Keya Chaudhuri

Sanguinarine: a monofunctional intercalating alkaloid

Sanguinarine (fig. 1), a benzophenanthridine alkaloid, has been reported to possess anti-tumour and anti-microbial activity [ 1 ]. It inhibits both photosynthetic phosphorylation associated with ferricyanide reduction and cyclic photophosphorylation catalyzed by phenazine methosulphate [2]. The alkaloid increases the intrinsic viscosity of DNA and uncouples respiration and oxidative phosphorylation in rat liver mitochondria [3]. These physical and biochemical effects can be attributed to the formation of a complex of sanguinarine with DNA. We report here a quantitative aspect of the binding of sanguinarine to calf thymus DNA from the measurements of absorption spectrophotometry, equilibrium dialysis, melting behaviour, viscosity, circular dichroism and spectrofluorimetric titration. We also compare our experimental data with ethidium bromide under identical experimental conditions and conclude that sanguinarine (like ethidium) binds by monofunctional mode of intercalation.

The Indian Genome Variation database (IGVdb): A project overview

Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.

The anti-bacterial action of diclofenac shown by inhibition of DNA synthesis

Most strains of Gram-positive and Gram-negative bacteria were inhibited by 50-100 mg/l of the anti-inflammatory agent, diclofenac sodium (Dc). In vivo test using 30 or 50 microg Dc per 20 g mouse (Swiss Albino variety) significantly (P <0.001) protected the animals when challenged with 50 MLD of a virulent Salmonella typhimurium. The anti-bacterial action of Dc was found to be due to inhibition of DNA synthesis which was demonstrated using 2 micro Ci (3H) deoxythymidine uptake.

Association of cholera toxin with Vibrio cholerae outer membrane vesicles which are internalized by human intestinal epithelial cells

Cholera toxin (CT) is the major virulence factor of pathogenic Vibrio cholerae. The present study demonstrates that a fraction of CT is associated with the outer membrane vesicles (OMVs) released by V. cholerae. Atomic force microscopy (AFM) and also transmission electron microscopy (TEM) of purified OMVs from toxigenic V. cholerae O395 revealed spherical shaped vesicles of size range 20–200 nm. Immunoblotting of purified OMVs with polyclonal anti‐CT antibody and GM1‐ganglioside dependent ELISA suggest that CT is associated with OMVs. CHO cell assay indicated that OMV associated CT is physiologically active. OMVs labeled with fluorescent dye interacted with intestinal epithelial cells via the CT‐receptor and were internalized increasing the cAMP level. Thus OMVs may represent an important vehicle in delivering CT to epithelial cells.

Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India

A study was conducted to explore the effect of arsenic causing conjunctivitis, neuropathy and respiratory illness in individuals, with or without skin lesions, as a result of exposure through drinking water, contaminated with arsenic to similar extent. Exposed study population belongs to the districts of North 24 Parganas and Nadia, West Bengal, India. A total of 725 exposed (373 with skin lesions and 352 without skin lesions) and 389 unexposed individuals were recruited as study participants. Participants were clinically examined and interviewed. Arsenic content in drinking water, urine, nail and hair was estimated. Individuals with skin lesion showed significant retention of arsenic in nail and hair and lower amount of urinary arsenic compared to the group without any skin lesion. Individuals with skin lesion also showed higher risk for conjunctivitis ((odds ratio) OR: 7.33, 95% CI: 5.05–10.59), peripheral neuropathy (OR: 3.95, 95% CI: 2.61–5.93) and respiratory illness (OR: 4.86, 95% CI: 3.16–7.48) compared to the group without any skin lesion. The trend test for OR of the three diseases in three groups was found to be statistically significant. Again, individuals without skin lesion in the exposed group showed higher risk for conjunctivitis (OR: 4.66, 95% CI: 2.45–8.85), neuropathy (OR: 3.99, 95% CI: 1.95–8.09), and respiratory illness (OR: 3.21, 95% CI: 1.65–6.26) when compared to arsenic unexposed individuals. Although individuals with skin lesions were more susceptible to arsenic-induced toxicity, individuals without skin lesions were also subclinically affected and are also susceptible to arsenic-induced toxicity and carcinogenicity when compared to individuals not exposed to arsenic.

Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression.

Arsenic is a well-established human carcinogen; however molecular mechanisms to arsenic-induced carcinogenesis are complex and elusive. The present study identifies a potential biomarker of arsenic exposure, and redefines arsenic-induced signaling in stimulation of cell proliferation. The effect of arsenic exposure on gene expression was evaluated in PBMC of arsenic-exposed individuals selected from a severely affected district of West Bengal, India. A novel, un-documented biomarker of arsenic exposure, CyclinA was identified by microarray analysis from the study. Non-transformed cell lines HaCat and Int407 when exposed to clinically achievable arsenic concentration showed significant increase of CyclinA substantiating the clinical data. An associated increase in S phase population of cells in cell cycle, indicative of enhanced proliferation was also noticed. On further investigation of the pathway to arsenic-induced proliferation, we observed that arsenic resulted: ROS generation; activated Erk signaling; stimulated AP-1 activity, including immediate early genes, c-Jun and c-Fos. N-Acetyl-l-cysteine, a ROS quencher, blocked the arsenic-induced effects. Our study underlines a previously undefined mechanism by which arsenic imparts its toxicity and results in uncontrolled cell proliferation.

Involvement of in vivo induced icmF gene of Vibrio cholerae in motility, adherence to epithelial cells, and conjugation frequency.

Previously, using global transcription profile approach icmF gene of Vibrio cholerae was identified as an in vivo induced gene. In the present study, the icmF gene of V. cholerae O395 was cloned, sequenced, and used to construct an icmF insertion mutant. This IcmF is homologous to Legionella pneumophila IcmF, belonging to the icm cassette responsible for macrophage killing and intracellular survival of the organism. The icmF insertion mutant exhibited reduced motility and increased adherence to human intestinal epithelial cells. The presence of ATP-GTP-binding site suggests further a possible role of IcmF as a signaling molecule. Triparental-mating assay, with the mutant as a recipient, showed higher conjugation frequency than wild type. We propose that the increased adherence to epithelial cell line and increased conjugation frequency of the mutant result from some sort of cell surface reorganization.

MAOA promoter polymorphism and attention deficit hyperactivity disorder (ADHD) in indian children

Attention deficit hyperactivity disorder (ADHD) is a highly disabling, early onset childhood neurobehavioral disorder with a higher occurrence in boys as compared to girls. Pharmacological and molecular genetic studies have revealed the influence of dopaminergic and serotonergic systems in the etiology of the disorder. Monoamine oxidase A (MAOA) is a mitochondrial enzyme that regulates the dopaminergic signals in the pre‐synaptic region. Polymorphism in the promoter region of the MAOA gene, which comprises of 30 bp repeats with repeat number varying between 2.5, 3.5, 4.5, and 5.5, has been shown to be associated with various neurobehavioral disorders including ADHD. This is the first study on Indian ADHD cases to validate an association between transmission of MAOA promoter polymorphism and risk of ADHD. We have analyzed the MAOA promoter polymorphism in a group of ADHD probands, their parents and ethnically matched controls by UNPHASED. Our findings indicate significant difference in the frequency of 3.5 repeat allele (P = 0.02) between cases and controls and preferential transmission of the short allele (3.5 repeat) from mothers to male ADHD probands (P = 0.005). We conclude that the short 3.5 repeat allele of the MAOA‐u VNTR is probably associated with ADHD in our population and could be the reason for making boys prone to ADHD as compared to girls.

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

IntroductionResistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated.Materials methods and resultsMenadione synergized NaAsO2 to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO2 and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC.ConclusionThis study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.

THE EFFECT OF pH ON THE ABSORPTION AND FLUORESCENCE SPECTRA OF SANGUINARINE

Abstract— The influence of pH in the range 1.0 to 13.0 on the structure of sanguinarine has been investigated by spectrophotometric and spectrofluorimetric measurements. The data on absorption maxima, molar extinction coefficient, fluorescence emission maxima, relative fluorescence intensity and fluorescence quantum yield of the sanguinarine under various pH are presented. It is suggested that the pH dependent absorbance and fluorescence property of sanguinarine is due to the formation of the carbinolamine by hydroxylation at C‐6 at alkaline pH.