Biplab Giri

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom.

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% alpha helix, 19% beta sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD(50) was 2.5 mg kg(-1) in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC(5)(0) on U937 and K562 cells were 3.5 microg/ml and 1.1 microg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p<0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flow-cytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity.

Experimental osteoporosis induced in female albino rats and its antagonism by Indian black scorpion (Heterometrus bengalensis C.L.Koch) venom.

The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion (Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca(2+), PO(4)(3-), CRE, OH-P levels, serum/plasma Ca(2+), PO(4)(3-), TRAP, IL1, IL6, TNFalpha and PTH level, bone Ca(2+), PO(4)(3-), Mg(2+), Zn(2+) and decreased level of serum/plasma ALP, EST and PTH, bone Na(+). In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract.

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC₅₀ values of 49 μg/ml and 30 μg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21(cip1) and p27(kip1)) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature.

A high molecular weight protein Bengalin from the Indian black scorpion (Heterometrus bengalensis C.L. Koch) venom having antiosteoporosis activity in female albino rats

This study reports the presence of a high molecular weight protein (Bengalin) from the Indian black scorpion (Heterometrus bengalensis) venom having antiosteoporosis activity in experimental osteoporosis developed in female albino Wister rats. Bengalin was purified through DEAE-cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the Bengalin was found to be 72kDa and the first 20 amino acid sequence was found to be G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin exhibited significant antiosteoporosis activity in experimental female rats, which was confirmed through analysis of urine Ca(2+), PO(4)(3-), CRE & OH-P. Bengalin (3 microg and 5 microg/100g rat/i.p.) antagonized osteoporosis by restoring urinary Ca(2+), PO(4)(3-), CRE and OH-P, serum/plasma Ca(2+), PO(4)(3-), ALP, TRAP, PTH, T(3), TSH, Osteocalcin, IL1, IL6 and TNF alpha and bone minerals Ca(2+), P, Mg(2+), Zn(2+), Na(+), as compared with the sham operated control rats. Bone minerals density of osteoporosis female rats was improved due to Bengalin, observed through DEXA scan. Subacute toxicity studies in male albino mice, Bengalin showed cardiotoxicity. In vivo experiments, Bengalin showed cardiotoxicity on isolated guinea pig heart, guinea pig auricle, and neurotoxicity on isolated rat phrenic nerve diaphragm preparation. Further detail studies on the toxicity, antiosteoporosis and structural identity of Bengalin are warranted.

Anti-inflammatory and Anti-tumor Activities of Parthenolide: An Update

Parthenolide (PTL), the secondary metabolite of feverfew plant (Tanaceum parthenium), has been used in various medical purposes globally. Inflammation represents a physiological response to injury and helps to restore tissue homeostasis. Inflammation and cancer both are associated with genotoxicity, invasion, metastasis, and abnormal tissue repair mechanisms. PTL inhibit major cellular inflammatory and proliferation pathways like NFκB, STAT3, and MAPK along with the activity and expression of several inflammatory mediators including COX. NFκB pathway plays a key role in controlling cell cycle progression and apoptosis together with metastasis and cancer of various types. Elevated NFκB, Wnt/β-catenin pathways are crucial factors of tumorogenesis. PTL inhibits NFκB and Wnt/β-catenin pathways, and thereby promotes apoptosis and suppresses cell proliferation. Experimental data showed that PTL protects normal cells from apoptosis; whereas in cancer cells it induces apoptotic cell death. Hence, parthenolide could be useful in controlling inflammatory diseases alone or together with tumorogenesis due to its evident anticancer potency and anti-inflammatory nature.

In Vitro Activity Screening of Snake Venom against Multi Drug ResistantTuberculosis

The re-emergence of multidrug-resistant tuberculosis (MDR-TB) events has brought to light the importance of screening effective novel drugs. In the present study, in vitro activities of different snake (Naja naja, Bungarus fasciatus, Daboia russelli russelli, Naja kaouthia) venoms have been investigated against clinical isolate of MDR-TB strains. All the venoms inhibited the mycobacterial growth for at least a week in common and two of them (Naja naja, Naja kaouthia) showed significantly longer inhibition up to two weeks against the MDR-TB strain with single dose and one repetition of those two venoms exhibited inhibition up to more than 4 weeks.

Double Edge Effect of DPP4 Inhibitor Sitagliptin, a Type-2 Anti-Diabetic Drug, on Inflammation, Injury and Cancer

Maintaining a healthy glycaemic condition is associated with so many more other protective/precautionary attributes. Recent advancement in the field of drug discovery have led us find more and more options to choose from. DPP-4 inhibitor sitagliptin makes sustained expression of incretins GLP-1 and GIP that in turn establishes glycaemic control. But recent reports claimed pro-inflammatory as well as pro-carcinogenic action of the drug after prolonged use in T2DM patients. Interestingly, in those cases also, the effector molecule responsible is said to be the incretin GLP-1. The same molecule has been hypothesized for being responsible to cause thyroid C-cell carcinomas. In vitro investigations using human cell lines did not show any clues in support of carcinoma formation. Instead, the DPP-4 inhibitory action of sitagliptin serves in other complications associated with T2DM. In any tissue injury due to hypoxic stress in T2DM, sitagliptin serves in the tissue repair mechanisms via its DPP-4 inhibiting property. Consequently, DPP-4, for not being able to be active in presence of sitagliptin, fails to truncate the CXCL-12/SDF-1 and therefore helps in the homing of HPCs/EPCs to repair the injured tissue. In conclusion, it can be said that more research is definitely required to be confirmed about the pre-cancerous activity of the drug, and to make the drug more usable in favour of the other beneficial effects of it.

Is it possible to avert arsenic effects on cells and tissues bypassing its toxicity and suppressive consequences of energy production? A hypothesis

Arsenic, a sulfhydryl reactive metalloid, found primarily in two forms: arsenite and arsenate, causing several human health problems, is considered as a dreaded agent against public health. It mainly spreads through groundwater contamination and affects human mainly through drinking water. Arsenic contaminated groundwater is now a major threat in some parts of India (the river basin of Ganga and Brahmaputra) and Bangladesh. The current authors belong to the region where arsenic poisoning and its consequences are spreading in an uncontrolled way. We are helpless to stop the spreading of geogenic groundwater arsenic contamination at present. Although most of the research on arsenic removal from drinking water and on toxicity profile has been carried out, very few preventive measures have been reported till date to balance the arsenic-induced cellular energy deficiency and oxidative stress-mediated cell death and cellular senescence. And, therefore, we need to think about alternative remedial to address such problems, which propel us to propose the current hypothesis that the adverse effects of energy imbalance due to arsenic toxicity in cells could be dodged by intake of moderate amount of alcohol. While pyruvate dehydrogenase complex is blocked by arsenic, glucose cannot be utilized through Krebs cycle. However, alcohol can produce energy by bypassing the aerobic adenosine triphosphate (ATP) production machinery. In addition, arsenic poisoning incurs cellular oxidative stress which needs to be scavenged further. So to meet this secondary problem, we also suggest consuming red grape juice (a potent antioxidant and cytoprotective agent) in addition to alcohol (as per International Center for Alcohol Policies (ICAP) Drinking Guidelines) in our second part of the hypothesis. In conclusion, it can be suggested that the red wine which contains moderate amount of alcohol and high levels of red grape polyphenols, galic acid, resveratrol, and other antioxidants could be the best alternative to tackle the arsenic-induced cellular aging, senescence, and death.

Molecular modeling of NK-CT1, from Indian monocellate cobra (Naja kaouthia) and its docking interaction with human DNA topoisomerase II alpha.

A 6.76 kDa molecular weight cardio and cytotoxic protein of 60 amino acids in length called NK-CT1, was purified from the venom of Indian monocellate cobra (Naja kaouthia) by ion-exchange chromatography and HPLC as described in our earlier report. Therefore it is of interest to utlize the sequence of NK-CT1 for further functional inference using molecular modeling and docking. Thus homology model of NK-CT1 is described in this report. The anti-proliferative activity of the protein, binding with human DNA topoisomerase-II alpha was demonstrated using docking data with AUTODOCK and AUTODOCK MGL tools. Data shows that M26, V27 and S28 of NK-CT1 is in close contact with the nucleotides of the oligonucleotide, bound with topoisomerase-II alpha complex.