellar dynamics and the diseases caused by defect in the proteins that constitute its architecture. The intricate ‘9+2’ tubulin-based architecture of ~650 proteins is made up of the central pair, 9 outer doublet microtubules, radial spokes, connecting links, dynein arms and accessory proteins called as the flagellar associated proteins (FAPs) that altogether make the Motility and Sensory apparatus of the micromachine. FAPs belong to a wide class of proteins carrying domains in their primary sequences that suggest functions such as signaling, metabolism and motility. The role of several FAPs in both the motility and sensory paradigm of the flagella remain elusive. The aim of the current study is to identify the role of one such FAP (FAP174) in the flagella of C. reinhardtii. In silico analyses of FAP174 revealed it to be a homologue of the Associate of c-MYC-1 (AMY-1) found in humans and plants. AMY-1 is known to interact with A-Kinase anchoring proteins (AKAPs); these proteins form a complex with AMY-1 and Protein Kinase A (PKA) resulting in the inhibition of PKA activity. Studies in human cilia and C. reinhardtii show that PKA negatively regulates motility. Thus, we hypothesize that FAP174 may act as a kinase inhibitor in flagella thereby regulating flagellar motility. Parallel studies in the laboratory have shown its interaction with a flagellar AKAP (RSP3). The ORF of fap174 gene was cloned, the protein over-expressed and the His-tagged FAP174 protein purified to near homogeneity. Using CD spectroscopy the recombinant protein showed intact secondary structure. This protein was therefore meted suitable for further characterization. Using MALDI-TOF-MS and Gel filtration techniques we have shown that it forms a stable dimer. Furthermore, denaturing and native electrophoretic techniques as well as DLS studies in the presence and absence of DTT have confirmed the dimer and revealed higher oligomers. To gain insights into the tertiary structure, NMR of the 13C and 15N- labelled FAP174 protein was carried out. The amino acid residues that may possibly contribute towards dimer and oligomer formation have been predicted. These residues have been mutated such that they would aid in understanding the dimerization and oligomerization nature of the protein.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 145 PP- 88 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

BIOPHYSICAL BASIS OF THE 5’ MRNA CAP RECOGNITION BY HUMAN 5’- DEPENDENT 3’-POLY(A)-SPECIFIC RIBONUCLEASE (PARN)

Anna Niedźwiecka 1,2, Małgorzata Lekka 2, Remigiusz Worch 1, Per Nilsson 3, Edward Darżynkiewicz 4, Anders Virtanen 3 1 Laboratory of Biological Physics, Institute of Physics PAS, 02-668 Warsaw, Poland; 4 Division of Biophysics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland; 2 Laboratory of Biophysical Microstudies, Institute of Nuclear Physics PAS, 31-342 Kraków, Poland; 3 Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Sweden

Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that plays a key role in 3’ deadenylation, is involved in nonsense-mediated mRNA decay, and also in regulation of cytoplasmic polyadenylation. The activity of PARN is stimulated by the presence of the mRNA 5’ cap (m7GpppN) that enhances the enzyme processivity. The goal of the study was to find structural requirements for the PARN affinity to the 5’ cap, as well as to gain an insight into the complete structure of the full length protein which was not resolved by typical methods such as crystallography and NMR. Kinetic parameters of the interaction estimated by surface plasmon resonance together with equilibrium dissociation constants for PARN mutants and the PARN RNA Recognition Motif (RRM) obtained by fluorescence titrations revealed that molecular recognition of the 5’ cap structure by poly(A)-specific 3’ ribonuclease is different than known for translation initiation factors. The results show that PARN binds the cap with micromolar affinity, without electrostatic steering, and that the RRM is responsible for this interaction. Contrary to all known RRMs, the PARN RRM does not exploit the canonical β-sheet but recognizes the mRNA 5’ cap by Trp475 at the outer surface of the domain. We have also investigated the global architecture of human PARN by atomic force microscopy imaging in liquid and report the dimensions of the full length protein at subnanometre resolution, confirmed by DLS and gel filtration. Acknowledgements: Polish National Science Centre grant N N301 267137, Swedish Research Council, Linneus Support and Lennanders Foundation at Uppsala University.

146 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 89

BIO-ACTIVITY INFORMATION TO ORGANIC CHEMISTS (BAITOC)

#,¤ #,¤ ¤ ,#,¤, Abhilash Jayaraj , Goutam Mukherjee ,Shashank Shekher and B.Jayaram* ǂ #Department of Chemistry & ¤Supercomputing Facility for Bioinformatics & Computational IL- 4 Biology & ǂSchool of Biological Sciences, Indian Institute of Technology, Hauz Khas, New Delhi – 110016, India Website: www.scfbio-iitd.res.in Tel. No.: 91-11-2659 1505; 91-11-2659 6786; Fax: 91-11-2658 2037 Email: [email protected]

India is well known for its expertise in organic synthesis. The number of new drug molecules coming out of India however, is not commensurate with this expertise. There is an urgent need to inform the organic chemist of the bioactivity/therapeutic potential of his/her molecule. BAITOC aims to fill this void, through literature collation and rapid scans against a database of protein structures and passing on this information to the scientist who can then attempt to elaborate his/her scaffold to develop lead molecules. BAITOC identifies biological targets showing the best bio-activity for a given organic molecule. It uses RASPD1 methodology to scan a user specified organic molecule to a list of human pathogen associated proteins. The protein structures are adapted from RCSB Protein Data Bank2 and disease and organism information from KEGG3. The active site prediction software AADS4-7 developed in-house, is used to predict and store, list of active-sites for each protein. Each active site is scanned for bioactivity with the user defined organic compound. The result of BAITOC is a list of pathogen proteins in decreasing order of bio-activity to the molecule under investigation. It also provides information on the associated disease and organism. The software is freely available at SCFBio website (http://www.scfbio- iitd.res.in/software/drugdesign/baitocnew.jsp). References: 1. Mukherjee, G; Jayaram, B.; “A Rapid Identification of Hit Molecules for Target Proteins via Physico-Chemical Descriptors” (Manuscript in preparation, 2012). 2. Berman, H.M.; Henrick K.; Nakamura H. Nature Structural Biology 2003, 10, 980. 3. Kanehisa, M.; Goto, S.; Sato, Y.; Furumichi, M.; Tanabe, M. Nucleic Acids Res 2012, 40, D109. 4. Singh, T.; Biswas, D. Jayaram, B.; J Chem. Inf. Modeling 2011, 51, 2515. 5. Jain, T.; Jayaram, B. FEBS Letters 2005, 579, 6659. 6. Jain, T.; Jayaram, B. Proteins: Structure, Function and Bioinformatics 2007, 67, 1167. 7. Gupta, A.; Gandhimathi, A.; Sharma, P.; Jayaram, B. Protein and Peptide Letters 2007, 14, 632.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 147 PP- 90 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

MOLECULAR IDENTIFICATION OF Amherstia nobilis

Meenakshi K and Indu George Department of Life Sciences, University of Mumbai

Amherstia nobilis, which is considered as the ‘Queen of the flowering trees’ is a native in Burma. It was introduced in India in the then Royal Botanical Gardens at Calcutta. It is a tropical tree with exceptionally beautiful flowers and belongs to Caesalpiniaceae family. This tree is is difficult to propagate and thus conservation of this tree is important. DNA barcoding is a molecular identification approach that may be useful in conservation strategies of Amherstia nobilis. This method could help in identification of species as corroboration to morphological observation. In plants, the search for suitable genomic regions has proven more challenging. Several regions in the plastid genome (eg.: rbcL, rpoC1, rpoB, ycf5, psbA – trnH, trnL, atp F – atp H, psbk – psb1) as well as the internal transcribed spacer (ITS) of the ribosomal nuclear DNA have emerged as good candidates for plant DNA barcoding. In the present study, we sequenced partial gene sequences of rbcL & matK to test their applicability in the identification of Amherstia nobilis and sequence similarity search done to a query sequence by using BLAST tool. Our analysis confirmed that the sequences belongs to Amherstia nobilis and further supports that rbcL & matK genes could be used as molecular markers to assign Amherstia nobilis at species level.

148 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 91

HYDROXAMIC ACIDS COMPLEXES AND THEIR APPLICATION IN BIOCHEMICAL PROCESSES

Dr.Aftab Ahmad Sulaiman M.Sc. MBA B.ED FICS Ph.D IL- 4 Maulana Azad National Urdu University, University.Department of Polytechnic,Darbhanga,Bihar,India [email protected],Mob:9973200231,Ph:06212274379

Hydydroxamic acids, are weak organic acids of general formula RC(=O)N(R')OH, are widespread in the tissues of plants, in metabolites of bacteria and fungi, including complex compounds.Hydroxamic acids complexes and their derivatives fulfill a variety of important roles in biology and medicine; here we provide a comprehensive brief review of the most basic medicinal chemistry and pharmacology of hydroxamate molecules. The design and synthesis of ligands for biomedical applications in fields such as anticancer applications has become of great importance. One of these important ligands is hydroxamate molecules. Key Words: Hydroxamic acids, biological activities, complex compound, biochemical processes. hydroxamate molecule. References : 1. Frey M, Chomet P, Glawischnig E, Stettner C, Grün S, Winklmair A, Eisenreich W, Bacher A, Meeley RB, Briggs SP, Simcox K, Gierl A : Analysis of a chemical plant defense mechanism in grasses. Science, 277, 696, 1997. 2. Melanson D, Chilton MD, Masters-Moore D, Chilton WS : A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype of bxbx maize. Proc Natl Acad Sci, USA, 94, 13345, 1997. 3. Argandoña VH, Luza JG, Niemeyer HM, Corcuera LJ : Role of hydroxamic acids in the resistance of cereals to aphids. Phytochemistry, 19, 1665, 1980. 4. Klun JA, Robinson JF : Concentration of two 1,4-benzoxazinones in dent corn at various stages of development of the plant and its relation to resistance of the host plant to the European corn borer. J Econ Entomol, 62, 214, 1969.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 149 PP- 92 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

UNDERSTANDING BIOPHYSICAL INTERACTIONS OF NATURAL FLAVONOIDS WITH MODEL MEMBRANES: A MOLECULAR DYNAMICS SIMULATION STUDY

Manoj Gadhwal a, Akshada Joshi a, Aarti Anantram a, Urmila Joshi a,Ragini Sinha b and Girjesh Govil b aPrincipal K M Kundnani College of Pharmacy, Cuffe Parade, Mumbai 400005 bNational Facility for High Field NMR, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005

Flavonoids are a group of naturally occurring, low molecular weight benzo–γ–pyrone derivatives and possess significant antioxidant and antitumour activities. These activities are proposed to be associated with their interaction with membrane bound enzymes. Membranes constitute a meeting point of proteins and lipids. Lipids are increasingly being recognized as regulators of location and activity of many membrane proteins. It is therefore important to understand the molecular mechanism of interaction of flavonoids with model membranes. Many physical techniques are used to study flavonoid-membrane interaction. These, however represent a static situation and do not provide atomistic details. MD Simulation is considered as a powerful tool to gain an insight into interactions of flavonoids with lipid membranes and provide a dynamic picture apart from providing atomistic details. Choice of DPPC as model membrane was a consequence of it being ubiquitous in nature in many biological membranes.

The simulation studies were carried out using GROMACS 4.5.3 and the system consisted of 128 DPPC molecules. The flavonoids included were quercetin, chrysin, genistein, luteolin and baicalein. The simulations were run for 20 ns. Results of simulation clearly show that the flavonoids have a strong affinity for the lipid bilayer via formation of extent of Hydrogen bonds. Flavonoids act as hydrogen bond donors. Majority of the times, the location of H- bond acceptor in the membrane was the phosphate group of the headgroup region. The diffusion coefficients for different flavonoids were calculated using simulation apart from the preferential location in the bilayer. Order parameter calculations indicated different orientation of different flavonoids.

150 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 93

PROBING THE IN–VITRO ACTIVITY OF QUERCETIN AND ITS MODIFIED ANALOGUES a a a b b* Urmila J.Joshi, Amol S.Gadge, Priscilla D’Mello, Ragini Sinha, Sudha Srivastava IL- 4 and bGirjesh Govil aPrin K M Kundanani College of Pharmacy, Cuffe Parade, Mumbai 400005, India bNational Facility for High Field NMR, Tata Institute of Fundamental Research, Mumbai-5, India

Based on molecular dynamics simulation results, structurally modified analogues of quercetin were synthesized and tested for antioxidant activity by TBARS assay, anti- inflammatory activity by carragenan induced rat paw edema and a possible anticancer activity in PC3, HepG2, HCT15, MCF7 cancer cell lines. It is observed that the structural modifications result in a significant reduction in the anti-inflammatory activity and antioxidant activity of these compounds. The anticancer activity of Q–Cl is comparable to quercetin in HepG2 cell lines and lesser in other cell lines. It is proposed that these compounds may be better bioavailable and need to be explored by further structural modifications for a better activity. Based on these results and by correlating the bioactivity of quercetin and its analogues, a possible structure activity relationship has been deduced.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 151 PP- 94 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

MECHANOBIOLOGICAL REGULATION OF EMBRYONIC STEM CELL SELF- RENEWAL AND DIFFERENTIATION

Anuj Rastogi, Kavitha Sthanam, Shamik Sen Cellular Biophysics Lab, Department of Biosciences and Bioenginnering, Indian Institute of Technology, Bombay

Measurement of the mechanical properties of single cells is of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this study, we introduce the significance of the tensional homeostasis in controlling the self renewal and differentiations states of the embryonic stem cells. ESCs are typically grown on a feeder layer of mitotically inactivated mouse embryonic fibroblasts. It is thought that MEFs secrete various factors that regulate the fate of ESCs. We have gone further to elucidate if the MEFs also provide mechanical stimulus for maintenance of pluripotency of ESCs. To quantify the contractile properties of MEFs, trypsin de-adhesion induced cell shape dynamics were studied. When treated with trypsin, mitotically inactive MEFs showed higher contractility than mitotically active cells. This highlights the change in cellular stiffness on becoming mitotically inactive. To find out the role played by the extra-cellular matrix proteins in influencing the mechanical phenotype of MEFs, the cells were seeded on 0.1% and 1% gelatin coated dishes. Altering the ligand density showed increased contractility at higher ligand density. Accutase treatment of ESCs showed that MEFs in contact with ESCs exhibit faster de-adhesion compared to those with no contact.

152 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 95

SPION (SUPER PARAMAGNETIC IRON OXIDE NANOPARTICLES)-ENHANCED POLYMERASE CHAIN REACTION.

1 1 2 Priyanka Kambli , Varsha Kelkar Mane and Jacinta D’souza . IL- 4 Department of Biotechnology, University of Mumbai. University of Mumbai Centre for excellence in Basic Science

Enhancing the specificity and efficiency of the Polymerase Chain Reaction (PCR) using metallic and non-metallic nanoparticles is an emerging area of research. Varying concentrations of titanium oxide, carbon nanotubes, iron oxide, silver, zinc oxide as well as gold nanoparticles are reported to inhibit or amplify the polymerase chain reaction. Iron oxide nanoparticles are of special interest due to their biocompatibility as well as super paramagnetic properties. The present study for the first time documents the enhancement in the PCR amplification efficiency using chemically synthesized SPIONSs. The aqueous uniformly dispersed super paramagnetic iron oxide nanoparticles (ferrofluid) were synthesized by controlled chemical co-precipitation. The characterization studies with UV- Visible Spectroscopy, X-ray Diffraction, AFM, Differential Light Scattering and FTIR

techniques confirmed presence of Fe3O4 nanoparticles with an average size of 60 nm. A varying concentration of the aqueous ferrofluid was tested for template amplification efficiency with alteration in GC content, chain length of the templates as well as the annealing temperature of the reaction. The Gaussian curve obtained in the series of reactions statistically concluded that ferrofluids (from milligram to pictogram per millilitre concentration) are capable of enhancing the PCR amplification by 10-15% at specific concentrations over the controls. This property could be attributed to the thermal conductivity of the ferrofluid as well probable enzyme nanoparticle interaction. The study is a step in the direction of maximizing the PCR product obtained using a limited amount of template that is usually the restricting parameter in some instances especially in forensic analysis.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 153 PP- 96 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STRUCTURAL DAMAGE OF DNA AND PROTEIN BY CARBONYLS AND DAMAGE REVERSAL BY VITAMINS AND NATURAL COMPOUNDS

Ahmad Ali, Rajeev Sharma, Subramanian Sivakami University Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai Email: - [email protected]

Non-enzymatic glycation (NEG) refers to the covalent binding of carbonyl groups of reducing sugars to amino groups of proteins, lipids, nucleic acids resulting in the formation of glycosylamines which rearrange themselves to get converted to advanced end glycation products (AGEs). This reaction is also known to produce free radicals. We studied the effect of glycation on DNA and proteins. DNA damage was induced by the glycation of lysine with methylglyoxal in the presence of metal ion. A comparison was made between different sugars and their potential in causing damage to DNA. In the presence of Lysine and metal ion, ribose and ribose-5-phosphate caused maximum damage to DNA as compared to other sugar and sugar phosphates. Some natural antioxidants like vitamins (B-complex), curcumin and jasad bhasma were used to check their effect and mechanism in reversing the glycation induced damage to DNA. Proteins were used as another structural model to study the differences in altering the conformation by carbonyls like glucose and methylglyoxal. SDS- PAGE was performed to analyze the glycation-induced damage to proteins and it was found that methyl glyoxal caused more damage than glucose. It can be concluded that free radicals generated via AGEs damaged DNA to a large extent and this damage was reversed by antioxidants and free radical scavengers. The damage to protein was also decreased in the presence of antiglycating agents and antioxidants. This study is significant for understanding the mechanism of formation of glycation related products and their alleviation by natural agents.

Keywords; AGE, Antioxidants, Dicarbonyls, DNA damage, Glycation

154 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 97

DECOLORIZATION OF METHYLENE BLUE BY ISOLATED MICROORGANISMS

1 Dr.Nilima Lankeshwar , Shruti Aiyar and Pallavi Chidraval IL- 4 1Department of Life Science, Ramnarain Ruia College, Matunga, Mumbai-19.

Wastewater containing dyes is very difficult to treat, since the dyes are recalcitrant organic molecules resistant to aerobic digestion and are stable to light. In present study, microorganisms were isolated from effluent samples. The dye decolorizing isolates were Streptococcus sp., Bacillus sp.and Escherichia coli. We prepared different concentrations of methylene blue, decolorization of methylene blue was observed fastest in the lowest concentrations of the dyes used i.e. 0.0001mg/ml. With increasing concentrations 0.01mg/ml,0.1mg/ml and 0.2mg/ml decolorization was slower as compared with that of 0.0001mg/ml. This study suggests that high decolorization show the potential for this microbial strain to be used in biological treatment of dyeing mill effluent.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 155 PP- 98 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

STUDY ON SYNTHESIS OF NANOCRYSTALLINE In2O3 POWDER USING ALOE VERA PLANT EXTRACT AND ITS STRUCTURAL PROPERTIES.

S. C. Kulkarnia, aDepartment of physics, S.P.H. Mahila Mahavidyalaya, Malegaon-Camp (Nashik),India

Nanocrystalline In2O3 powder was synthesized by simple, cost effective and environmental friendly route using Aloe vera plant extracted solution and Indium (III)2,4 - pentanedionate.Nanocrystalline powder was formed after calcination the dried precursor of 0 0 In2O3 in air at350 C-550 C for 2 h. The structural, morphological and compositional properties of synthesized nanocrystalline powder were characterized using XRD, SEM and

EDX. The morphology and size of In2O3 material were affected by calcination

temperature.The prepared In2O3 nanocrystalline powder shows strong PL emission in UV region.Aloe vera plant extracted solution synthesis is useful method using cheap precursor for

preparation In2O3 nanocrystalline powder.

Keywords: Aloe vera, thick film.

156 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 99

NEW INSIGHT INTO PH INDUCED CATALYTIC RATE REDUCTION OF HIV-1 PROTEASE

$ Amit Das, J.L. Ferrer and M. V. Hosur IL- 4 Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-4000085. $ Institut de Biologie Structurale J-P Ebel, Groupe Synchrotron, CEAEA-CNRS-UJF, F-38027, Grenoble, Cedex 1, France

HIV-1 protease is an important target for design of mechanism-based drugs against AIDS. The observed drastic reduction in the catalytic activity of HIV-1 protease below pH 3.0 and above pH 7.0 have been attributed, in the literature, to di-protonated and di-ionised states respectively, of the catalytic aspartates. We have undertaken crystallographic study of HIV-1 protease as different pH values to investigate pH-activity profile of this enzyme. Crystal Structures to higher than 1.8 Å resolution have been determined of unliganded HIV-1 protease, at three pH values, 2.0, 6.2 and 7.5. While the rest of the structure remains the same (average RMSD of Cα atoms = 0.23 Å), there is a difference in the water structure at the catalytic centre. At pH 6.2, there is one water molecule, hydrogen bonding to catalytic aspartates, whereas at pH 2.0 and at pH 7.5 there are two water molecules, which are hydrogen bonded to each other at 2.6 Å, and to the two catalytic aspartates. Interestingly, our hydrogen bonding analyses indicate that the catalytic aspartates at these pHs are mono- + protonated, but at pH 2.0, a water molecule exists as hydronium ion (H3O ). Conclusion: The structures of unliganded HIV-1 protease at pHs of 2.0 and 7.5 suggest a new explanation for activity reduction at low and high pH values. The water dyad in the active site can cause steric hindrance to the incoming scissile peptide bond and disrupt the stereochemical requirements for water attack. Further, proper positioning of a water molecule for activation may not happen when two water molecules are bound in the active site, and this also could contribute to loss of activity.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 157 PP- 100 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

A NEW CLASS OF ALKALINE PHOSPHATASES REVEALED THROUGH X-RAY CRYSTALLOGRAPHY

Subhash C. Bihani@, Shree Kumar Apte$, Jean-Luc Ferrer% and Madhusoodan V. Hosur@ @Solid State Physics Division, $Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400085, India, %Institut de Biologie Structurale J-P Ebel, Groupe Synchrotron, CEAEA-CNRS-UJF, F-38027, Grenoble, Cedex 1, France.

The alkaline phosphatases (AP) are metallo-enzymes, which hydrolyze phosphate monoesters to yield inorganic phosphate and water. APs are part of an enzyme super family which includes Nucleotide Pyrophosphatases (NPP), co-factor independent Phosphoglycerate Mutases (iPGM), phosphonate monoester hydrolases (PMH) and aryl sulfatases (AS). Two Zn2+ ions and one Mg2+ ion are proposed to facilitate various steps in the catalytic mechanism of APs. The hydrolysis reaction by APs is proposed to proceed via an intermediate in which the substrate phosphoryl group assumes trigonal bipyramidal conformation. An active site arginine residue, which is conserved in APs isolated from bacteria such as E.coli, and human placenta stabilizes this intermediate through bi-dentate hydrogen bonding to the substrate phosphoryl group. However, in the AP isolated from the bacterium Sphingomonas sp. (SPAP), which is more efficient in the bio-precipitation of uranium from alkaline wastes, this arginine residue is totally absent. Here we report three high-resolution crystal structures of SPAP-substrate/product complexes, determined using X-ray diffraction data collected on the FIP beamline at the European Synchrotron Radiation Facility (ESRF). The crystallographic phase problem was solved using Multi-wavelength Anomalous Dispersion method. The crystal structures reveal following unique features of SPAP: 1) threonine replacing serine as the catalytic residue, 2) absence of Mg2+ ion binding pocket, and 3) lysine residue in the active site to play the role of the Mg2+ ion. The substrate phosphoryl group is bound in a manner not observed before in any other AP. Structural superposition has been employed to analyse evolutionary relationships between SPAP and other APs. SPAP is found to represent a new class of APs, which has evolved along a route different from that of other APs. Because of the direct contact between the active site lysine with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The three dimensional structure has suggested how SPAP can be engineered for higher activity through site-directed mutagenesis. Details will be presented.

158 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 101

ANOMALIES IN THE MOTION DYNAMICS OF LONG FLAGELLA MUTANTS OF Chlamydomonas reinhardtii

†∆ † ∆ † Dolly K. Khona, Venkatramanan G. Rao, Mustafa J. Motiwalla, P. C. IL- 4 Shreekrishna Varma,† Anisha R. Kashyap, ‡ Koyel Das,¶ Seema Shirolikar,§ Lalit Borde,§ Jayashree A. Dharmadhikari,§ Aditya K. Dharmadhikari,§ Sapna,@ Siuli Mukhopadhyay,¶ Deepak Mathur,§ and Jacinta S. D’Souza†*

†UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina campus, Santacruz (E), Mumbai 400 098, India; ‡K. J. Somaiya College of Commerce and Science, Vidyavihar, Mumbai 400 077; ¶Department of Mathematics, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India; @National Institute of Pharmaceutical Education and Research (NIPER), EPIP campus, Industrial Area, Jandaha Road, Hazipur (Vaishali) – 844102, India; §Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India. ∆Equal Contribution; *Author for correspondence: [email protected]

Although C. reinhardtii has long been used as a model organism in cell motility studies and flagella dynamics in particular, the motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using real-time movies and morphological analyses, we have characterized long-flagella mutants (lf1, lf2, lf3 and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies and trajectory of swimming. These mutants, although aberrant in signalling proteins involved in the regulation of flagellar length, bring about a phenotypic variation in this length. Our results reveal that, the flagella beat frequency and swimming velocity are negatively correlated with length of the flagella, and when compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26 – 57%) and beat frequencies (by 8-16%) compared to values pertaining to wild-type cells. The cells seem to have an in-built mechanism that computes the length of the flagella required for maximal swimming speed. Any increase in this length compromises the swimming velocity either by reduction of beat frequency or by an alteration in the waveform of the flagella. We demonstrate that the mutants have no apparent ultrastructural deformities in their organelles, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells. It appears that either there are aberrations in the flagellar proteome or the ratios of dynein isoforms across the flagellar length are erratic. The latter is currently being investigated.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 159 PP- 102 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

Α-AMYLASE FROM Spirulina platensis; BIOCHEMICAL PURIFICATION, CHARACTERIZATION AND MOLECULAR CLONING.

Mosami Galvankar*, Rutwik Thengodkar*, Deepak Modi# and S. Sivakami* * University Department of Life Sciences, University of Mumbai, Mumbai-400098. # Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health, Parel, Mumbai-400012. [email protected]

Cyanobacteria, are Gram negative photosynthetic prokaryotes and have received worldwide attention due to their use in pharmaceuticals to produce various secondary metabolites including vitamins, toxins, food, fuel, colorant, apart from pollution abatement, mariculture, and fertilizer. The cyanobacterium Spirulina platensis is capable of adapting to diverse habitats like soil, sand, marshes, brackish water, seawater, and freshwater, tropical waters, thermal springs, salt pans and fish ponds. Growth and survival under diverse and stressful conditions is facilitated by enzymes. Screening for enzymes from Spirulina may lead to enzymes with possible potential in biotechnological use. Thus this study was initiated, also Spirulina has a potential of being an inexpensive and commercial source of industrially important enzymes which have not yet been explored. Objectives were to develop a biofermentor and downstream processing unit for efficient growth and biomass recovery of Spirulina. Screen for enzymes like α-amylase, catalase, urease and thioredoxin reductase; to biochemically characterize α-amylase upon purification by ion-exchange and gel filtration chromatography. Prepare a recombinant α-amylase from Spirulina gene sequence for easy industrial preparation. Spirulina platensis demonstrated the presence of α-amylase, catalase, urease and thioredoxin reductase. α-Amylase was selected for purification in purview of its industrial potential. A 40 fold induction of α-amylase activity was achieved by inclusion of 1% starch. Amylase purified to electrophoretic homogeneity by DEAE-Cellulose and Sephadex G-200 exhibited optimum activity at 50°C and was stable over a wide range of pH 6-11 and utilizes Ca2+ as a cofactor. These properties could be of interest to industrial processes running under alkaline conditions to employ amylases from Spirulina platensis. 1470 bp α-amylase gene was amplified from Spirulina platensis. Translation of the amplified sequence demonstrated that the highly conserved residues namely, Asp206 and Asp297 are located in the active site and play a key role in catalysis. The recombinant protein was successfully overexpressed and purified. The studies conducted herein have important implication in understanding the biology of S. platensis, development of tools for its mass production, induction of α-amylase and characterization of the enzyme for industrial applications.

160 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 103

DIFFERENTIAL ACETATE METABOLIC FLUX THAT LEADS TO TRIACYLGLYCEROL IN Chlamydomonas reinhardtii: AN IN-CELL NMR STUDY

Himanshu Singh1*, Manish Shukla2, B J Rao2 , KVR Chary1 1Department of Chemical Sciences, Tata Institute of Fundamental IL- 4 Research, Mumbai-400005 2Department of Biological Sciences, Tata Institute of Fundamental Research,Mumbai-400005.

In recent times, Chlamydomonas reinhardtii a unicellular algae has been a model organism for studying metabolic changes associated with biofuel production. Besides, C. reinhardtii offers duality of metabolism being both phototrophic and hetrotrophic in nature. The major metabolic flux it follows in various conditions is not yet clearly understood. On the other hand, live cell NMR, a non invasive technique, has been extensively used in the past to detect metabolites in the cells. Against this backdrop, we set out to monitor live metabolic changes in C. reinhardtii cells during light (mixotrophic) and dark (heterotrophic) phases of growth as well as following the UV/nutritionally stressed stationary phase cultures. The in-cell metabolic changes in C. reinhardtii cells were followed by monitoring [1, 2- 13C]-labelled acetate assimilation over a period of 8 days. For the initial 24 hours of detailed kinetic measurements, the assimilation in dark phase of growth was faster than light phase of growth, reflecting higher heterotrophic metabolic efficiency in dark over mixotrophic metabolism in light. While bocarbonate was the predominant metabolite observed both in light and dark phases of growth, Carbon dioxide was observed only during the dark phase of growth, reflecting a differential metabolic flux in growth. UV stress decelerated acetate assimilation and metabolite formation in cells. The UV effect also resulted in an increased bicarbonate accumulation perhaps due to reduced efficiency of carbon concentrating mechanisms operating in the cell. When the cells were incubated in light or dark for up to 8 days and monitored at intervals of about 48 hours, further remodeling in metabolism took place where in bicarbonate and Carbon dioxide were routed towards lipogenic pathway leading to lipid body production containing triacyl glycerate (TAG) metabolites. We explain this metabolic flow by a model for acetate assimilation in the cell.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 161 PP- 104 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

A MODEL FOR Α→Β TRANSITION IN PROTEINS: INSERTION/DUPLICATION MUTANT OF BACTERIOPHAGE T4 LYSOZYME

Harpreet Kaur and Yellamraju U. Sasidhar* Department of Chemistry, Indian Institute of Technology Bombay Powai, Mumbai 400 076, India E-mail: [email protected]

An α→β transition underlies the first step of disease causing amyloidogenesis in many proteins. In view of this, many studies have been carried out using peptide models to characterize these secondary structural transitions. In this paper we show that an insertion/duplication mutant ‘L20’ of bacteriophage T4 lysozyme (Sagermann, M.; Baase, W. A.; Matthews, B. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6078-6083) displays an α→β transition. We performed molecular dynamics (MD) simulation of L20, using GROMACS package of programs and united atom GROMOS 53a6 force field for a time period of 600 ns at 300 K, in explicit water. Our MD simulation demonstrated that the transition occurs in a duplicated α-helical region inserted tandemly at the N-terminus of the ‘parent’ helix. We show that a C-terminal β-sheet anchors the parent helix while the loosely held N-terminal loop in duplicate region is vulnerable to solvent attack and thus undergoes an α→β transition. Main chain-solvent interactions were seen to stabilize the observed β-structure. Thus L20 serves as a good protein model for characterization of α→β transition in a full length protein.

162 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 105

ROLE OF ELECTRICAL PROPERTIES OF EXOGENOUS DOPAMINE AS NEUROTRANSMITTER

1 1 2 Mugdha Patki , Vidya Patil , Prajakta Borgaonkar IL- 4 1. Department of Physics, D.G.Ruparel College, Mahim, Mumbai 2. Department of Physics, Vikas College Vikhroli, Mumbai Address for correspondence:D.G. Ruparel College, Senapati Bapat Marg, Mahim, Mumbai 400 016 . e-mail id: [email protected] Tel.No. 022-28372543, Fax No. 022-24713450

In human body, a neuro-signal is transmitted from one neuron to the next neuron across a synaptic cleft through a biomaterial called neurotransmitter. In vitro study of a small bio- molecule which is one of the biogenic amine, that is dopamine, was conducted. The purity of the selected material is endorsed by FTIR spectroscopy. Various electrical properties like dielectric constant, conductivity of exogenous dopamine were studied. Variations of capacitance and conductivity with concentration and temperature were studied and dielectric constant was calculated. The experimental value of dielectric constant indicates that the material under consideration is in a ferroelectric range. The non-linear rise in the dielectric constant can be attributed to structural reorientation of neurotransmitter. Frequency response for different concentrations in tune with variation in capacitance was observed. This study may lead to understanding the physiological and psychological behavioral responses of human body with temperature variations. This study of biofeedback can be used in the complementary and alternative medicine therapy.

Keywords : Dopamine, dielectric constant, conductivity, alternative medicine.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 163 PP- 106 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

MOLECULAR DOCKING, MD SIMULATIONS AND FREE ENERGY CALCULATIONS OF DENV INHIBITORS FOR A NOVEL BINDING SITE

Kshatresh Dutta Dubey and Rajendra Prasad Ojha Biophysics Unit, Department of Physics, DDU Gorakhpur University, Gorakhpur, 273009, India. Email- [email protected], [email protected]

The Dengue hemorrhagic fever is one among most fatal human pathogen that causes thousands of death annually worldwide. Entry of dengue virus is mediated by conformational change in envelope protein due to change in endosomal pH. The structural study of dengue envelope protein (DENV) reveals that domain-III of envelope protein exhibits largest conformational change during entry of virus. Hence, a drug which may block this conformational change will be relatively more effective.

In the present work we aim to explore the hot spots and key interacting residues using some known drugs using molecular docking, molecular dynamic simulations and free energy calculations. We have investigated the conformational changes in envelope protein in presence of different drugs and pointed out those residues which commonly participate in binding of all drugs. The binding of these drugs was calculated by free energy methods and molecular docking methods and their relative accuracy were also compared. We have found several amino acids commonly interacting to all five drugs. In addition, the results of molecular dynamic simulations was more reliable than molecular docking. The binding free energy of R1 is better than other drugs which is analogues to the experimental observations. The root mean square deviations for R1 is better than other drugs which also supports a stable (less flexible) binding of this drug.

164 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 107

IN-SILICO DESIGNING OF POTENT LEAD MOLECULES DHPM DERIVATIVES AGAINST NOVEL PROTEINVOLTAGE-DEPENDENT P/Q-TYPE CALCIUM CHANNEL SUBUNIT ALPHA-1A ISOFORM 1 IL- 4 Afaque Momin,YashodhanChipkar,S K Sahoo, ManojRathod,GN khalsa college, Matunga, Mumbai and SVNIT Surat, Gujrat

Hypertension is the term used to describe high blood pressure.Blood pressure is a measurement of the force against the walls of arteries as heart pumps blood through the body. Hypertension is caused due to the high intake of calcium through the blood vessels by the Voltage Gated Calcium channels.Voltage-dependent calcium channels (VDCC) are a group of voltage-gated ion channels found in excitable cells (e.g., muscle,glial cells, neurons, etc.) with a permeability to the ion Ca2+.The Novel protein sequence for the calcium channel protein was found on the NCBI website. The name mentioned was Voltage-dependent P/Q- type calcium channel subunit alpha-1A isoform 1 [Homo sapiens]” (Uniprot ID: - NP_000059.3). The 3D Model for the protein was searched throughout the online databases but no valid model was found on any of the databases, so we decided to model the protein. The length of the protein was 2266 amino acids. The Swiss Automated Modeller, Salilab Modeller Software 9.3 were used to perform the basic homology modelling but it resulted in low sequence similarity. Therefore the model was rejected. We tried the online server i- Tasser for ab-initio modelling but due to the length it was rejected by the server. Finally, the @tome Meta server was used to perform the Advanced homology modelling using Multiple template sequence alignment with the templates 3RVY, 2R9R, 3RVZ, 3BEH and 2KAV which resulted in a good model and the result accuracy was checked by Ramachandran plot. The present drugs against hypertension are the DHPM derivatives(Di-Hydro Pyrimidinone Derivatives) We designed 30 different analogs from the 2D structure of the drug using Chemdraw Ultra software, checked the drug likeness using PASS Software and minimized the energy and calculated bond angles and torsion angles and stabilized the structures using Hyperchem Software. The Docking process was performed using Maestro Schrodinger 2012 (Glide Dock). The docking of the DHPM analog no.5(2R,4S)-methyl2-amino-1,2,3,4- tetrahydro-4-methyl-6-((S)-7-methyl-7Hcyclopenta[c][1,2]oxathiin-3-yl)pyrimidine-5- carboxylate),Drug Likeliness of 0.919 was the best with the protein binding site. Adomet Toxicity of the designed analogs was checked using Medchem Designer Software and all the analogs were found to be non-toxicto absorption, distribution, metabolism, and excretion in human body. The Glide Score (G-Score) obtained was -7.84, which showed promising results. The strong interactions were found with the Amino acids THR1461, GLU668 and GLN663. Other interactions were found with the Amino Acids LEU1366, LEU494, LEU600, GLU1463, TYR596, THR594, THR666, PHE1370, PHE1457, VAL1369, and TRP597. We can conclude that the novel protein which we modeled is responsible for the disease hypertension and the improved drugs which we have designed in the form of the DHPM derivatives could be used as improved and better drugs against hypertension. Keywords: - Hypertension, Voltage-dependent calcium channels,DHPM derivatives.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 165 PP- 108 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECTS OF MECHANICAL VIBRATIONS ON GROWTH AND PHOTOSYNTHETIC YIELD IN MUNGBEAN (Vigna radiata L.) SEEDLINGS

V. K. Ghodake, S. S. Jagtap, G. R. Kulkarni, P. B. Vidyasagar Biophysics Laboratory, Department of Physics, University of Pune, Pune 411007, India. E-mail: [email protected]

Natural or artificial vibrations such as earthquake, blasting etc affect adjacent living and non living things. Due to mechanical vibrations seeds undergo physiological and developmental changes arising from mechanical stress. Effects of mechanical vibrations on Arabidopsis thaliana plant, Cucumber seed, rice seed etc have been already reported. The objective of the present study is to study the effects of low frequency vibrations on growth of mungbean seedlings, chlorophyll content and photosynthetic yield in shoots. Mungbean (Vigna radiata L.) seeds were exposed to sinusoidal vibrations of low frequency i.e. 10 – 60 Hz, amplitude of 2 mm for 10 minute duration using Electrodynamic Shaker system. Then the seeds were soaked in distilled water for 24 hours and grown on 0.8% agar- agar for five days using a standard seed germinator. Fluorescence spectra of shoot chlorophyll, Performance Index (PI) of photosynthesis showed significant differences between control and vibration exposed samples. Measurements with Handy PEA instrument showed an increase in maximum quantum efficiency of photosystem II (Fv/Fm) after five days in mungbean seedlings when exposed to 10 Hz mechanical vibration.

Keywords: Mechanical Vibration, Mungbean, Frequency, Amplitude, Chlorophyll fluorescence, Photosynthetic yield

166 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 109

THERMODYNAMICS OF PROTEIN-LIGAND INTERACTIONS: BINDING OF THE PHENAZINIUM DYES PHENOSAFRANIN AND SAFRANIN O WITH HUMAN SERUM ALBUMIN IL- 4 Jhimli Bhattacharyya, Ishita Saha, Gopinatha Suresh Kumar Biophysical Chemistry Laboratory, CSIR-Indian Institute of Chemical Biology Kolkata 700 032 Email: [email protected]

Elucidating the thermodynamics of small molecule-serum protein interactions and correlating to the structural aspects have multifaceted implications for the development of more effective agents for chemotherapy and photodynamic therapy. In this study, thermodynamics of the binding of two phenazinium dyes, phenosafranin (PSF, 3,7-diamino-5-phenyl phenazinium chloride) and safranin O (SO, 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride) with human serum albumin (HSA) was correlated with structural aspects. Along with the calorimetric techniques, fluorescence and circular dichroism spectroscopy was used to understand the interaction in details. Binding parameters revealed that PSF has higher affinity (K = 1.60 × 105 M-1) compared to SO (K = 0.97 × 105 M-1) to HSA. The binding of both dyes was favoured by negative enthalpy and a stronger favourable entropy contribution. The o negative values of ∆Cp in both cases ndicated the involvement of similar hydrophobic forces in the complexation.and suggested that the binding is specific and accompanied by burial of nonpolar surface area. Enthalpy-entropy compensation was also observed for both dyes. The fluorescence quenching studies suggested a static quenching mechanism. Forster resonance energy transfer (FRET) data showed that the specific binding distances between Trp (donor) residue of the protein and the dye (acceptor) molecules were 3.95 and 4.07 nm, respectively, for PSF and SO. Competitive studies with site markers revealed that both dyes bind to same site, site I (subdomain II A) of HSA but SO with bulkier groups binds weakly due to steric hindrance. The binding of the two dyes altered the protein conformation by reducing the α- helical content. This work presents an in-depth understanding of the structural and energetic aspects of the interaction of the phenazinium dyes with the serum protein HSA, that may be helpful for futuristic application of these molecules in drug therapy.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 167 PP- 110 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECT OF SIZE OF SUGAR OSMOLYTES ON THE STABILITY AND THE FUNCTIONAL ACTIVITY OF LYSOZYME

*Asimul Islam, Ilyas Beg and Faizan Ahmad Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Correspondence: Phone: +91-11-26983409. E-mail: [email protected]

Many organisms survive in various stresses by synthesizing and accumulating low molecular weight organic compound called osmolytes that arose by natural selection and have the ability to stabilize intracellular proteins against the environmental stress. Stabilization by osmolytes induces folding of aberrant proteins and therefore, it is of therapeutic use for a large number of protein misfolding diseases. It is generally accepted that stabilizing osmolytes are the selective advantage for organism to adapt to environmental stresses, less is known about the interrelationship between the stability provided by most of the common osmolytes and the effect of enzyme activity. In order to test the relationship between the function and stability of proteins in the presence of sugars, we measured enzyme activity and o thermodynamics stability (ΔGD ) of lysozyme in the absence and presence of different concentrations of sugars near physiological conditions. To investigate the effect of monomeric sugars on the stability of lysozyme, we chose glucose, galactose and fructose from hexose sugars. Glucose and galactose are epimer to each other and have aldehyde groups whereas fructose has keto group. Our results showed that sugar osmolytes increased o Tm and
GD of lysozyme at each pH. The lysozyme stabilization by sugar osmolytes showed

that the change in the Gibbs free energy (ΔGD) associated with the equilibrium, N (native) state ↔ D (denatured) state of the protein in the presence of equimolar mixture of glucose and fructose is larger than that of sucrose which is disaccharides of glucose and fructose.

However, at the molar scale, ΔGD obtained in the presence of sucrose is much higher as

compared with ΔGD obtained using individual monosaccharide. In order to understand kinetics, we studied the activity of lysozyme in the presence and absence of different concentrations of various sugars. Sugar polyols at lower concentration were found to be

minimal perturbation on the kinetic parameters, Km and kcat. At higher concentrations, sugar

polyols were found to decrease the kinetic parameters, Km and kcat of lysozyme. Moreover,

both Km and kcat of the protein were found to decrease with the increasing sizes of co- solvents. Our results suggest that crowding of co-solvents favour more number of N states of the protein which lead to increase of enzyme-substrate affinity but at the same time it decreases the catalytic rate of the enzyme. This shows that crowding of co-solvent effects on global protein stability may be distinct from effects on mobile regions of the protein that undergo conformational changes during catalysis.

168 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 111

STRUCTURAL BASIS OF LIGAND RECOGNITION IN MAMMALIAN HEMEPEROXIDASES.

Punit Kaur, Rashmi P. Singh, Amit K. Singh, Nisha Pandey, Mau Sinha, Sujata Sharma IL- 4 and Tej P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

Mammalian heme peroxidases constitute a group of proteins that consists of four members including lactoperoxidase (LPO), myeloperoxidase (MPO), thyroid peroxidase (TPO) and eosinophil peroxidase (EPO). They catalyze peroxidation of halides and pseudohalides with the help of hydrogen peroxidase. They also catalyse single electron oxidation of various physiologically important organic substrates including phenols, catecholamines and catechols. They play an important role in non-immune host defense against infectious organisms.We have determined several structures of LPO in unbound state and in complexed states with halides, pseudohalides and aromatic compounds. The complexes with SCN¯ have shown that SCN ion binds to LPO at the distal heme site at a favourable distance from catalytically important water molecule W1. The structure also revealed an additional SCN¯ binding pocket which is part of the substrate diffusion channel to keep second SCN¯ ion ready for diffusion to the catalytic site. The structures with halides have revealed the mode of binding and provided insights on the preference for the binding of halides. The structures of complexes with aromatic compounds have shown two types of modes of binding where aromatic compounds are either held at the substrate binding site at a hydrogen bonding distance with conserved water molecule W1 or they replace W1. The compounds act as substrates when they do not displace W1 and inhibitors when W1 is replaced. The alignment of compounds to act as substrates in LPO is guided by Gln 423 while such a selection criterion is not available in MPO where Gln423 is replaced by Glu423. As a result MPO shows poor preference for aromatic substrates

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 169 PP- 112 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SILVER NANOPARTICLES SYTNHESIZED UNDER CLINOROTATION AND ASSESMENT OF THEIR CYTO-GENOTOXICITY

Avinash J. Aher†, Vijay L. Markad††, Sagar S. Jagtap†, K. M. Kodam††, Suresh W. Gosavi†, Pandit B. Vidyasagar† †Department of Physics, University of Pune, Pune-411007, India. ††Biochemistry Division, Department of Chemistry, University of Pune, Pune-411007, India.

From past several years microgravity has been used as a tool in field of biological and physical processes. Microgravity environment provieds a unique window to gain a better understanding of how gravity driven phenomena like sedimentation, bouyancy driven convection, solidification and crystal growth get affected. Microgravity allows researchers to study underlying events free from these effects. Materials science research in microgravity can lead to a better understanding of how materials are formed and how the properties of material are influenced by their formation in microgravity. In this regard, present study deals with studying the effects of clinorotation (simulated microgravity) on biosynthesis of silver nanoparticles using bacterium Escherichia coli (DH5α). Microgravity condition was simulated by using 1-dimensional horizontal axis clinostat. Silver nanoparticles synthesized under simulated microgravity condition were characterized by using UV-Vis spectroscopy, Energy dispersiveX-ray spectroscopy (EDX), Transmission Electron Microscopy (TEM) and X-ray Diffraction (XRD) techniques. These AgNPs were further screened for their cyto- genotoxic activity against cervical cancer cell lines and human skin keratinocytes (HaCaT) using alkaline comet assay. Results indicated that AgNPs caused dose dependent reactive oxygen species production that leads to DNA damage and decreases cell viability.

170 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 113

HIGHLY SENSITIVE ELECTROCHEMICAL BIOSENSOR PLATFORM USING AU-POLYANILINE NANOCOMPOSITE

Ankan Dutta Chowdhury, Rupali Gangyopadhyay, Amitabha De IL- 4

Development of a biosensor based on Au-Polyaniline nanocomposites is described in the present work. In the present work, gold nanoparticles (AuNP) incorporated Polyaniline nanowires (PAn-NW) were used for fabrication of a novel biosensing platform. PAn-NW with AuNP (8-10 nm) was treated with β-mercaptoethylamine (MEA) that leads to the formation of the -NH2 functionalized PAn-AuNP-β-MEA in dispersed form. Immobilizing the functionalized dispersion on the Pt electrode makes a biosensing platform suitable for attachment of different biomolecules via -NH2 functionality and sensing of the target elements in turn. This immobilization offers an easy but efficient technique for addition of functional groups to conducting polymers and eliminated the hazards of functionalizing biomolecules. These biomolecules can be varied by any enzyme like glucose oxidase to monitor glucose concentration or any oligonucleotide to monitor DNA concentration. Detailed characterizations of the synthesized materials by TEM, UV-vis and FT-IR spectroscopy have revealed the presence of Au nanoparticles on PAn surface and covalent attachment of GOx to the electrode for glucose analysis. This system can be used for amperometric detection of glucose in aqueous solution over a wide concentration range (1µM-20mM) with a very good sensitivity. The Pt||PAni/Au-PAni/GOx biosensor exhibits a good linear relationship with the total concentration of glucose in the range of 1 to 20m M. The response displays a linear range in figure 5B from 1mM to 20mM with a correlation coefficient of 0.996 (n=7) and a slope of 0.012 µAmM-1. The sensor exhibits good stability (robustness), sensitivity of 14.63 µAmM-1cm-2 with a low limit of detection of 1µM (measured with the chronoamperometry and the flow cell techniques) and selectivity with respect to common interferences (AA, UA) in blood. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents avoiding generation of an overlapping signal from such interferers. All results including the stability test show that the proposed system presents a promising electrode for glucose detection.

Ref. : (1) Functionalized polyaniline nanowires for biosensing, Rupali Gangopadhyay, Ankan Dutta Chowdhury, Amitabha De; Sensors and Actuators B: Chemical, Volumes 171–172, August–September 2012, Pages 777–785. (2) Electrochemical Biosensor for Low Level Glucose Detection Based on Glucose Oxidase Covalently linked to Polyaniline- Gold Nanocomposite; Ankan Dutta Chowdhury, Rupali Gangopadhyay, Amitabha De, (Communicated).

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 171 PP- 114 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EFFECT OF HYPERGRAVITY AND GAMMA IRRADIATION ON WHEAT SEEDLINGS

S. Singh, S. S. Jagtap, P. B. Vidyasagar Department of Physics, University of Pune, Pune 411007 Email: [email protected]

Studying the Physiological response of plants to altered gravity is very significant, as gravity plays an important role in overall growth of plants, and studies related to radiation effect on plants also has a great purpose in space-based farming research. Thus bearing this in mind, several experiments considering effect of hyper gravity (300g-1500g), gamma irradiation (20Gy-100Gy)and combined effect of hyper gravity(300g-1500g) with gamma irradiation(40Gy) were carried out on pre-soaked wheat genotype(lokvan) seeds and the physiological response of wheat seedlings was studied. Root length, shoot length, chlorophyll content, fluorescence, proline content and photosynthetic activity were measured on 5th day. The results obtained showed that root and shoot length, as well as chlorophyll content and fluorescence decreased with increasing radiation dose however net photosynthetic rate, stomatal conductance and proline content increased with increasing radiation dose. Similar effects were observed in pre-soaked wheat seeds exposed to hyper gravity for short interval and grown in normal conditions. Further, combined effects of hyper gravity and gamma irradiation will also be discussed.

Keywords: Gamma, altered gravity, wheat

172 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 115

“EFFECT OF ELECTROMAGNETIC FIELDS EMITTED BY CELLULAR PHONE BASE STATION ON HUMA HEALTH”

Dr. Rajendra V. Joshi* & Dr. H.D. Khanna** IL- 4 *Lecturer in Biophysics, Department of Physiology, SMIMER, Surat **Professor, Department of Biophysics, IMS, Banaras Hindu University, Varanasi.

In today’s world Globalization is the new mantra. It is very difficult not to have technology. But with technology, come certain hazards. The only way to beat these is again, better technology. Electromagnetic radiation is everywhere. More and more wireless communication services (cellular phones, wireless internet, etc.) are expected to come up and it seems that there is no way to reverse this trend. A cell phone technology is one of them, which introduce in India few years back, but now its need of society. It also works on electromagnetic radiation. Therefore, the aim of the present study is to find out the effect of cellular phone base station radiation, due to frequent exposure, on human health. The present study was conducted over a period of three year. This covers urban as well as rural areas of Surat districts. The city of Surat was selected as the urban area while villages surrounding it represented the rural areas. Study obtains information regarding radiation exposure to individuals by means of cellular phone base stations and their effect on human health. Study was undertaken in 66 subjects, including control group. The study includes anthropometric parameters, a detailed clinical examination and standard cardiovascular autonomic function tests. We are not at all against any technologies coming up, we are welcoming all the technologies, but we maintain that health precautions and safety precautions must be taken. Even if effects are of small amplitude and do not seem to be detrimental. But once the energy is absorbed by the biological matter, can cause severe and long lasting damage to human health and effects will enhance due to frequent exposure to source. It might take years for the damage to produce noticeable symptoms.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 173 PP- 116 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ACTIVATOR BINDING SITE OF SOLUBLE GUANYLATE CYCLASE

Tajith B. Shaik, M.G. Meena Lakshmi, M.M. Idris and Biswajit Pal Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Uppal Road, Hyderabad – 500007. E-mail : [email protected]

Soluble Guanylate Cyclase (sGC), a heterodimeric heme protein, is an emerging therapeutic target for cardiovascular disease, erectile dysfunction, etc. This protein contains a large (alpha1) subunit and a small (beta1) subunit. Upon binding of NO to the heme iron, heme- protein covalent bond is cleaved and structural reorientation occurs. This signal is transferred to the catalytic domain, which converts GTP into cGMP, which has several downstream regulatory functions such as smooth muscle relaxation, activation of gated ion channels, etc. Failure in the NO/sGC/cGMP pathway leads to different pathological conditions like hypertension, heart disease, erectile dysfunction, etc. This pathway is targeted by blocking the degradation of cGMP using phosphodiesterase inhibitor drugs (e.g. Viagra) for the treatment of these clinical conditions. A new series of compounds, like riociguat, cinaciguat, etc. are on clinical trials to treat pulmonary hypertension, heart failure, etc. These compounds target sGC in heme-dependent as well as heme-independent manner. However, the binding site(s) of these compounds are not well characterized. Based on docking studies, it was proposed that YC-1 (3-[5′- hydroxymethyl-2′-furyl]-1-benzylindazole), a heme-dependent exogenous activator, binds to the N-terminal domain (NTD) of alpha1 subunit of sGC (Pal & Kitagawa, BBRC, 2010, 397, 375-379). To experimentally validate the hypothesis, alpha1-NTD from zebrafish was cloned, over- expressed in E. coli and purified. Purified recombinant protein was used to verify the binding of YC-1 using “reverse titration” in Isothermal Titration Calorimetry. Our results clearly demonstrate that YC-1 binds in the NTD of alpha1 subunit of sGC. This finding would help in future structural studies, which in turn, would help to design more specific activators of sGC.

174 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 117

DOLASTATIN 15 ENHANCES THE CHEMOPREVENTIVE POTENTIAL OF CELECOXIB BY INHIBITING PI3-K/Akt EFFECTORS PATHWAY IN COLON CARCINOGENESIS IL- 4 Honit Piplani and S N Sanyal Department of Biophysics, Panjab University, Chandigarh 160 014, INDIA Email: [email protected]

Recent studies in the past decade had shown Phosphoinositide 3-kinase (PI3-K) to be an important regulator of apoptosis during the chemoprevention of various types of cancers including Colorectal Cancer using various Natural and Synthetic Compounds. Also, combinatorial strategy of using NSAIDs and natural compounds can bring down the side effects of NSAIDs while enhancing the chemopreventive potential of the drug. In the present study, we have explored the combinatorial strategy of using NSAID, Celecoxib along with Dolastatin 15, a natural compound isolated from Dolabella auricularia to suppress the colorectal cancer in the early stage and also examined the underlying mode of action of these drugs by studying the PI3-K effector pathway. In vivo, after 6 weeks of DMH administration, apoptotic cell index was found to be lowered as compared to the normal. However, both the drugs had increased the apoptotic cell index while inhibiting the PI3-K and Akt expression as assessed by Western blot and Immunofluorescence analysis. Molecular docking studies reveal both the proteins to be the direct targets of the drugs via their interaction with the ATP binding site. The effect was accompanied by an increased expression of GSK-3β, Bcl2 family protein Bad, transcription factor Egr-1 and important tumor suppressor protein PTEN. Also, during the chemoprevention, G1- associated cell cycle protein, Cyclin D1 expression was lowered along with an increase in the level of intracellular calcium as found out by FURA-2 and CTC analysis. The level of reactive oxygen species was also found to be higher in case of Celecoxib and Dolastatin 15 treated groups. The present study also revealed that the number of cells having higher mitochondrial membrane potential was lowered during chemoprevention as assessed by Rhodamine 123 flow cytometric and spectrofluorimetric analysis.

Keywords: Celecoxib, Dolastatin, Egr-1, Molecular Docking, PI3-K.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 175 PP- 118 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

COMPARATIVE STABILITY MEASUREMENT OF C-PE IN WILD TYPE AND NATURAL TRUNCATED FORMS

Khalid Anwer1*, Safikur Rahman1, Avani Kaushal2, Datta Madamwar2, Faizan Ahmad1 and Md. Imtaiyaz Hassan1 1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi 110 025, India 2BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, [email protected]

Studies based upon folding/unfolding of proteins are of great interest and importance since a long time because, the mystery of folding is still remaining unsolved. Here we are reporting the folding studies on cynaobacterial phycoerythrin from Phormidium tenue. We observed that the α-subunit of 164 amino acid residues (19 kDa) of C-phycoerythrin (C-PE) undergoes degradation in the starved condition and released a truncated α-subunit of 133 amino acid residues (14 kDa) only, that is devoid of 31 N-terminal residues. We studied the denaturation pattern of the α-subunit of C-PE in the intact as well as in truncated (structure recently determined) forms using GdmCl, Urea, LiCl along with pH and temperature dependent, using UV-visible absorbance, CD & fluorescence spectroscopy and DSC. The results showed an o appreciable difference in ΔGD (~1.0 kcal mol-1), m (~ 0.5 kcal mol-1 M-1) and Cm (~0.25 in o M GdmCl) between intact and truncated proteins. The difference in ΔGD suggests that the two proteins are almost equally stable and the natural deletion of 33 N residues do not alter its structure and thus functions. The pH-induced denaturation infers that the optimum pH of both the forms is 7 - 8.5, however stable from pH 5 - 9. The pH induced denaturation curves further suggest that the transition of melting starts from pH 5 and lasts around pH 3.0 in both the cases. Thus, the two proteins are almost equally stable concerned to pH-induced denaturation. Structural analysis concludes the presence of 3 crucial hydrogen bonds (between Met1 and Glu109, Lys2 and Gly103 & Asn30 and Arg33), the absence of which in o truncated form is responsible for difference in ΔGD value and stability. The proteins are irreversible through thermal denaturation but reversible using GdmCl-induced denaturation.

Moreover the transition curves of different probe are precisely overlapping on fd plot, suggests that GdmCl-induced denaturation is two state (N & D) and one step (ND) process in truncated as well as in intact wild type forms of C-PE.

176 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 119

IDENTIFICATION OF TRACE ELEMENTS IN BUFFALO HORN SAMPLE BY SPECTRO CHEMICAL ANALYSIS

1 2 3 D. Saira Khanam S.M.D. Shoaib R. Jeevan Kumar IL- 4 and Adeel Ahmad 1 1 Biophysics Unit, Department of Physics, Nizam College (Autonomous), Osmania University, Hydearabad-1, A.P., India. 2 School of Physics, Maulana Azad National Urdu University, Polytechnic Branch, Darbhanga- 846 001, Bihar State, India. 3 Molecular Biophysics Laboratory, Department of Physics, S.K. University, Anantapur-515 055, A.P., India. Email: [email protected]

Horn is highly organized material from the gross macroscopic to the molecular level. As for as organic matter is concerned, the major portion of the horn is made up of albuminoidal proteins called Keratins. Horns are the mixture of both polar and non-polar organic (Keratins) and inorganic (Calcium phosphate) materials. Horn is natural composite material, which by weight contains about 55% to 60% mineral, 30% to 40% matrix and 5% to 10% water. Spectro chemical method is described for the determination of major, minor trace elements in animal horn. In the present study we analysis the buffalo horn samples by spectro chemical analysis using Jerrel Ash 1.5 M plane grating spectrograph (Model No. 19-300 USA) and we trace out few major and minor constituents present in it. Analytical methods are described for the determination of major, minor trace elements in horn. Zn, K, Fe, Mg, Ba, Al, Ca, Sb and Cd are determined in the buffalo horn as major trace elements and the following 17 elements are determined as minor trace elements i.e., Mn, Cr, Ni, Pb, Cu, Na, W, V, Ti, Tl, Rh, Mo, Co, Sn, Be, Bi, and Ag. It is interesting to note that very low amount of these trace elements have been shown to be essential to the growth of animal horns. The results are further discussed in detail.

Key Words: buffalo horn sample, keratin, major and minor trace elements.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 177 PP- 120 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

DEVELOPMENT OF SPR BIOSENSORS FOR VIRAL PATHOGENS

Praveen Singha, R P Singha , K K Rajaka ,Takeshi Onoderab, V.P.Singha, Satish Kumara aIndian Veterinary Research Institute Izatnagar, Bareilly-2432122, UP, India bBioelectronics Lab, Department of Electronics, Kyushu University, Fukuoka, Japan

Biosensors are electronic devices which transduce biological recognition event to a measurable electrical signal. They are rapid, real-time, label-free and specific and highly sensitive. Surface plasmon resonance biosensors are optical biosensor; detect the change of refractive index at interacting interface sensitively. They offer the most popular and promising biosensor technology to date. In the work, reported here, we have attempted to develop SPR biosensor surfaces for two prominent viruses of small ruminant Goat and Sheep namely the PPR virus and Blue Tongue virus. Gold sensor chip were prepared by layer by layer self assembling process. Carboxy-EG6-hexadecane thiol and 16-mercapto hexadecanoic acid self assembled monolayers based surfaces were found to be superior to amino terminated surfaces. PPR antibody(4G6) was immobilized on sensor surface for detection of target antigen. BTV peptide was also self assembled on gold surface and had shown its efficacy in detection of positive serum samples. Surface has shown high affinity and high resonance angle shift on interaction with concentrated PPR antigen. Surface behaved consistently and repeatedly shown stable response with similar antigen. SPR biosensor has shown distinct response in terms of angle shift when cell culture grown antigen was flown without treatment. Biosensor surface was regenerated in 5 minutes with injection of 65 ul of 50 mM NaOH for next antigen cycle.

PPR Antigen injection over

) Fig. 1: Interaction of 4G6 antibody surface 16-MHDA Surface-4G6 PPR g

e 68.1 monoclonal-PPR Ag Interaction PPR antigen with 4G6 d ( e

l ligand immobilized g

n over MHDA-SAMs. A

e The 4G6 monoclonal c 68 n

a antibody based SPR n o

s sensor surface is being e

R tested for 67.9 reproducibility and storability. 0 2000 4000 6000

Time(s)

The SPR sensorgram over the sensor surface is shown in Fig. 1. Detection limit of sensor surface is being tested and efforts are on to enhance it through surface manipulation and higher loading of ligand.

178 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 121

IN-SILICO STRUCTURE MODELING AND CHARACTERIZATION OF HYPOTHETICAL PROTEIN PRESENT IN HUMAN FETAL BRAIN (Q14166)

Parakh Sharma, Komal Patil, Devangi Sarang IL- 4 Department of Bioinformatics, G.N.Khalsa College,Mumbai, INDIA

Human fetal fore brain is formed of sub plate, thalamic reticular nucleus (TRN) complex and ganglion eminence. Next to the TRN, within the fibres of internal capsule a group of neuron is present in early developmental stage called as perireticular thalamic nucleus.The perireticular nucleus (PN) is described thin sheet of small cells. PRN role in brain development is guiding fibers towards their end stations or serving to rearrange the complex mapped projections linking thalamus and cortex. After the axons have formed their connections, the PN rapidly decreases in size and disappear in adult. All three structures are essential for the formation of adult projections indevelopmental neuropathology as these structures may be damaged in disorders that preferentially occur in preterm infants.One of the hypothetical protein(HP) in the PN isHP KIAA0153 (Q14166).It is predicted to serve role in cell cycle, DNA-condensation, neurogenesis, or apoptosis and important in human brain development. Modeling and characterizationis necessary to understand crucial function of protein, as its structure is not available in any online structure databases. Q14166 is like Tubulin tyrosine ligase protein 12 (644 amino acid AA) contains TTL domain from 340-644 amino acid residues. Thesequence for Q14166 was retrieved from UniProt database. TheSwiss Modeller server, Geno 3d,and PS2 server gave low sequence similarity so the model was rejected. I-tesser server modeled Q14166and the resulted structure was evaluated with Evaltool, which shown significant values. The Ramachandran plot gave 86% allowed region and 5% disallowed region. Insilico bioinformatics tools like CDD, Pfam, and Prosite, InterPro, String database characterized further HP Q14166 protein. The resultant protein has multidomains from (1-32 AA)it belong to clan 07657gp45 sliding clamp, C terminal superfamilyessential for the interaction with polymerases. They adopt a DNA clamp fold; consisting of two alpha helices and two beta sheets the fold is duplicated and has internal pseudo two-fold symmetry. (From 32-224 AA) isP-Loop-NTPase Superfamily, clan 09099.This family has conserved nucleotide phosphate-binding motif (Walker A and Walker B motif)and hasdiverse cellular functions. Lastly from (225-339 AA) isclan 03778includes a diverse set of enzymes that possess ATP-dependent carboxylate-amine ligase activity. There were no results from Pfam, InterPro, Prosite, String tool for (1-340 AA). Lastly structure of resultant protein was compared from VAST, DALI server with other similar proteins. We concluded that (32-224 AA) that has same domain asP-loop NTPase superfamily and similar to protein (PDBID: 1QBK B). For other domain there was no exact match found.Q14166 has multi domains and same functions like their resultant domainsuperfamilies. Further this domain prediction can help to study protein-protein interactions, at the brain level disorders that preferentially occur in preterm infants.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 179 PP- 122 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

INTERACTION STUDIES OF PYRAZOLO[3,4-D]PYRIMIDINES WITH DNA

Umesh Yadavaa and Ramesh Kumar Yadavb aDepartment of Physics, DDU Gorakhpur University, Gorakhpur -273009 bDepartment of Physics, B.R.D. Post Graduate College, Deoria -274001

The interaction studies of pyrazolo[3,4-d]pyrimidine compounds with DNA has been carried out through molecular docking and MD simulation methods. Glide Standard precision and Extra precision modules are used for docking. The docking energy, glide score and hydrogen bonding interactions as observed from both methods show that docking of pyrazolo[3,4- d]pyrimidine moieties connected with a trimethylene linker recognizes both the strands of DNA through hydrogen bonding interactions within the minor groove of DNA. To study the stability of the docked complex, Molecular Dynamics simulation of the best docked complex was also carried out for 12.0 ns using DESMOND. RMSD calculations, energy variations and hydrogen bonding distance variations with time shows that the complex remain stable during the course of dynamics and Pyrazolo[3,4-d]pyrimidine compounds may be used as minor groove binders.

180 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 123

PYRAZOLO[3,4-D]PYRIMIDINES AS ANTI-COAGULATION AND ANTI- INFLAMMATION AGENT: INSIGHT FROM MOLECULAR DOCKING AND MD SIMULATION STUDIES IL- 4 Umesh Yadava, Maheshwer Singh and Mihir Roychoudhury Department of Physics, DDU Gorakhpur University, Gorakhpur -273009

Phospholipase A2 (PLA2), isolated from Daboia russelli pulchella (Russell’s viper), is enzymatically active as well as induces several pharmacological disorders including neurotoxicity, myotoxicity, cardiotoxicity, anti-coagulant, haemolytic, and platelet effects. Indomethacin reduces the effects of anti-coagulant and pro-inflammatory actions of PLA2. Pyrazolo[3,4-d]pyrimidines constitute a class of naturally occurring fused uracils that posses diverse biological activities. Nine trimethylene-linker molecules based on pyrazolo[3,4- d]pyrimidine cores, whose crystallography was reported previously, have been used for docking studies with the X-ray crystal structure of russelli's viper PLA2 (PDB ID: 3H1X), using AUTODOCK and GLIDE (Standard precision and Extra precision) modules. Docking results through each method demonstrate that pyrazolo[3,4-d]pyrimidine molecules can bind with both anti-coagulation and enzymatic regions of PLA2 and may be used as anti- coagulation and anti-inflamatory agents. To study the stability of the docked complex, Molecular Dynamics simulation of the best docked complex was also carried out for 15.0 ns using DESMOND. RMSD calculations, energy variations and hydrogen bonding distance variations with time shows that the complex remain stable during the course of dynamics.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 181 PP- 124 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

PROBING THE MOLECULAR MECHANISM OF CATALYTIC COUPLING AND AMMONIA CHANNELING IN THE PURL PROTEIN USING STATISTICAL COUPLING ANALYSIS

Deepanshu Choudhary, Ajay Singh Tanwar, Venuka Durani Goyal, Ruchi Anand Chemistry Department, IIT Bombay

The Formylglycinamide Ribonucleotide Amidotransferase enzyme, also known as PurL, is a 140 kD protein that converts formylglycinamide ribonucleotide (FGAR) to formylglycinamidine ribonucleotide (FGAM). While its biological role is known in that it catalyzes the fourth step in the biosynthesis of purines, several aspects of its molecular mechanism are still not clear. It has two active sites on two opposite ends of the protein and even though the crystal structure of the protein is solved, it is not known how reaction intermediates travel from one active site to the other and how the different domains communicate with each other to carry out the complete reaction. We are using statistical coupling analysis of multiple sequence alignments to highlight the functionally and structurally important amino acids so that we can focus the research in those areas. This method takes advantage of patterns of consensus and correlation in amino acid sequences to identify important regions of a protein and classifies them as sectors. Upon applying this method to the PurL protein we have found sectors of coevolving amino acids that span the length and breadth of the protein and connect the two active sites via sparse yet mostly contiguous routes. These amino acids could be important for catalytic coupling and ammonia channeling. We are in the process of validating these results with more detailed bioinformatics analysis using concepts of consensus and correlation and also with experiments like Xenon trapping to identify cavities and channels in the protein and site directed mutagenesis to probe the importance of various identified residues.

182 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 125

MICRORNA AND ITS ASSOCIATION WITH THE PROMOTERS

Kalyani Korla University of Hyderabad, Hyderabad – 500046 IL- 4

MicroRNAs (miRNA) are ~22 nucleotides (22-nt) long RNA. They have established their role in regulating a number of pathways in the past decades via post-transcriptional regulation mechanisms in the cytoplasm. Recent studies have shown their presence in the nucleus, and few experimental results indicate their role in transcriptional regulation. Findings about the association of miRNA with the transcription factors indicate their collaborative action in promoter recognition. In the present study we have taken the promoter region (1000- nucleotides upstream from transcription start site - TSS) from ten human Toll-Like Receptor (TLR) genes and looked for the presence of exact matching sequences within mature miRNA from human. We have found that a number of 6-nt sequences are common between miRNA and TLR promoter, however this number decreases when we consider longer sequences. We have found >11-nt matches which account for more than 50% of the miRNA length (~22-nt) and thus can be considered strong enough to resist displacement by other factors. Some of these matching sequences were found to be present in close proximity to TSS. These 12-nt matching sequences were found to be present in high frequency in the genome, whereas random probability based distribution expects them to be present around 200 times only. A 19-nt long matching sequence was found, whose expected frequency in the genome is ~0.01, but is found to be present several thousand times in the whole human genome. This emphasises on its role in regulatory aspects in promoter recognition and binding. MiRNA bind to their target in post-transcriptional regulation in a sequence-dependent manner and keeping in view a match of 12-nucleotides and more, the prospect of their transcriptional regulation can’t be overlooked. The approach used in the present work is simple and can be used for prediction of promoter sequences and open avenues for study of complemented action of transcription factors and miRNA in transcriptional initiation mechanism.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 183 PP- 126 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

EXPLORING PARAMETER SPACE OF LYSOZYME AGGREGATION

Mohanish Borana and Basir Ahmad University of Mumbai- Department of Atomic Energy- Centre for Excellence in Basic Sciences, University of Mumbai, Vidhyanagari, Kalina Campus, SantaCruz(E) Mumbai – 400098, Emails: [email protected] ; [email protected]

Protein aggregation, the process by which native proteins convert into insoluble fibrillar structures, has implication in human health, biotechnology and material science. Most studies on aggregation kinetics of proteins associated with diseases indicated accumulation of critical nucleus during the process. However, little is known about the conformation of aggregation nucleus and its precursor monomer. In spite of their importance these states are difficult to characterize mainly because they form transiently in the process. In this study, we have designed the conditions and method to stabilize the monomeric aggregation precursor state of lysozyme. We report that conformation of monomeric aggregation precursor state of lysozyme regulate the accumulation and conformation of critical nucleus of the aggregation. We would also show that the polymorphism in fibrils/protofibrils thus formed is ultimately controlled by monomeric aggregation precursor state. Small molecule inhibitor and accelerator of aggregation act by binding to native state of the protein and modifying the unfolding pathway of native state and conformation of aggregation precursor state. Our study offers opportunities to investigate the small differences existing in the mechanism of formation of different kind of aggregates.

184 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 127

RAMAN SPECTROSCOPY OF ORAL CANCER TISSUES: A CORRELATION WITH BIOCHEMISTRY

1 1 2 S.P.Singh , Atul Deshmukh , Pankaj Chaturvedi IL- 4 and C. Murali Krishna1* 1Chilakapati lab, ACTREC, Navi Mumbai, 410210, India, 2Department of surgical oncology, Tata memorial hospital, Mumbai, 400012, India , *Email: [email protected] Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy, worldwide [1]. Histopathology, the gold standard has limitations like subjectivity and time consumption. Optical spectroscopic methods are been widely explored as alternative/adjunct non invasive diagnostic methods. Earlier ex vivo and in vivo Raman spectroscopic studies have demonstrated that in normal conditions lipid features are predominant while tumors have proteins rich features [2-6]. Aim of the present study is to correlate Raman spectral features of normal and tumor oral tissues with biochemical assays. Raman spectra from 40 (20 normal and 20 tumor) oral biopsies were acquired using HE-785 commercial Raman spectrometer. Typical normal spectrum show Raman bands at -1 -1 -1 1750 cm , strong CH2 bend at 1450 cm , and two sharp features around 1330 cm which can

be attributed to lipids. Dominating protein bands indicated by amide I, broad δCH2 and broad features in the amide III were seen in mean malignant spectrum. Ratio of lipid and protein was high for normal tissues while low for tumor tissues, corroborating earlier reports. Estimation of total lipid, total protein, and phospholipid was performed by Folin-Lowry, Floch and Rouser method, respectively [9-11]. Biochemical estimation yielded similar results i.e. high lipid and low protein for normal tissues while high protein and low lipids for tumor tissues. Significant differences between ratio of protein and lipid for normal and tumor tissues were observed. Findings of the study suggest that major spectral features under normal condition consist of lipids and while proteins are predominant in tumors. This can be attributed to the fact that in case of tumors or other pathological conditions there is loss in architectural arrangement of different layers, therefore, loss of lipid features is expected as contents of different layers are mixed. Additionally, cells of pathological conditions have large amounts of surface proteins, receptor proteins, enzymes, antigens, and anti-bodies which may give rise to a protein-dominated spectrum. References: 1. D. M. Parkin et al. CA Cancer J Clin, 2005, 55, 74–108. 2. K. Venkatakrishna et al. Curr Sci, 2001, 80, 101–05. 3. R. Malini et al. Biopolymers, 2006, 81,179–93. 4. S. P. Singh et al. J Cancer Res Ther,2012, 8, S126-S132. 5. S. P. Singh et al. Proc. of SPIE, 2012, 8219, 82190K1-K6. 6. S. P. Singh et al. J Biomed Opt 2012, 17, 105002. 7. Folch et al, J Biol Chem 1957, 226, 497 8. R. Subapriya et al. , Clinical Biochemistry 2002, 35489–93. 9. Rouser et al., Lipids 1970, 5, 494-6.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 185 PP- 128 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IN VIVO RAMAN SPECTROSCOPY: CLASSIFICATION OF SUB-SITES IN ORAL CAVITY

Aditi Sahu1, Atul Deshmukh1, Pankaj Chaturvedi2 and C. Murali Krishna1* 1Chilakapati lab, ACTREC, Navi Mumbai, 410210, India, 2Department of surgical oncology, Tata memorial hospital, Mumbai, 400012, India , *Email: [email protected]

Oral cancer is the most common cancer in males in India, where about 75-80,000 new cases are reported each year. Early detection of cancerous changes may be the best and most cost- effective means to improve survival and quality of life for oral cancer patients from all socioeconomic communities [1,2]. Thus, there is a need to develop alternative methods for early diagnosis of oral cancers. Raman spectroscopy (RS) is a vibrational spectroscopic technique based on inelastic scattering of light. Potential of in vivo Raman spectroscopy in classifying healthy, contralateral, premalignant and malignant conditions as well as in identifying tobacco and malignancy related effects in oral buccal mucosa has already been demonstrated [3-5]. However, tongue and lip are other two predominant sites associated with oral cancer, especially in South Asian countries. Therefore, efficacy of this methodology has to be evaluated for these sub sites to answer the question if multiple anatomic sites can be combined and analyzed or inherent anatomic differences at these sub sites should be taken in to consideration. Aim of the present study is investigate the relationship between spectral contrast and anatomic differences. In vivo Raman spectra from 72 healthy volunteers and 82 oral cancer patients using fiber-optic probe coupled HE-785 commercial Raman instrument were acquired. Spectra were recorded from different sites, namely buccal mucosa, lip, tongue, retromolar trigone, hard palate, floor of mouth and gingiva from both, healthy subjects and contralateral site, premalignant patches and tumors in oral cavity of patients. The spectral acquisition details

were: λex-785 nm, laser power-80 mW, spectra were integrated for 3 seconds and averaged over 3 accumulations. Spectral data was analyzed using Principal Component-Linear Discriminant Analysis (PC-LDA). Analysis was restricted to the three most common sites of oral cancer, buccal mucosa, lip and tongue. Preliminary findings of the study indicate that buccal mucosa, lip and tongue can be efficiently classified in healthy subjects, while classification decreases from increase in severity of pathological condition. Thus there may be a need to consider buccal mucosa, lip and tongue as separate entities for building of robust models and eliminating the influence of spectral contrasts due to anatomic differences that may possibly confound normal and cancer classification. References: 1. J. Ferlay et al. IARC Scientific Publication 5 (2004) 2. R. Sankaranarayanan et al. IARC Scientific Publication 145 (1998) 3. S. P. Singh et al. J Cancer Res Ther 8, S126-S132 (2012) 4. S. P. Singh et al. J Biomed Opt 17:105002 (2012) 5. S. McGee et al. J Biomed Opt. 13(6): 064034 (2008)

186 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 129

RAMAN SPECTROSCOPY OF GAMMA IRRADIATED HELA CELLS: AN EXPLORATORY STUDY

Priyanka Sathe1, Donil Domnic2, Jayant Sastri Goda2, 2 1* IL- 4 Supriya Chopra , C.M. Krishna 1Chilakapati lab, 2 Clinical biology lab, ACTREC, Navi Mumbai, 410210, India, *Email: [email protected]

Raman spectroscopy (RS) is a non-destructive, rapid and high information yielding vibrational spectroscopic method that can detect subtle biochemical perturbations within cells or tissues [1,2]. Potentials of RS in identifying normal cancerous conditions and HPV +ve & HPV –ve cell lines have already been demonstrated [3-6]. Present study aims at classifying Raman spectra of HeLa cells irradiated at different doses and to correlate it with different radiobiological assays (Clonogenic assay, γ-H2AX assay). HeLa cells were grown under standard conditions to achieve 70% confluency. Cells were irradiated with single fraction (0 Gy, 2Gy, 4Gy, 6Gy, 8Gy and 10Gy) by Co-60 radiotherapy source. Raman spectra were acquired using 40x objective coupled HE-785 commercial Raman instrument at 1 hr and 24 hr time point. Spectral acquisition parameters were: λex-785 nm, laser power-40 mW, integration for 15 seconds and averaged over 3 accumulations. A total of 90 spectra were acquired over 3 independent experiments (conducted for both time points). Pre-processed spectra (in region 800-1800 cm-1) were subjected to Principal Component Analysis (PCA). Parallely clonogenic and γ-H2AX assay were also performed. Exclusive clusters for HeLa cells irradiated at low doses i.e. 2Gy, 4Gy and control for both time points were obtained after PCA. Overlapping clusters were observed for cells irradiated at higher doses i.e. 6Gy, 8Gy and 10Gy. In-vitro clonogenic assay correlates well with Raman spectroscopic analysis as surviving fraction of HeLa cells decreased in a dose dependent manner till 6Gy, after that plateauing of survival curve was observed. Similar results were obtained with γ-H2AX analysis as mean γ-H2AX foci intensity increased linearly with dose administered till 6Gy and after that saturation in the intensity and mean number of γ-H2AX foci was observed. Significant differences between γ-H2AX foci intensity at 1 hour and 24 hours post irradiation were also observed. Findings of the study suggest HeLa cells irradiated at different doses can be classified with RS and have good correlation with biochemical assays. References:

1. Kelly J. et al; Journal of Proteome research (2011) 2. Murali Krishna C. et al; Journal of Cancer research and Therapeutics (2008) 3. Kanter E. et al; Journal of Biophotonics (2009) 4. Jess P. et al; International Journal of cancer (2007) 5. Meade A. et al; Analyst (2010) 6. Ostrowska K. et al; Analyst (2010)

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 187 PP- 130 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IN SILICO STUDIES OF SELECTED STEROIDS Γ-SITOSTEROL AND ANTCIN-A COMPLETELY INHIBITION OF TRYPANOTHIONE REDUCTASE OF LEISHMANIA INFANTUM: TARGET FOR ANTILEISHMANIAL ACTIVITY

Ravi Kumar Gundampati1, Medicherla V. Jagannadham1, Shradda Sahu2, Kirti Sonkar1, Rajasekhar Chikati3 1Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. 2School of Biochemical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi-221005, India 3DBT-Bioinformatics Infrastructure Facility (BIF), Department of Biochemistry, Sri Krishna Devaraya University, Anantapur-515003, India. E-mail addresses: [email protected], [email protected]

The theoretical docking study, conducted on a sample of previously reported for Anticancer, Anti-diabetic & antioxidant potential of γ-sitosterol and Antcin-A at the binding site of Leishmania infantum Trypanothione reductase (Try R) examine interaction energy. Our studies indicate that γ-sitosterol and Antcin-A displays potent activity against TryR with lowest binding energy and RMSD values to be -9.34 Kcal/Mol for γ-sitosterol, -8.36 Kcal/Mol for antcin-A and 2.0 Å. Docking analysis of TryR with ligands enabled us to identify specific residues viz. Pro-59, Ala-200, Ala-205, Glu-203, Lue-62, Asp-218, Val-64, Cys-193 and Gln-280 within the TryR and Val-58, Leu-95, Ala-181, Val-201, Isoleu-206, Asn-266, Asp-277 and Met-282 binding pocket to play an important role in ligand binding affinity. The study contributes towards understanding mechanism of antileshmanial effect on the basis of our in silico studies we hypothesize that this compounds into γ-sitosterol and Antcin-A can be inhibitory effect on against leishmaniasis. Keywords: Autodock, Trypanothione reductase, γ-sitosterol and Antcin-A, Leishmania infantum, antileishmanial activity.

188 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 131

RESIDUE LEVEL DESCRIPTION OF IN-VIVO-SELF-ASSOCIATION OF PLASMODIUM FALCIPARUM P2

1 2 1, 31 Pushpa Mishra , Shobhona Sharma and Ramakrishna V. Hosur* Department of IL- 4 Chemical sciences, Tata Institute of Fundamental Research, Mumbai, India 2Department of Biological sciences, Tata Institute of Fundamental Research, Mumbai, India 3UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai, India

Plasmodium falciparum P2 (PfP2) is a ribosomal stalk protein. It also performs extra ribosomal novel functions which seem to be associated with homo oligomerization. Previous in-vitro studies have demonstrated that the protein has a high tendency to self-associate predominantly into an 8-mer; interestingly, this is the same size as that of the ribosomal stalk. In-vitro HSQC of the pure protein and its in-cell (E.coli) HSQC spectrum are very similar, indicating that the protein, both inside the cell and under in-vitro conditions is in a similar state. In view of this, we have characterized here the folding and concomitant self-association of the recombinant chain (rPfP2), using an in-vitro dissociation-association strategy. We observed that the residue stretch, (Met31-Leu44) of the recombinant protein, mapping to Met1-Leu14 of the native PfP2 protein, acts as a nucleation site for helix formation and subsequent self-association. Further association appears to be driven by hydrophobic and complimentary electrostatic charge interactions on the surfaces formed. One stretch, (Ile97- Ala116), always remains floppy, and this may serve as “hinge” for protein segmental motions. Based on these we have proposed a possible model for rPfP2 self-association into an 8-mer. Keywords: PfP2, Folding and self-association, NMR

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 189 PP- 132 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CSF BIOMARKERS OF ALZHEIMER’S DISEASE

Pratibha Sharmaa, Pallavi Manrala, G. Hariprasada, Chandralekhab, Manjari Tripathic and A. Srinivasana aDepartment of Biophysics, bDepartment of Anaesthesiology, cDepartment of Neurology, All India Institute of Medical Sciences, New Delhi, India

Alzheimer’s disease is the most common cause of dementia in elderly persons culminating in cognitive impairment and memory decline. This disease has a complex pathology and etiology. Early diagnosis of Alzheimer's disease will allow treatments that may help slow its progression. There is an urgent need for an early and definitive diagnosis of disease. The correlation between CSF parameters and progression of Alzheimer's disease is higher than other risk factors such as age, sex, education, etc. CSF samples of clinically diagnosed Alzheimer’s disease patients were compared with age and gender matched non demented subjects. CSF samples were depleted of high abundant proteins using antibody based affinity chromatography. Depleted CSF protein profiles were compared using 2D-DIGE for differentially expressed proteins. These were identified by NanoLC-ESI-Q-TOF mass spectrometer. Apolipoprotein E, clusterin, complement C4b, complement factor B and isoforms of hemopexin were identified as differentially expressed proteins with p value ≤ 0.05 and a ≥ 1.5 fold difference. Pathway analyses show that these proteins have interacting partners in Alzheimer’s and Apoptotic pathways. The possible roles of these proteins in relation to the disease are discussed. Keywords: biomarker pathways, diagnosis, neurodegenerative disease

190 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 133

COMPARATIVE STUDY OF MODIFIED TFO

Rakesh Kumar Tiwari* and Rajendra Prasad Ojha Department of Physics, DDU Gorakhpur University, IL- 4 Gorakhpur, U.P. India. 273009 *[email protected]

A comparative study of triple helix formation by natural oligonucleotides and PNA, as a third strand, is presented here. The parameters of PNA are prepared using G-6-31(d) basic set. The RESP charges are calculated and used for the MD simulation. The initial structure of duplex is generated by LEAP and NAB under AMBER11 and Na ions are placed near to phosphate to neutralize the system. In this study we have taken two sets of purine rich mixed sequence, in which first and second strand is common and the third strand is parallel to the first strand of natural oligonucleotides and made of natural oligonucleotides and PNA. Common method is applied to both the sets of complexes i.e. the ions are randomized with water before starting the equilibration of 1 ns dynamics and after this, system is simulated for 20 ns dynamics. DNA parameters are calculated using PTRAJ by using ff10 force-field under AMBER11 software. The free energy calculations show that PNA can form stable complex with the mixed sequence of DNA and structure is stabilized during dynamics with “O” type of hydrogen bindings in base triplets. The MD results support a dynamically stable model of the modified DNA triplex over the entire length of the trajectory in both cases. We observed that the bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding in mixed sequences but they form hydrogen bonds with the bases of both the strand of DNA duplex. RMSD results and atomic fluctuations have been calculated and discussed which suggest that PNA strand has potential to use as drug.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 191 PP- 134 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IN SILICO TRUNCATION OF N-TERMINAL FRAGMENT OF BORRELIA BURGDORFERI OSPA: TOWARDS DESIGNING A SECOND-GENERATION VACCINE AGAINST LYME DISEASE

Mutangana Dieudonné, Ramesh K V and Maithri S K Department of Biotechnology, Center for Postgraduate Studies, Jain University, 18 / 3, 9th Main, Jayanagar 3rd Block, Bangalore – 560011 *Email ID: [email protected] Phone: +91 97 39 888 926 Fax No: +91 80 4121 0692

Here, we describe a structure-based approach to truncate the size of an antigen protein for a second generation vaccine. Our method consists of (i) remodeling the three-dimensional structure of an antigen and assessing the post translational modification state of the protein before truncation (ii) truncating the primary structure of an antigen prior to remodeling its three-dimensional structure, (iii) docking the truncated structure onto an antibody, (iv) generating a stable antigen fragment that contains the bioactive epitope after truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from Borrelia burgdorferi, the spirochete causing Lyme disease elicits protective immunity that blocks transmission of this organism from the tick vector to the vaccinated animal and thus has been current focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer β-sheet. All anti- OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of truncating the N- terminal domain alone to produce a recombinant protein-based vaccine. Removal of ineffective parts from OspA antigen may reduce side effects and thereby lead to a safer vaccine. Among various truncations tried (i. e, 5, 10, 15, 20, 15, 30, 35, 40 and 45%), 20% retained the conformation of the native protein. Also, the results of docking studies revealed there was no reduction in the affinity of the OspA antigen to its antibody in relation to the full-length OspA- antibody complex. However, further truncations (beyond 20 %), which removed the second glycosylation site, caused reduction in the affinity of the protein towards its antibody, demonstrating the importance of glycosylation site for antigen stability. Our strategy should be useful for the refinement of OspA-based vaccines and developing second generation vaccines for other diseases. Keywords: outer surface protein A; second generation vaccine; protein truncation, protein stability.

192 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 135

CONFORMATIONAL PROPENSITIES AND DYNAMICS OF AN INTRINSICALLY UNSTRUCTURED

-CRYSTALLIN USING REPLICA EXCHANGE MOLECULAR DYNAMICS AND NMR IL- 4 Sunita Patel, V. Ramanujam, K.V.R. Chary Dept. of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India

A well-defined structure of a protein has so far been considered as to perform its functions. However, recently many proteins have been identified to function without having a definite structure and such proteins are classified as intrinsically unstructured proteins. They lack single folded conformation, instead, they have interconverting, dynamic ensemble of structures either of the whole or parts of the protein. On the other hand, βγ-crystallins are a group of proteins mostly found in eye lens. Most of these proteins are structural proteins with high intrinsic stability, which is further enhanced by Ca2+-binding. A marine bacterial homolog from Hahella chejuensis, which displays structural topology similar to the βγ- crystallins, has been characterized by NMR spectroscopy as an intrinsically unstructured protein, which upon Ca2+-binding undergoes conformational transformation and acquires a typical βγ-crystallin fold. In the present study, we have characterized the intrinsically unstructured state of Hahellin by NMR and replica exchange molecular dynamics and found it to be in a highly flexible molten globular state. Network analysis and clustering on the observed conformational ensemble show a heterogeneous mixture of multiple conformations. Many of the observed conformational clusters display an increased helical propensity and decreased β-strand propensity with respect to the protein in Ca2+-bound state. This is similar to the observations made on the Ca2+-free state of the protein by NMR. The computed radii of individual clusters thus derived are in good agreement with the hydrodynamics radius (Rh) of the unstructured Hahellin as measured by dynamic light scattering. The clusters with larger populations show 50-60% native contacts in their structural topology, similar to that of the Ca2+-bound form of Hahellin, while the rest show a completely altered conformational state. Taken together, this study provides an in-depth understanding of conformational dynamics of an intrinsically unstructured protein.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 193 PP- 136 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

CHEMICAL SYNTHESIS OF QUANTUM DOTS, THEIR CHARACTERIZATION AND FURTHER OPTIMIZATION USING TAGUCHI METHOD

Sunil Pandey1, Vaibhav Patil, 1, 2 Goldie Oza1 and P. M. Dongre2 1N. S. N. Research Centre for Nanotechnology and Bionanotechnology, Ambernath (W), Maharashtra, India 2Department of Biophysics, University of Mumbai, Mumbai Author to whom correspondence should be addressed; E-Mail: [email protected]

We have designed a new approach to synthesize Trioctylphosphine oxide (TOPO) capped high quality CdSe Quantum Dots using the hot injection method. These size-tunable CdSe Qdots were characterized by UV-vis absorption spectroscopy and photoluminescence spectroscopy. The relationship between the synthesis conditions and spectroscopic properties was studied. Several factors affect the absorbance, photoluminescence (PL) spectra and colours of Qdots produced, for example temperature of reaction solution, TOPO concentration, Cd as well as Se concentration, etc. Furthermore to optimize these conditions and get maximum value of S/N ratio we used Taguchi methodology. For this we selected four parameters such as temperature of solution mixture, TOPO concentration, Cd:Se ratio and Paraffin: Oleic acid ratio. Experiments were carried out by following specially designed orthogonal table, as per Taguchi method, in a series of L-1 to L-9. The data support the idea that specific combination of all above parameters modulates synthesis of high quality Qdots with maximum S/N ratio.

194 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 137

STUDIES ON INTERACTION BETWEEN DNA AND AMINO ACIDS, POLY AMINO ACIDS AND PROTEINS UNDER THE INFLUENCE OF MOLECULAR CROWDING. IL- 4 Priyanka Pal, P. M. Dongre and A. V. Chitre* Dept. Of Biophysics, University Of Mumbai, Vidyanagri, Santacruz (E), Mumbai – 400098, India.

Basically DNA wound around a protein core is a fundamental and structural subunit of eukaryotic organism which is a stable protein complex. Ample studies have been reported about the DNA and its interaction with its binding proteins which have emphasized the significance of DNA, its stability, reactivity and organization with respect to its role in cell transformation. The studies in molecular biology have been mostly done in dilute solutions. However in living cells there is considerable high concentration of macromolecules/ salts, & other constituents, effectively reducing the volume available causing increase in concentration resulting in ‘crowding’ which radically alters the behaviour of molecules. This aspects needs to be given the due significance in any studies on behaviour of cell components and their function. Nucleosomes being colloidal in nature these aspects assume more significance. A wide variety of physiological processes are the reflection of ligand interactions with macromolecules especially with proteins and nucleic acids. Upon reflection it is clear that virtually all biological phenomenons depend on one or more ligand interaction. It is not surprising therefore that a large amount of biochemical and physiological research have been directed on exploring these interactions in depth. In the present preliminary work we have attempted to study the molecular crowding effects on interactions of amino acids, poly amino acids and a few proteins with DNA using some related and unrelated polymers as crowding agents by fluorescence enhancement of probe dyes and 2-d gel electrophoresis. These results are presented in the text.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 195 PP- 138 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

GREEN SYNTHESIS OF SILVER NANOPARTICLES AND ITS APPLICATION AGAINST MULTIDRUG RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)

Rebecca Thombre1*, Glory Francis1, Parvathi Laxminarayan1, Fenali Parekh1, Neeta Patil2 and 1 Dept. of Biotechnology, 2 Dept of Botany, Modern College, Shivajinagar,Pune-411005 *Corresponding author: [email protected]

Nano technology is an emerging field of Science which involves synthesis and applications of nano particles. Bioinspired synthesis of nanoparticles has advantages over chemical and Physical methods as it is cost effective and environment friendly. Silver nanoparticles are a metal of choice as they have large spectra of applications in different fields due to their unique optical, electrical properties as well as medicinal properties. The use of environmentally friendly materials like plant leaf extract, bacteria and fungi are used for the synthesis of silver nanoparticles as they offer numerous benefits of eco-friendliness and compatibility for pharmaceutical and biomedical applications as they do not use toxic chemicals in the synthesis protocols. In the present investigation, a common weed called Parthenium hysterophorus (Congress grass), Lemon extract (Citrus limon) and Neem extacts (Azadarichta indica) have been exploited for synthesizes silver nanoparticles as a step towards green technology. The nanoparticles were characterized by UV-Vis spectroscopy, SEM, EDX, XRD and FTIR. The AgNp’s demonsrated potent antibacterial activity against MRSA. Keywords: Silver nanoparticles, Parthenium hysterophorus, weed utilisation

196 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 139

HPLC-MS AND HPTLC FINGER PRINTING OF FRACTIONS OF ASTERACANTHA LONGIFOLIA LEAF EXTRACT

1 1 Amit Kumar Dash*, G. K. Dutta, G. Sahoo , A. Maity , IL- 4 K. K. Sardar2 and M. K. Panda3 Department of Veterinary Physiology and Biochemistry College of Veterinary Science and A.H., Anjora, Durg, Chattishgarh, 491001, India 1Department of Veterinary Biochemistry, 2Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and A.H., OUAT, Bhubaneswar, Odisha, 751003, India 3Officer in Charge, Central Laboratory, OUAT, Bhubaneswar, Odisha, 751003, India Corresponding author: E-mail id. [email protected]

The present study was designed to figure out the HPLC-MS and HPTLC finger printing pattern of three fractions (water soluble, methanol soluble and residual) of hot water leaf extract of Astercantha longifolia Linn. Nees, a promising medicinal plant with great economic potential found in moist places throughout India. Methanolic and residual fractions were fractionated into two peaks each by HPLC with retention times of 1.07, 4.72 min and 0.95, 1.15 min, respectively. Only one peak was observed in aqueous fraction with a retention time of 0.91 min. Considering the m/z value and comparing it with the finding of other workers it could be attributed that methanolic fraction may contain glycine betaine, isotope of cadmium, β-alanine betaine, alkaloids (conhydrinone or N-methyl coniine), Phe-leu- , N3 alkylated fragment of phenyl glycidyl ether with uracil along with other components; aqueous fraction may contain glycine betaine, Phe-leu- and other components; residual fraction may contain glycine betaine, isotope of cadmium, along with other components . Scanning of developed HPTLC plates of different fractions at 254 nm attributed to the presence of nine, six and five peaks in methanolic, aqueous and residual fractions, respectively. However, scanning at 366 nm showed presence of only three, four and two peaks in these fractions, respectively. Thus, 254 nm was found to be optimal wavelength for identification of more compounds compared to 366 nm. Both HPLC-MS and HPTLC finger printing confirmed that although some compounds are similar in all the three fractions, the overall composition differs from each other.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 197 PP- 140 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

MEMBRANE FUSION INDUCED BY SMALL DRUG MOLECULES: A NEW AND ALTERNATE FUNCTION OF NSAIDS.

Sutapa Mondal Roy and Munna Sarkar* Chemical Sciences Division Saha Institute of Nuclear Physics, Kolkata – 700064.

The principal functions of NSAIDs (Non Steroidal Anti-Inflammatory Drugs) are controlling pain and inflammation. Apart from their usual functions, a new and alternate function has been established by our group for NSAIDs, as inducer of membrane fusion. This property is shown by three oxicam group of NSAIDs viz. meloxicam (Mx), piroxicam (Px) and tenoxicam (Tx). Unlike proteins and peptides, these drug molecules, being small in size, do not have the advantage of providing enough energy by conformational reorganization, required to overcome the energy barriers of intermediate steps of fusion. These oxicam NSAIDs can induce fusion at concentration even below their peak plasma concentration, which makes our work exceedingly significant and worth studying. The effects of the following parameters on fusion process have been elucidated here. They are, a) Lipophilicity of the drugs b) Concentration of the drugs c) Temperature of the system d) Effect of orientational order of the lipid tails and increased head group spacing e) Effect of lipid head- group size mismatch. Simple model membranes viz. small unilamellar vesicles (SUVs) made up of dimyristoylphosphatidylcholine (DMPC) have been used in the studies. It is found that the process of Mx, Px and Tx induced membrane fusion is strongly guided by the hydrophilicity of the drugs and there exists a particular threshold concentration of these drugs, up to which drug induced fusion increases and then decreases. These drugs, getting partitioned in the membrane bilayer, cause an optimum perturbation and induce fusion following a sequential model. At high concentration of the drugs, the perturbation of the bilayer becomes so high that the vesicles are ruptured leading to the lowering of fusion. Studies on the effect of different physico-chemical parameters of both the participating drugs and the membranes on these oxicam NSAIDs induced membrane fusion is the subject of this presentation, which will make the better use of these drug molecules to induce membrane fusion in a controlled manner for several biotechnological and biomedical procedures.

Reference:

1. Biophysical Chemistry 2008, 137(1), 28-34. 2. The Journal of Physical Chemistry B 2009, 113, 16323-16331. 3. Langmuir, 2010, 26, 18967-18975. 4. Langmuir 2011, 27, 15054-15064.

198 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 141

IN VITRO ANTIBACTERIAL, MEMBRANE DAMAGE, ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF BARLERIA PRIONITIS L EXTRACT ON UTI CAUSING MULTIDRUG RESISTANT E.COLI IL- 4 C N Khobragade1, Rashmi M Bhande2, P.M.Dongre3, Jessy M.4 1&2School of Life Sciences, Swami Ramanand Teerth Marathwada University Nanded 431 606, India 3&4Department of Biophysics,Mumbai University Mumbai 400 032,India

Resistance to antibiotics is a ubiquitous clinical problem and is compounded by a dearth of new therapeutic agents. A variety of compounds from natural sources that modify membrane permeability are employed in management of multidrug resistant organisms. In the present investigation total phenol, flavonoid contents (0.33±0.1 mg of Gallic acid and 0.9±0.5mg of

Quercetin equivalent per gram of dry extract respectively), anti-oxidant (IC50 0.3±0.02 mg/ml compared to standard Ascorbic acid 0.5±0.01 mg/ml) and anti-inflammatory activity (in terms of albumin percent inhibition was 85.77% as compared to standard Ibuprofen 90.0% at 50μg/ml) of the extract were evaluated along with the effect of extract on membrane potential of E.coli by fluorescence spectroscopy method. Extract of B.prionitis L effectively disrupted E.coli cell membrane by depolarization peak at 516.62 nm with intensity 210.91 a.u. suggesting decrease in membrane potential after the treatment with extract. The presence of reactive oxygen scavenging agents in the extract effectively exerted their antibacterial action through membrane perturbations. Thus B.prionitis L extract containing bioactive compounds can be studied as future alternative to treat UTI infections caused by E. coli.

Keywords: E.coli, UTI, Barleria prionitis L, Membrane potential, Antioxidant and Anti- inflammatory activity

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 199 PP- 142 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

POTENTIAL ANTI-TUBERCULAR HITS: DESIGN, SYNTHESIS AND EVALUATION OF 2,5 DI-SUBSTITUTED BENZIMIDAZOLES

Sandeep Kamble1, Ravi D. Wahavle1, Manoj Avghade2, Dhiman Sarkar2, Premlata K. Ambre1, Raghuvir R. S. Pissurlenkar1† and Evans C. Coutinho1 Pharmaceutical Chemistry Division, Bombay College of Pharmacy, Kalina, Santacruz (East) Mumbai 400098 Combichem-Bioresource Center, OCD, National Chemical Laboratory, Dr. HomiBhabha Road,Pune 411 008 †Corresponding author: [email protected]

Tuberculosis is one of the most devastating bacterial diseases, characterized as a chronic bacterial infection caused by Mycobacterium tuberculosis an aerobic acid fast bacillus. Although the recommended Directly Observed Treatment Short-course therapy (DOTS) provides a cure for TB, need for effective TB drugs that have short duration of treatment period and that can target multi drug resistant strain still prevails. One such target identified is mycobacterial glutamine synthetase (MtGS) which catalyzes the conversion of glutamate to glutamine in the presence of ammonium ion with accompanied hydrolysis of ATP as an energy source in the mycobacterial nitrogen metabolism. Present research work aims at the design, syntheses and evaluation of novel compounds as competitive inhibitors of nucleotide binding site of glutamine synthetase. The novel compounds are identified by fragment based ligand design approach using BROOD 2.0(Open Eye Molecular Modeling Suite). The fragment based de novo study was carried out on purine analog inhibitor bound to the nucleotide binding site of glutamine synthetase where the purine core was initially replaced by benzimidazole. After which diverse substitutions were identified for the dichlorobenzyl and morpholine moiety substitution to generate a diverse set of benzimidazole analogs. The designed analogues were docked using GLIDE 5.6 of Schrödinger Suite 2010 to short list candidates for synthesis. Eight different analogs of benzimidazole were synthesized and characterized. The synthesized compounds were evaluated for inhibitor activity against mycobacterial glutamine synthetase, where in,5- benzoyl-2-(3-methoxyphenyl)-1H-1,3-benzdiazole showed 40% inhibition.

200 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 143

INTERACTION OF OFLOXACIN WITH BOVINE SERUM ALBUMIN: SPECTROSCOPIC AND FÖRSTER RESONANCE ENERGY TRANSFER (FRET) STUDIES IL- 4 B. Ahmad, N. Agarwal K.J. Karthika, V. Kumar, N. Mothi, Sanoj, P. Sheokhand UM- DAE Centre for Excellence in Basic Sciences, University of Mumbai, Vidhyanagari,Kalina Campus, Santa Cruz(E)Mumbai – 400098, Emails: [email protected]

Serum albumin being the major transporters binding protein for the drugs and other physiological substances, it is considered as a model for studying drug–protein interaction in vitro. The interaction of ofloxacin with Bovine Serum Albumin (BSA) has been studied by spectroscopic methods. In this work, it was proved that the fluorescence quenching of BSA by ofloxacin is a result of the formation of an ofloxacin–BSA complex. Binding studies concerning the number of binding sites and apparent binding constant were investigated. The binding of ofloxacin quenches the BSA fluorescence, revealing a 1:1 interaction with a

5 binding constant of about 10 M ˡ. Finally, the binding distance r between donor (BSA) and

⁻ acceptor (ofloxacin) was estimated on the basis of the Förster resonance energy transfer (FRET) method.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 201 PP- 144 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

IMPACT OF CLASSICAL MUSIC ON ELECTRICAL SIGNALS OF BRAIN

Martina David, Saira Shahid and P M Dongre Department of Biophysics, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098

Music interconnection with society can be seen throughout history. It has been thought that music stimulates brain’s organization ability through rhythm. The effects seems to be in terms of memory improvement, control pain, reduce anxiety, boost IQ, enhance creativity, increase motivation etc. It is also been seems that music can be used to create and maintain different states of consciousness, from highly alert and energized to sleep, as well as the all- important relaxation/meditation state where person can get rid of stress. Based on these observation we wish to predict scientifically whether music has any impact on EEG (Electroencephalogram) signals. EEG is the recording of electrical activity along the scalp. EEG measures voltage fluctuations resulting from ionic current flows within the neurons of the brain. In the present investigation we have conducted study of EEG patterns of 40 volunteer subjects who were made to listen to Indian classical music (raga) through audio compact disc (CD) of Science of Music, Mumbai. The suitable EEG electrodes were placed on scalp for recording of surface potential (EEG). The data acquisition system has been used for recording and analysis of EEG pattern. EEG signal were recorded of each subject up to15 minutes with and without classical music. The EEG pattern was analyzed using in build software of data acquisition system. Statistically significant changes were noticed in alpha and beta waves during listening of classical music.

202 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 145

NANOPARTICLE INDUCED CONFORMATIONAL CHANGES OF BSA CORRELATE WITH ITS FUNCTIONS IN PRESENCE OF UV RADIATION

Meena Pandey Pant, Jessy John, Amruta Joshi and P M Dongre IL- 4 Department of Biophysics, University of Mumbai, Santacruz (E), Mumbai 400098 INDIA

With the advent of nanotechnology the interaction between nanoparticles (NPs) and biological materials including living cells has became an important areas of basic and applied research at the interface of biology. Here, we report here the effect of UV radiation (254 nm) on Bovine Serum Albumin (BSA) in presence of silver nanoparticles. Ultraviolet absorption spectroscopy indicated the fragmentation and aggregation of BSA in presence of UV radiation. Fluorescence spectroscopy results indicated radiation and silver nanoparticles quenched the emission intensity of BSA excited at 280 nm. CD spectra changes with respect UV radiation dose. The remarkable changes in the functional properties such as esterase activity, free thiol groups and copper binding assay were noticed. The possible mechanism of interaction between silver nanoparticles and BSA have been exploited.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 203 PP- 146 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SIMPLE, EFFECTIVE AND INEXPENSIVE MEMBRANE MODEL FOR BIOPHYSICAL INVESTIGATION

Shalaka Haryan, Shamli Satpute, Mahesh Samant P M Dongre and D C Kothari* Department of Biophysics, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098 *Department of Physics, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098

The purpose of the study was to develop simple, effective and inexpensive biological membrane model to investigate the biophysical parameters such as membrane permeability (P), diffusion coefficient (D) and diffusion rate (J) of biomaterials. First time we are reporting a such kind of investigation. For this purposes, we have used Chicken eggs shell membrane to study the diffusion of silver nanoparticles (SNP) and amino acids (tryptophan, tyrosine and phenylalanine). The SNP’s were synthesized using chemical degradation method and characterized by UV visible, Atomic force Microscopy (AFM) and Dynamic light scattering (DLS). Scanning electron microscopy (SEM) has been used for study the images of eggs shell membrane. Approximately 50 nm sizes of silver nanoparticles were obtained. The eggshell membrane was placed in such way that it will make two compartments having partition of membrane. One compartment containing silver nanoparticles, amino acids and other having only buffer. The change in concentrations of silver nanoparticles and amino acids alone and combinations of SNPs in another compartment was monitored at particular interval till the saturation takes place. The diffusion coefficient, membrane permeability and diffusion rate of SNP and amino acids was estimated. SEM images of membrane model shows that silver nanoparticles bind/adsorb on membrane proteins. The mechanisms and nature of diffusion of silver nanoparticles and amino acids through cell membrane will be discussed.

204 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 147

IMPEDANCE SPECTROSCOPIC CHARACTERIZATION OF MERCAPTOPROPIONIC ACID CAPPED PT NANOPARTICLES/POLYPYRROLE COMPOSITE FILM BASED IMMUNOSENSOR IL- 4 Sujeet K. Mishra, V.K. Tanwar, Ashok M. Biradar and Rajesh* Polymer and Soft Materials Section, CSIR-National Physical Laboratory Dr. K.S. Krishnan Road, New Delhi-110012, India.

We report, 3-mercaptopropeonic acid capped gold nanoparticles, Pt (MPA), embedded polypyrrole (PPy) composite film electrochemically grown onto an indium-tin-oxide (ITO) glass plate, wherein Pt (MPA) were used as conjugates for the efficient binding of C-reactive protein antibody, αCRP-Ab, for immunosensor fabrication. The Pt (MPA)-PPy nanocomposite was characterized by Scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR) and electrochemical techniques. The impedimetric response of the electrode towards the detection of biomarker was analyzed in terms of constant phase element (CPE), electron transfer resistance (Ret) and Warburg impedance (WR) using Randles model as the equivalent circuit. The immunosensor showed Ret dominant impedance at low ac frequency, indicating a good biocompatible electrode with high impedimetric sensitivity towards protein antigen [αCRP-Ag] in phosphate buffer solution. The immunosensor exhibited a linear impedance response to [αCRP-Ag] in the range of 10 ng to 10 µg mL-1.

Keywords: cyclic voltammetry, electrochemical impedance spectroscopy, polypyrrole, protein immobilization

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 205 PP- 148 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ZNS NANOPARTICLES FUNCTIONALIZED REDUCED GRAPHENE OXIDE COMPOSITE BASED IMMUNOSENSOR FOR THE DETECTION OF CARDIAC BIOMARKER

Rajesh*,Sujeet K. Mishra, Vikash Sharma and Ashok M. Biradar Polymer and Soft Materials Section, CSIR-National Physical Laboratory Dr. K.S. Krishnan Road, New Delhi-110012, India

We report, a 3-mercaptopropeonic acid capped ZnS(MPA) nanoparticles functionalized reduced graphene oxide over a silane modified indium-tin-oxide (ITO) glass plate for the construction of an immunosensor. The graphene oxide was electrostatically attached over silane modified ITO glass plate and was subsequently reduced by electrochemical technique. The reduced graphene was functionalized with ZnS(MPA) through a crosslinker, 1 pyrene methylamine and cardiac protein antibody, cMb-Ab, respectively, using carbodiimide coupling reaction for the fabrication of the immunosensor. The resulting immunosensor was characterized by scanning electron microscopy, transmission electron microscopy (TEM) and electrochemical techniques. The ac frequency dependence impedance studies were conducted on the immunosensor for the quantitative detection of target cardiac protein antigen, cMb-Ag, in phosphate buffer solution at pH 7.4. The immunosensor exhibited a linear impedance response to [cMb-Ag] in the range of 10 ng to 900 ng mL-1 with high selectivity and sensitivity of about 800 Ω per decade.

Keywords: cyclic voltammetry, electrochemical impedance spectroscopy, nanoparticles.

206 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 149

PHARMACOPHORE MODELLING AND 3D-QSAR STUDIES OF PROTEIN TYROSINE PHOSPHATASE 1B INHIBITORS

Sonali H. Tikhele, Raghuvir R.S. Pissurlenkar and Evans C. Coutinho† IL- 4 Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santacruz (East), Mumbai 400 098, India †Corresponding Author: [email protected]

The pursuit for agents that can become potential treatment for Type II diabetes continues to be a major research focus worldwide. As impaired insulin action is an underlying mechanism in progression of Type II diabetes, the insulin signalling pathway has been the heart of research for identification of significant therapeutic target/s for drug intervention against the disease. Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of the insulin signalling pathway, has emerged as an attractive therapeutic target for the treatment of Type II diabetes and also for obesity. Pharmacophore hypotheses were built on a set of potent and diverse inhibitors which were substantially validated for specificity, sensitivity and selectivity using active and inactive ligand sets. Based on validation studies the pharmacophore hypothesis chosen for virtual screening has features viz. acceptor, hydrophobe, negative ionisable and ring features. The NCI and ZINC databases with ~14 million molecules were screened for possible hits. The virtual hits were docked in restrained conformations in the active site of PTP1B enzyme to rank the hits based on binding energies. Statistically significant and robust 3D-QSAR models (CoRIA, CoMFA and CoMSIA) also were developed on a training set of PTP1B inhibitors to predict the virtual screen hits. The screened molecules were short listed based on ligand efficiency, ADMET/Tox.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 207 PP- 150 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ASSEMBLING NEUROSPHERES: DYNAMICS OF NEURAL PROGENITOR/STEM CELL AGGREGATION PROBED USING AN OPTICAL TRAP

Uma Ladiwala1, Himanish Basu2, and Deepak Mathur2 1 UM-DAE Centre for Excellence in Basic Science, University of Mumbai, Kalina Campus, Mumbai 400 098, India 2 Tata Institute of Fundamental Research, 1 Homi Bhabha Road, Mumbai 400 005, India

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (~5 s) and most probable minimum distance of approach (4-6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane- mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (~18 pN). Our findings have important implications for the neurosphere assay: neurosphere formation by cell aggregation is a robust process that cannot be readily disrupted without the use of external forces whose magnitudes are larger than tens of picoNewtons. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, Cytochalasin-D, an actin polymerization inhibitor, disrupted the dynamics of adhesion events such that filopodia- mediated adhesion was inhibited but not membrane-membrane adhesion.

208 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 151

DOCKING STUDIES OF PHENOTHIAZINE DRUGS WITH SNAKE VENOM TOXINS

Vrushali Hingane, Aruna Vinchurkar*, P M Dongre Department of Biophysics, University of Mumbai, IL- 4 Vidyanagari, Santacruz (E), Mumbai 400 098. *Department of Biophysics, Govt. Institute of Science, Aurangabad, 431004

The present investigation explore the neutralization of snake venoms using phenothiazines drugs on animal model and docking of these drugs with toxins of Naja Oxiana & Vipera russelli. The four phenothiazine drugs viz. Promethazine (PMZ), Prochlorparazine (PCP),Chlorpromazine (CPZ), Triflupromazine (TFZ) and the toxins of Naja Oxiana - Phospholipase A2, Cytotoxin I, Cytotoxin II, Neurotoxin long, Neurotoxin II & Vipera russelli - Anticoagulant class II PA2 Viperotoxin (RV-4/RV-7) Serine proteinase (RVV-V) have been used for docking study. The PDB file of toxins were downloaded from RCSB Protein Data Bank. The docking results reveal that various amino acids, Vander wall and hydrogen bonding are involved in interaction between Phenothiazines drugs and toxins. In case of Naja oxiana venom, The experimental results on animal model (Mus musculus) for Naja oxiana venom indicate that postponement of death was observed in PMZ treated animals than other phoenothiazine drugs. The order of delay in death time of drugs was PMZ>TFZ>PCP, however CPZ didn’t showed delay in death time in animals but in docking its showed interaction with toxins of Naja oxiana. In case of Viperid venom, all experimental animals were found to be survived when treated with TFZ, CPZ, PCP however, PMZ treated animal showed 3-6 hrs postponement in death. The correlation between docking study, involvement of amino acids of toxins and death postponement will be discussed at molecular level

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 209 PP- 152 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

PP- A DATABASE RESOURCE ON RADIOMODIFIERS

Amruta M Joshi, P M Dongre Department of Biophysics, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai-400098 E-mail: [email protected]

Discovery of radiomodifying agents has been a great breakthrough in radiation therapy of various cancers. There are two classes of radiomodifiers one who protect (radioprotector) normal cells from ionizing radiation harm other enhances radiation effect (radiosensitizer) in malignant cells. This area has been actively researched upon since long and still continues to be a dynamic one with several new strategies surfacing. Numerous data have been generated and yet there is no systematic representation of it which would help for further research. Hence our laboratory has decided to develop bioinformatics database resource on radiomodifiers. This database has been designed in such a way that one can retrieve information on parameters like physico-chemical properties, Dose modifying factor (DMF),

Clinical studies, target sites, pharmacokinetic studies, and LD50 studies with a single click on a computer through internet. Further the information is connected to its e-sources via a hyperlink as a result an investigator could access the full text/abstract. Further, there is a provision for advanced search where a user can select the criteria to achieve a specified result/s. This database will prove useful for researchers, clinicians & others who work in related areas.

210 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 153

DEVELOPING AN ATOMISTIC VIEW OF ENZYME CRTISO: MAPPING PROTEIN-SUBSTRATE INTERACTIONS

Asha Parmar, Pushpa Mishra, K Vijaya Lakshmi, IL- 4 R.V. Hosur and Jyotishman Dasgupta * Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India *Corresponding author: [email protected]

Carotenoids, the tetraterpenoid organic molecules containing 40 C-atoms with extended conjugation, are synthesized via the mevalonic acid (MVA) pathway forming the first stable colourless carotene, 15-cis-phytoene. However, the further branching differs in prokaryotes and eukaryotes implying an evolutionary significance. In prokaryotes and fungi, the conversion of first carotene 15-cis-phytoene to all-trans-lycopene utilizes a single flavoenzyme carotene desaturase (CRTI) whereas plants and cyanobacteria recruit two desaturases and two isomerases for the same chemical transformation. One of the key regulatory steps in the biosynthesis, the isomerization of 7, 9, 9’, 7’-tetra cis-lycopene (prolycopene) to all-trans-lycopene is catalyzed by the flavoenzyme CRTISO. This reaction fundamentally maintains the equilibrium between cis and trans carotenoids. Therefore understanding the mechanism underlying the conversion of 7, 9, 9’, 7’-tetra cis-lycopene (prolycopene) to all trans lycopene in absence of light and in presence of a redox partner is of fundamental importance. In this work, we aim to characterize the molecular nature of the substrate-protein interactions using a structural probe. From the structural point of view the important questions to be addressed are the substrate-protein molecular interactions and FAD-substrate coupling during catalysis. To extract the structural information on CRTISO, we initiated NMR characterization, crystallization and optical measurements so as to elucidate key chemical players in catalysis. The information learned from the above methodologies will be discussed in the context of the initial step in the catalytic cycle

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 211 PP- 154 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

KINETICS OF CELLULASE BY IMMOBILIZATION ON SILVER NANOPARTICLES EMBEDDED CHITOSAN BEADS.

1Shilpa Patil,2P.M.Dongre,3M.N.Fawde. 2Department of Biophysics,University of Mumbai, 1,3Department of Biochemistry, Babasaheb Ambedkar Marathwada University.

Cellulase is an enzyme complex which breaks down cellulose to beta-glucose.This enzyme refers to a family of enzymes which act in concert to hydrolyze cellulose. Cellulases are widely distributed throughout the biosphere and are most manifest in fungal and microbial organisms. The chief objective of this study is to improve the stability of the immobilized cellulase on silver nanoparticles embedded chitosan beads. SNPs were synthesized using microwave irradiation and characterized by UV spectrophotometer. The size of SNP was obtained approximate 50nm. The epichlorohydrine used as cross-linking agent and then cross-linked chitosan-silver nanoparticle composite bioadsorbent was prepared. The kinetics was studied at the different substrate concentrations, pH, temperature and time. The results obtained indicate that the enzyme was stable at optimum temperature substrate concentration and time, however, the enzyme was unstable at optimum pH. The results would be useful towards various industrial applications like paper, detergent and cosmetics.

212 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 155

MULTI-MICROSECOND MOLECULAR DYNAMICS SIMULATION OF META- RHODOPSIN-II: MEDIATED OPENING OF THE INTER-DOMAIN INTERFACE IN THE ALPHA SUBUNIT OF TRANSDUCIN IL- 4 Ravindra Venkatramani †, Parag Mukhopadhyay††, Andrew J. Tebben††, and David Beratan# † Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, Maharashtra, India. †† Computer Assisted Drug Design, Pharmaceutical Research Institute, Bristol Myers Squibb, Princeton, NJ 08540. # Departments of Chemistry, Biochemistry, and Physics, French Family Science Center, Duke University, Durham, North Carolina 27708, U.S.A.

The structural basis for how an active-state G protein coupled receptor (GPCR) causes the release of a bound GDP in its cognate G-protein located 35-40 Å from the receptor-G protein interface remains unknown. We report a molecular model of receptor induced conformational changes in a G protein through multi-microsecond molecular dynamics (MD) simulations of a membrane bound active-state GPCR, metarhodopsin II, in complex with its cognate GDP bound heterotrimeric G protein, transducin (Gt). Three large scale conformational changes were captured during the course of MD simulations: 1) motion of the coiled-coil N- terminus helices in Gt away from the helical domain of the Gt, 2), the active-state receptor-mediated opening of the inter-domain interface of Gt that solvates the GDP pocket, and 3) a rigid body rotation of transducin in the plane of the membrane surface. Our simulation results are consistent with recent double electron-electron resonance spectroscopy experiments on the rhodopsin-transducin system (PNAS, 2011, 108, 9420). Based on these simulation results we propose: 1) the motion of the coiled-coil N-terminus helices in Gt away from Gt to be a necessary step in receptor-mediated opening of the interface between the helical and the GTPase domains in Gt, and 2) multiple energetically similar conformational states of the G protein-activated GPCR interface culminates to GDP release from G(GDP). Testing these two ideas in experiments would further advance our understanding of activated receptor-G protein couplings necessary for triggering GDP release.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 213 PP- 156 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

ESTIMATION OF TUMOUR CONTROL PROBABILITY AND NORMAL TISSUE COMPLICATION PROBABILITY USING IN-HOUSE DEVELOPED SOFTWARE.

Dayananda S Shamurailatpam, Prabhakar Dongre#, Arun Oinam@, Suman Mallik. Department of Radiation Oncology, KDAH, Andheri, #Department of BioPhysics, University of Mumbai, Mumbai, @Departmentt of Radiotherapy PGI Chandigarh,Chandigarh.

Aim: I) To develop a computer program for estimating tumour control probability (TCP) and normal tissue complication probability (NTCP) II) To estimate TCP and NTCP of patients treated for head and neck (H&N) cancer. Introduction: Radiobiological model describe the dependence of tumour and normal tissue responses on the irradiated volume for a given time-dose-fractionation (TDF) schedule. It is increasingly used for ranking different radiotherapy plans based on predicted clinical outcome (TCP and NTCP). Software based on different radiobiological models is commercially available in latest treatment planning system (TPS) but are very expensive and difficult to integrate in the existing TPS. We developed a stand alone, user friendly software based on the most widely accepted Lyman-Kutcher-Burman (LKB) model for NTCP and Poisson-based model for TCP. The software was validated using standard normal tissue tolerance dose data and subsequently used to estimate the TCP and NTCP of patients undergoing intensity modulated radiotherapy (IMRT) of H&N cancer. Material and methods: Theory: According to LKB model of NTCP, the probability of complication after uniform irradiation of a specified volume of organ follows a sigmoid dose-response relationship and can be expressed by the cumulative normal distributio

  t 2  t   1 2 NTCP   e   dx ………………..(1) 2    D  TD v  t  50 ………………..(2) m .TD 50 v 

n TD50 v  TD50 ).1( v ………………..(3)

Where, ‘D’ is the dose to uniform irradiation of volume fraction ‘v’ (v=V/Vtotal), TD50 (v) is the dose at which probability of complication becomes 50% in 5 years. Parameters “m”

describes the slope of the NTCP curve at TD50 and “n” determine the equivalent uniform dose (EUD) of inhomogeneous irradiation using dose volume histogram (DVH) reduction method. The most widely accepted TCP is Poisson-based model represented by

N   D   .Exp2 1 i / ln 2 i1vi  50   1    TCD50   TCP    …………………(4)  2 

Where, TCD50 is the tumour dose required to produce 50% TCP and  50 is the slope of the dose response at 50% TCP.

214 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

The effect of fractionation and dose per fraction size was taken in to account in the software

using equivalent physical dose of 2 Gy per fraction (EQD2) given by

  D  ………………(5)      n  EQD  D  f  2   IL- 4   2     Where, are the tissue specific LQ parameters of the target and normal tissue.  Software program: Based on the LKB and Poisson models, a computer program was developed in METLAB (software V7.1). This program can be run in any PC having METLAB installed and support any kind of DVH from any TPS. The accuracy of the TCP and NTCP calculated by this in-house software was validated using normal tissue tolerance dose data compiled by Emami et al. After validation, the software was used to calculate TCP of various tumour and NTCP of parotid gland in 16 patients treated for various H&N cancer using IMRT. For this the cumulative DVH of tumour (various PTVs), parotids (Left and right) and spinal cord of each patient was exported from Eclips TPS in ASCII format in dose bin size of 1 cGy. Data after converting to MS Excell was imported in to in-house developed software to estimate different DVH parameters, TCP and NTCP. The parameters used to calculate the NTCP (m=0.40, n=1, TD50=39.9 for the endpoint of reduction in stimulated salivary flow below 25% in 12 months after RT) were selected from a recent publication by

Houweling et al. TCP was estimated using TCD50 and  50 value compiled by Okunieff et al. Results:

The variation in DVH parameters (D100, D95, D2/3, D1/2, D1/3, D1cc, Dmean, Dmax, Dmin) calculated using TPS and in-house developed software agrees within 0.4%. Validation of NTCP of various organs calculated using in-house software showed good agreement with that of Emami data. The TCP of each 37 tumours except two tumour was more than 91% with mean (SD) of 96.3% (2.9), 96.5% (2.3) and 92% (4.7) for high, intermediate and low risk tumour respectively. The NTCP of the 32 parotid glands ranges from 1.3% to 23.1% with a mean (SD) of 7.3% (5.2) for right and 8.1% (7.3) for left parotid. Conclusion: A user friendly radiobiological model program was developed using MATLAB platform. The software which can be operated in any PC or laptop can support any kind of DVH from any TPS. DVH parameters, NTCP and TCP calculated using in-house developed software was in good agreement with TPS calculated values, Emami and Okunieff data. The software was successfully used to estimate TCP and NTCP of head and neck cancer patients treated using IMRT.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 215 PP- 157 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

BUOYANCY DRUG DELIVERY SYSTEMS

K.Ravi Kumar , P.Mahaboob Alikhan Dr.K.V.Subba Reddy College Of Pharmacy , Kurnool , A.P.

The controlled gastric retention of solid dosage form may be achieved by the mechanism of mucoadhesion ,flotation , sedimentation , expansion , modified shape systems are by the simultanious of pharmalogical agents that delay gastric emptying based on these approaches classification of floating drug delivery systems(FDDS) has been evaluated.several recent exampels have been reported showing the efficacy of systems for bioavailability.Low density approach the globular shell apperently having low density of gastric fluid can be used as carrier for drug controlled release .In coated shellspopcorn , poprice and polysterol have been exploited as a drug carriers.The polymer of choice can be either ethyl cellulose or hydroxy propyl cellulose depending on type of release desired .The fluid filled floating chambered type of dosage formsinclude incorporation of a gas filled floatation chamber into a microporous component that houses a drug reservoir. Hydrodynamically balanced systems(HBS) designed toprolong the stay of dosage form in the gastro intestinal tract and aid in enhancing the absorption. The success of capsule as a better system is best exemplified wilth chlordiazepoxipide . HBS of chlordiazeopoxide hydrochloride had camparible blood level time profile as 10mg commercial capsules .The bilayered and matrix tablets have shown buoyancy characteristics some of novel polymers used in the formulation as hydroxy propyl cellulose ,cross povidine .The three layered principle has developed of an assymetrlic configuration delevery system in order to modulate the release extent for achieve zero order release kinetics by moniter a constant area at the diffussing front with subsequent dissolution toward the complication of the release process. Over all the experiment We develop a swellable tripled layered tablet with buoyancy abillity to prolong the gastric residency time of triple drug regimen in Hellicobacter Pylori .The floatation was accomplished by incorporating gas generationlayer consist of sodium bicarbonate.

216 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS” PP- 158

Structural and Functional Characterization of an Archaeal


crystalline

A.L. Susmitha and K.V.R. Chary Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai-400005. IL- 4 Eye lens evolution occurred through rational modification of precursor single domain Greek- key motif containing proteins. Though lens and non-lens proteins are structurally similar, there is a vast functional diversity owing to the hierarchy in their domain organization. In this context, we designed human eye lens like crystallins from an archeal protein (M- crystallin) through structure based domain duplication. Magnetic resonance spectroscopy has been used to characterize the Ca2+ binding properties and function of M- crystallin in single domain form. Dimer form of this protein is made to functionally mimic the human
D crystallin, involved in formation of cataracts in the eye. The relevance of Ca2+ binding in microbial proteins, gain in stability and associated function will be discussed. Residue level detail of the initial aggregation of crystallins will be presented. Also, the rationale behind the necessity of domain duplicates and concomitant increase in the refractive index in case of eye lens proteins will be outlined.

37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013 | 217 PP- 159 NATIONAL SYMPOSIUM ON “FRONTIERS OF BIOPHYSICS, BIOTECHNOLOGY & BIOINFORMATICS”

SYNTHESIS OF NOBLE BIMETALLIC NANOPARTICLES FROM THE AQUEOUS STEM EXTRACT OF Ceriops tagal Larkins Ramteke*, Poonam Gawali and B.L.Jadhav Department of Life Sciences, University of Mumbai, Mumbai- 400 098

Background: C.tagal, a true mangrove, has wide range of medicinal properties. Synthesis of nanoparticles using C.tagal as a reducing agent may enhance its biological activity. We have synthesized stable bimetallic nanoparticles using aqueous stem extract of C.tagal. UV–visible spectroscopy, transmission electron microscopy, X-ray diffraction and Fourier transmission Infrared spectroscopy were used to characterize the synthesized nanoparticles. Results: The kinetics of particle formation was time and temperature dependent. An effect of temperature on rate of synthesized nanoparticles was studied at RT, 40ºC, 50ºC and 60ºC. Optimization and characterization studies indicate that the maximum rate of synthesis is

achieved by challenging 5% aqueous stem extract with 0.9mM HAuCl4 and 1mM Ag2So4

(1:1) and 0.9mM HAuCl4 and 2mM AgNO3 (1:1) at R.T in 40 min. Pink and grey colour is visualized after complete bio-reduction of HAuCl4 : Ag2So4 and HAuCl4 :AgNO3 respectively. Further, the UV-visible absorption spectroscopy confirmed the formation of bimetallic nanoparticles and the peaks were observed at 535 nm. The TEM result revealed the spherical shape of nanoparticles. XRD data showed characteristic peaks for Ag-Au bimetallic nanoparticles. Before and after bio-reduction changes in functional group of plant extract was recorded by FTIR spectra. Conclusion: Rapid eco-friendly bimetallic nanoparticles are synthesized at R.T. using 2mM

AgNo3: 0.9mM HAuCl4 and 1mM Ag2SO4: 0.9mM HAuCl4. This is the first report on synthesis of bimetallic nanoparticles using C.tagal. Keywords: Bimetallic nanoparticles, Mangroves.

218 | 37th ANNUAL MEETING OF INDIAN BIOPHYSICAL SOCIETY, Jan 13-16, 2013