Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

Targeted Ablation of Nesprin 1 and Nesprin 2 from Murine Myocardium Results in Cardiomyopathy,Altered Nuclear Morphology and Inhibition of the Biomechanical Gene Response

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.     

Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca²⁺) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6^ΔC). Furthermore, stable overexpression of Anxa6^T356D, a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.     

Neurotransmitters in alcoholism: A review of neurobiological and genetic studies

Recent advances in the study of alcoholism have thrown light on the involvement of various neurotransmitters in the phenomenon of alcohol addiction. Various neurotransmitters have been implicated in alcohol addiction due to their imbalance in the brain, which could be either due to their excess activity or inhibition. This review paper aims to consolidate and to summarize some of the recent papers which have been published in this regard. The review paper will give an overview of the neurobiology of alcohol addiction, followed by detailed reviews of some of the recent papers published in the context of the genetics of alcohol addiction. Furthermore, the author hopes that the present text will be found useful to novices and experts alike in the field of neurotransmitters in alcoholism.     

Microtubule associated proteins of goat brain

The microtubule associated proteins of goat brain were separated from tubulin on the basis of their thermostability and then fractionated by chromatography on Sepharose 4B column. Analysis of the fractions by SDS-Polyacrylamide gel electrophoresis and assay of their tubulin-assembly-promoting activity indicate that this activity resides primarily in the tauproteins (mol. wt. 55,000–70,000) and a class of even lower molecular weight (25,000–35,000) proteins. Electrophoresis of the microtubule associated protein fractions separated from tubulin by phosphocellulose chromatography are in agreement with the results obtained from fractionation on Sepharose 4B columns.     

Immobilization of yeast cells containing beta-galactosidase

Cultural conditions optimum for beta-galactosidase production by Saccharomyces anamensis are pH 4.5, temperature 26 +/- 2 degrees C, and 30 h of incubation period. Addition of lactose at 24 h fermentation greatly increase the level of enzyme. Optimum pHl, temperature, pH stability, and thermostability of yeast beta-galactosidase are negligibly affected by immobilization. The K(m) values of enzyme in the native and immobilized cells are 102mM and 148mM, respectively. Glucose noncompetitively inhibits the enzyme activity. Addition of substances such as dithioerythritol, glutathione, and bovine serum albumin to the native cell during assay procedure and immobilized cell prior to immobilization have stimulatory effects on enzyme activity. Metal ions like Ca(2+), Mg(2+) enhance the beta-galactosidase activity for both intact and bound cells. Immobilized cells retain 68.6% of the beta-galactosidase activity of intact cells and there is no significant loss of activity on storage at 4 degrees C for 28 days.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.