Cletus J. M. D'Souza

The pharmacological role of nucleotidases in snake venoms

Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as nucleotidases (5′nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near‐ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)—a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established. Copyright

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N10‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.

The Pharmacological Role of Phosphatases (Acid and Alkaline Phosphomonoesterases) in Snake Venoms related to Release of Purines - a Multitoxin

Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.

MTNR1B gene polymorphisms and susceptibility to Type 2 Diabetes: A pilot study in South Indians.

Type 2 Diabetes (T2D) is the major health concern in the Indian subcontinent. A genome-wide association study carried out with non-diabetic Indians showed association of MTNR1B variants with fasting glucose. MTNR1B mediates the effect of melatonin on insulin secretion. In light of the growing importance of MTNR1B in the etiology of T2D, we sought to test its association with the disease in the south Indian type 2 diabetics. Five single nucleotide polymorphisms of MTNR1B (rs10830962, rs10830963, rs3847554, rs1387153 and rs2166706) were genotyped in 346 T2D patients and 341 non-diabetic controls. None of the SNPs differed significantly between patients and controls with respect to allele and genotype frequencies. Linear regression analysis after adjustment for age, sex and BMI showed a significant positive association of rs3847554 with fasting glucose under recessive model (β=14.98, p=0.012). Haplotypes constituted by minor alleles of rs3847554, rs1387153, rs2166706, rs10830963 and major allele of rs10830962 showed significant positive correlation with fasting glucose (p<0.05). Though the results obtained are suggestive of MTNR1B role in T2D etiology, they need to be confirmed with much larger sample sizes.

Identification of nuclear genes affecting 2-Deoxyglucose resistance in Schizosaccharomyces pombe.

Abstract 2-Deoxyglucose (2-DG) is a toxic glucose analog. To identify genes involved in 2-DG toxicity in Schizosaccharomyces pombe, we screened a wild-type overexpression library for genes which render cells 2-DG resistant. A gene we termed odr1, encoding an uncharacterized hydrolase, led to strong resistance and altered invertase expression when overexpressed. We speculate that Odr1 neutralizes the toxic form of 2-DG, similar to the Saccharomyces cerevisiae Dog1 and Dog2 phosphatases which dephosphorylate 2-DG-6-phosphate synthesized by hexokinase. In a complementary approach, we screened a haploid deletion library to identify 2-DG-resistant mutants. This screen identified the genes snf5, ypa1, pas1 and pho7. In liquid medium, deletions of these genes conferred 2-DG resistance preferentially under glucose-repressed conditions. The deletion mutants expressed invertase activity more constitutively than the control strain, indicating defects in the control of glucose repression. No S. cerevisiae orthologs of the pho7 gene is known, and no 2-DG resistance has been reported for any of the deletion mutants of the other genes identified here. Moreover, 2-DG leads to derepressed invertase activity in S. pombe, while in S. cerevisiae it becomes repressed. Taken together, these findings suggest that mechanisms involved in 2-DG resistance differ between budding and fission yeasts.

Plasmid loss in plasmid-carrying strains of Escherichia coli treated with phenoxazines and an approach to study their DNA binding properties.

The effect of subinhibitory concentrations of 2-trifluoromethyl-N10-substituted phenoxazines on plasmid-coded antibiotic resistance in Escherichia coli was investigated. Phenoxazine treatment resulted in the loss of resistance markers to an extent of 8-63% in all the strains tested, and the disappearance of plasmid DNA in phenoxazine sensitive colonies was evidenced by agarose gel electrophoresis. The resistant strains were sensitized in the presence of phenoxazines with a concomitant reduction in the MIC (minimum inhibitory concentration) values. The UV, fluorescence spectral, and ethidium bromide displacement agarose gel assay methods revealed that phenoxazines are intercalated with plasmid DNA. Progressive addition of DNA led to a significant reduction in the peak intensity of the absorption maximum of phenoxazine derivative. Further, destabilization of ethidium bromide-DNA complex as seen from fluorescence microscopy in the presence of phenoxazines was observed. The potency of phenoxazines to sensitize the resistant organisms follows the order butyl > propyl > acetyl derivatives.

Multifaceted effects of antimetabolite and anticancer drug, 2-deoxyglucose on eukaryotic cancer models budding and fission yeast

Glycolytic inhibitors are of interest therapeutically as they are effective against cancers that display increased glycolytic rate and mitochondrial defects. 2‐Deoxyglucose (2‐DG) is one such glycolytic inhibitor and was identified to be a competitive inhibitor of glucose. Studies from past few decades have shown that the mechanism of action of 2‐DG is complex involving several metabolic and signaling pathways. Budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are two important models for studying metabolism, cell cycle and cell signaling. These two unicellular eukaryotes are Crabtree positive yeasts exhibiting a metabolism similar to that of cancer cells. Effects of 2‐DG in yeast is of interest owing to these similarities and hence yeasts have emerged as ideal model organisms to study the mode of action and resistance to 2‐DG. In this review, we summarize the studies on biological effect and resistance to 2‐DG in budding and fission yeasts and give an insight into its possible mechanism of action as models for understanding cancer metabolism and drugs affecting cancer progression.

Vanillin Analog – Vanillyl Mandelic Acid, a Novel Specific Inhibitor of Snake Venom 5′-Nucleotidase

Snake venom 5′‐nucleotidase (5′NUC) plays a very important role in envenomation strategies; however, apart from its modulation of hemostatic functions, its other pharmacological effects are not yet well characterized. Several studies have used specific inhibitors of enzyme toxins as a biochemical or pharmacological tool to characterize or establish its mechanism of action. We report here for the first time vanillin mandelic acid (VMA), an analog of vanillin, to potentially, selectively, and specifically inhibit venom 5′NUC activity among other enzymes present in venoms. VMA is much more potent in inhibiting 5′NUC activity than vanillyl acid (VA). The experimental results obtained are in good agreement with the in silico molecular docking interaction data. Both VA and VMA are competitive inhibitors as evident by the inhibition‐relieving effect upon increasing the substrate concentration. VMA also dose‐dependently inhibited the anticoagulant effect in Naja naja venom. In this study, we report novel non‐nucleoside specific inhibitors of snake venom 5′NUC and experimentally demonstrate their involvement in the anticoagulant activity of N. naja venom. Hence, we hypothesize that VMA can be used as a molecular tool to evaluate the role of 5′NUC in snake envenomation and to develop prototypes and lead compounds with potential therapeutic applications against snake bites.

Production of oxygen free radicals by Ehrlich ascites tumour cells: effect of lipids

Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and platelet activating factor (PAF) stimulated the generation of oxygen free radicals (nitro-blue tetrazolium reduction) in Ehrlich ascites tumour (EAT) cells. PAF was effective at an optimal concentration of 4 μM, but was inhibited by BN 52021, a specific PAF antagonist. Lyso-PAF was ineffective. Inclusion of different lipids during incubation prior to the addition of PAF, resulted in the activation/inhibition of free radical generation. Among the phospholipids at a concentration of 50 μg/ml, the order of activation was phosphatidylserine > phosphatidylglycerol > phosphoinositides > phosphatidylinositol > phosphatidylethanolamine. Phosphatidylcholine was not effective, while sphingolipids were inhibitory. In addition, Ehrlich ascites tumour cells grown in mice under marginal vitamin A deficiency, showed an augmented production of free radicals compared to control cells. This was suppressed by exogenous addition of vitamin A or superoxide dismutase. These results suggest that membrane lipids and dietary factors like vitamin A probably function as physiological modulators in regulating the free radical generation.

PAF-Acether in AK-5 tumour cells

PAF-Acether fraction derived from stimulated AK-5 tumour cells, aggregated human platelets. The platelet aggregating ability increased linearly with increasing concentration of the stimulant, calcium ionophore A23187, and reached a maximum at 6 microM in 25 minutes. This factor had biological and chemical properties identical to authentic PAF-acether. Our results demonstrate that, although PAF-acether is produced mainly from pro-inflammatory cells, it appears to be produced even in tumour cells.