Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.     

Cloning and characterization of an eukaryotic initiation factor-2alpha kinase from the silkworm,Bombyx mori

Eukaryotic initiation factor 2alpha (eIF-2alpha) kinases are involved in the translational regulations that occur in response to various types of environmental stress, and play an important role in the cellular defense system operating under unfavorable conditions. The identification of additional eIF-2alpha kinases and the elucidation of their functions are necessary to understand how different eIF-2alpha kinases can specifically respond to distinct stimuli. Here, we report a novel eIF-2alpha kinase, termed BeK, from the silkworm, Bombyx mori. This gene encodes 579 amino acids and contains all 11 catalytic domains of protein-serine/threonine kinases. Most notably, it contains an "Ile-Gln-Met-Xaa-Xaa-Cys" motif, which is highly conserved from yeast to mammalian eIF-2alpha kinases. BeK does not show any significant homology in the NH(2) terminal regulatory domain, suggesting a distinct regulatory mechanism of this novel eIF-2alpha kinase. BeK is ubiquitously expressed in the various tissues throughout the final larval stage. Importantly, BeK is activated in Drosophila Schneider cells following heat shock and osmotic stress, and activated-BeK has been shown to phosphorylate an eIF-2alpha subunit at the Ser(50) site. However, other forms of stress, such as immune stress, endoplasmic reticulum stress and oxidative stress, cannot significantly elicit BeK activity. Interestingly, the baculovirus gene product, PK2, can inhibit BeK enzymatic activity, suggesting that BeK may be an endogenous target for a viral gene product. Taken together, these data indicate that BeK is a novel eIF-2alpha kinase involved in the stress response in B. mori.     

Synthesis and biological evaluation of pyrazole linked benzothiazole-β-naphthol derivatives as topoisomerase I inhibitors with DNA binding ability

A series of new pyrazole linked benzothiazole-β-naphthol derivatives were designed and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable cytotoxicity with IC₅₀ values ranging between 4.63 and 5.54?µM against human cervical cancer cells (HeLa). Structure activity relationship was elucidated by varying different substituents on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV–visible, fluorescence and circular dichroism studies showed that these derivatives exhibited good DNA binding affinity. Additionally, these derivatives can effectively inhibit the topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that the derivatives bind with the minor groove of the DNA.     

Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair

Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.     

Differences in the plasma membrane proteins of chronic alcoholic rat brain

Plasma membranes were isolated from the cerebral cortex of control and chronic ethanol-treated rat brains. Analysis of protein composition by SDS-PAGE and by two-dimensional gel electrophoresis (IEF-SDS-PAGE) revealed significant differences in the membrane protein patterns between control and ethanol-treated rat cerebral cortices, indicating the loss of several proteins in membranes from ethanol-treated rat brains. Plasma membrane-associated protein species are categorized into ethanol-sensitive and -insensitive proteins, based on their response to ethanol. This study reports that ethanol depletes certain intrinsic proteins of membranes that might be responsible for plasma membrane disruption by ethanol.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.