DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Synthesis and pharmacological activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(M)2(U)]2+, where M = 2,2'-bipyridine/1,10-phenanthroline and U = tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH3-tpl (Ru3), 4-CH3O-tpl (Ru4), and 4-NO2-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV-Vis, 1H NMR, 13C-NMR, FAB-Mass spectrophotometer, and elemental analysis. The complexes display metal-ligand charge transfer (MLCT) transitions in the visible region. The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's ascitic carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1-Ru6 were found to increase the life span of the tumor hosts by 19-52%, and decreased tumor volume and viable ascitic cell count. The results of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the compounds itself. The significant antibacterial activity was observed for Ru1-Ru4 against microorganisms like Vibrio cholera 865, Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033 and Shigella sonnai.     

Assessment of dominance hierarchy through urine scent marking and its chemical constituents in male blackbuck Antelope cervicapra, a critically endangered species

In ungulates the process of chemical communication by urinary scent marking has been directly related to reproductive dominance, territorial defense and proximity to resources. The differences in the frequency of urine marking and chemical composition of urine of males Antelope cervicapra before, during and after the dominance hierarchy period were assessed. The variations in the urine marking and its chemical profiles of dominant males (n = 9), bachelors (n = 5) and sub-adult males (n = 5) were compared to find out how the dominance hierarchy influences the confined blackbuck herd under semi-natural captive conditions. The frequency of urine marking is significantly higher (p < 0.001) in dominant males. Twenty-eight major constituents were identified in the urine of dominant males (before, during and after the dominance hierarchy period), bachelor and sub-adult males. Among these, three specific compounds namely, 3-hexanone (I), 6-methyl-5-hepten-2-one (II) and 4-methyl-3-heptanone (III) were seen only in dominant males urine during the dominance hierarchy period. Based on the behavioural observation and the unique chemical constituents in the urine, it is concluded that the dominant male scent odor suppresses aggression, scent marking, scent production and territorial patrolling activities of subordinate males, through which the dominant male establish their hierarchy and attains success in reproduction.     

Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.     

Evaluation of Neurotransmitter Alterations in Four Distinct Brain Regions After Rapid Eye Movement Sleep Deprivation (REMSD) Induced Mania-Like Behaviour in Swiss Albino Mice

A number of neurotransmitter systems have been implicated in contributing to the pathology of mood disorders, including those of dopamine (DA), serotonin (5-HT), norepinephrine (NE) and γ-aminobutyric acid (GABA). Rapid eye movement sleep deprivation (REMSD) alters most of the neurotransmitters, which may have adverse behavioural changes and other health consequences like mania and other psychiatric disorders. The exact role of REMSD altered neurotransmitter levels and the manner in which emerging consequences lead to mania-like behaviour is poorly understood. Thus, we sought to verify the levels of neurotransmitter changes after 48, 72 and 96 h of REMSD induced mania-like behaviour in mice. We performed modified multiple platform (MMP) method of depriving the REM sleep and one group maintained as a control. To measure the hyperactivity through locomotion, exploration and behavioural despair, we performed the Open Field Test (OFT) and the Forced Swim Test (FST). Quantitative determinations of DA, 5-HT, NE and GABA concentrations in four distinct brain regions (cerebral cortex, hippocampus, midbrain, and pons) were determined by the spectrofluorimetric method. These experiments showed higher locomotion and increased swimming, struggling/climbing and decreased mobility among REMSD animals as well as disrupted concentrations of the majority of the studied neurotransmitters during REMSD. Our study indicated that REMSD results in mania-like behaviour in mice and associated disruption to neurotransmitter levels, although the exact mechanisms by which these take place remain to be determined.     

The C1 and C2 domains target human type 6 adenylyl cyclase to lipid rafts and caveolae

Previous data has shown that adenylyl cyclase type 6 (AC6) is expressed principally in lipid rafts or caveolae of cardiac myocytes and other cell types while certain other isoforms of AC are excluded from these microdomains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we show AC6 is localized in lipid rafts of COS-7 cells (expressing caveolin-1) and in HEK-293 cells or cardiac fibroblasts isolated from caveolin-1 knock-out mice (both of which lack prototypical caveolins). To determine the region of AC6 that confers raft localization, we independently expressed each of the major intracellular domains, the N-terminus, C1 and C2 domains, and examined their localization with various approaches. The N-terminus did not associate with lipid rafts or caveolae of either COS-7 or HEK-293 cells nor did it immunoprecipitate with caveolin-1 when expressed in COS-7 cells. By contrast, the C1 and C2 domains each associated with lipid rafts to varying degrees and were present in caveolin-1 immunoprecipitates. There were no differences in the pattern of localization of either the C1 or C2 domains between COS-7 and HEK-293 cells. Further dissection of the C1 domain into four individual proteins indicated that the N-terminal half of this domain is responsible for its raft localization. To probe for a role of a putative palmitoylation motif in the C-terminal portion of the C2 domain, we expressed various truncated forms of AC6 lacking most or all of the C-terminal 41 amino acids. These truncated AC6 proteins were not altered in terms of their localization in lipid rafts or their catalytic activity, implying that this C-terminal region is not required for lipid raft targeting of AC6. We conclude that while the C1 domain may be most important, both the C1 and C2 domains of AC6 play a role in targeting AC6 to lipid rafts.     

β-Amyloid1-42, HIV-1Ba-L (Clade B) Infection and Drugs of Abuse Induced Degeneration in Human Neuronal Cells and Protective Effects of Ashwagandha (Withania somnifera) and Its Constituent Withanolide A

Alzheimer''s disease (AD) is characterized by progressive dysfunction of memory and higher cognitive functions with abnormal accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles throughout cortical and limbic brain regions. Withania somnifera (WS) also known as ‘ashwagandha’ (ASH) is used widely in Ayurvedic medicine as a nerve tonic and memory enhancer. However, there is paucity of data on potential neuroprotective effects of ASH against β-Amyloid (1–42) (Aβ) induced neuropathogenesis. In the present study, we have tested the neuroprotective effects of Methanol: Chloroform (3:1) extract of ASH and its constituent Withanolide A (WA) against Aβ induced toxicity, HIV-1_Ba-L (clade B) infection and the effects of drugs of abuse using a human neuronal SK-N-MC cell line. Aβ when tested individually, induced cytotoxic effects in SK-N-MC cells as shown by increased trypan blue stained cells. However, when ASH was added to Aβ treated cells the toxic effects were neutralized. This observation was supported by cellular localization of Aβ, MTT formazan exocytosis, and the levels of acetylcholinesterase activity, confirming the chemopreventive or protective effects of ASH against Aβ induced toxicity. Further, the levels of MAP2 were significantly increased in cells infected with HIV-1_Ba-L (clade B) as well as in cells treated with Cocaine (COC) and Methamphetamine (METH) compared with control cells. In ASH treated cells the MAP2 levels were significantly less compared to controls. Similar results were observed in combination experiments. Also, WA, a purified constituent of ASH, showed same pattern using MTT assay as a parameter. These results suggests that neuroprotective properties of ASH observed in the present study may provide some explanation for the ethnopharmacological uses of ASH in traditional medicine for cognitive and other HIV associated neurodegenerative disorders and further ASH could be a potential novel drug to reduce the brain amyloid burden and/or improve the HIV-1 associated neurocognitive impairments