Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi

Background  

Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium.     

Assembly and Disassembly of the Capsid-Like External Scaffold of Immature Virions during Vaccinia Virus Morphogenesis

Infectious poxvirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxvirus particles. In the case of vaccinia virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant virus particles. Furthermore, the morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.The assembly and morphogenesis of vaccinia virus (VACV) and other poxviruses occurs in specialized regions of the cytoplasm called factories. The first distinctive viral forms discerned by transmission electron microscopy are spherical immature virions (IVs) and their membrane crescent precursors, which appear to be covered by a layer of spicules (14). More-recent studies employing three-dimensional deep-etch electron microscopy revealed that the “spicule coat” of IVs is actually a continuous honeycomb lattice (20). The IVs enclose dense granular material comprising the core precursors and a DNA nucleoid. The “spicule coat” is lost as the IVs undergo a remarkable transition into dense, brick-shaped infectious mature virions (MVs).Several studies led to the identification of D13 protein trimers as the building blocks of the scaffold: (i) single amino acid changes in D13 are responsible for VACV mutants that are resistant to the drug rifampin (rifampicin) (4, 11, 42), which causes reversible formation of irregular membranes lacking the “spicule coat” (18, 29, 30); (ii) repression of D13 expression results in a phenotype identical to that caused by the drug rifampin (50); (iii) antibody to D13 labels IVs (40) on the outer surface (28, 41); (iv) in the presence of rifampin, D13 antibodies label cytoplasmic inclusions that are distinct from aberrant viral membranes (40); and (v) the results of physical and microscopic studies indicate that D13 exists as trimers of 63-kDa subunits arranged mostly in hexagons on the surface of IVs (41).Poxviruses are thought to share a common origin with members of the asfarvirus, iridovirus, phycodnavirus, and mimivirus families (23). These large DNA viruses, except for the poxviruses, have an icosahedral capsid surrounding an internal membrane (31, 47-49). Interestingly, a domain of VACV D13 has homology with the capsid proteins of these related large DNA viruses (24). Moreover, a parapoxvirus ortholog of D13 was shown to self-assemble in vitro and to have structural similarities with the capsid proteins (22). These findings, together with the honeycomb lattice structure of the IV scaffold, suggest that the infectious form of the ancestor of poxviruses may have had an icosahedral capsid and that the stages of morphogenesis recapitulate evolution (41).In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the IV membrane to form the lattice structure and how the scaffold is removed during morphogenesis.     

Utilization of various carbon sources for the growth of waterborne conidial fungi

Four isolates of waterborne conidial fungi (Tetracheatum elegans, Tetracladium marchalianum, Pestalotiopsis submersus and Flagellospora penicillioides) were investigated for their carbon requirement, using eight different carbon sources (viz. glucose, fructose, sucrose, xylose, starch, cellulose, dextrin and lactose). All fungi tested grew sparsely on the basal medium lacking in carbon, which was the control. However these fungi were found to vary in their ability to use the supplied sources of carbon. Glucose and sucrose were found to be suitable sources of carbon for all four fungal isolates, whereas fructose proved good for T. marchalianum and P. submersus. Starch and xylose also supported growth of T. marchalianum, P. submersus and F. penicillioides. Cellulose, a polysaccharide, was a poor source of carbon for the growth of these isolates. Four g/L of glucose was recorded as the most useful concentration that gives the maximum dry weight of selected fungi (262 mg and 400 mg for T. elegans and P. submersus respectively after 15 d).     

Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide)

End-functionalized poly(N-isopropylacrylamide) (PNIPA) was synthesized by living free radical polymerization and conventional free radical polymerization and was used to prepare graft copolymers with poly(ethylenimine) (PEI). The copolymers exhibited lower critical solution temperature (LCST) behavior between 30 and 32 degrees C and formed complexes with plasmid DNA. The LCST of the copolymers in the DNA complexes increased slightly to approximately 34-35 degrees C. Cytotoxicity of the copolymers was evaluated by measuring lactate dehydrogenase (LDH) release from cells. The copolymers exhibited temperature-dependent toxicity, with higher levels of LDH release observed at temperatures above the LCST. Cellular uptake and transfection activity of the DNA complexes with the PEI-g-PNIPA copolymers were lower than those of the control PEI/DNA complexes at temperature below the LCST but increased to the PEI/DNA levels at temperatures above the LCST.     

Focal Adhesion Kinase contributes to insulin-induced actin reorganization into a mesh harboring Glucose transporter-4 in insulin resistant skeletal muscle cells

Background  

Focal Adhesion Kinase (FAK) is recently reported to regulate insulin resistance by regulating glucose uptake in C2C12 skeletal muscle cells. However, the underlying mechanism for FAK-mediated glucose transporter-4 translocation (Glut-4), responsible for glucose uptake, remains unknown. Recently actin remodeling was reported to be essential for Glut-4 translocation. Therefore, we investigated whether FAK contributes to insulin-induced actin remodeling and harbor Glut-4 for glucose transport and whether downregulation of FAK affects the remodeling and causes insulin resistance.     

DNMT1 as a molecular target in a multimodality-resistant phenotype in tumor cells

We have previously shown that hydrogen peroxide-resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H(2)O(2), etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H(2)O(2), etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos-overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos-overexpressing cells were exposed to 5-aza-2'-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H(2)O(2), etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1(-/-)) or DNMT3B (DNMT3B(-/-)) were shown to be more sensitive to the cytotoxicity of H(2)O(2), etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H(2)O(2)) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy.     

BAT3 and SET1A form a complex with CTCFL/BORIS to modulate H3K4 histone dimethylation and gene expression

Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.     

Genetic diversity and functional characterization of endophytic <Emphasis Type="BoldItalic">Bacillus thuringiensis</Emphasis> isolates from the North Western Indian Himalayas

A total of 15 endophytic Bacillus thuringiensis isolates were obtained from root nodules of six legumes (soybean, ricebean, gahat, frenchbean, lentil and pea). All of these isolates were characterized by the presence of one of two different types of crystalline inclusions (spherical and bipyramidal) and tolerance to a wide pH range (4–10; optimum 7.0) and NaCl concentrations up to 8%. Genetic diversity among the B. thuringiensis isolates was determined by repetitive extragenic palindromic PCR assays (rep-PCR) using the Bacillus cereus-repetitive extragenic palindromic, BOX, enterobacterial repetitive intergenic consensus sequence and (GTG)₅ primers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteogram of the B. thuringiensis isolates revealed the presence of two major polypeptides (24.4 and 131.0 kDa). Maximum crystal protein profile was observed in the B. thuringiensis isolates producing the spherical crystal, while those isolates producing the bipyramidal crystal protein showed four four major polypeptides (24.4, 33.8, 81.2 and 131.0 kDa). The purified crystal protein profile of the B. thuringiensis isolates revealed the presence of only one major protein of 130 kDa mass. Isolates VRB1 and VLG15 possessing the cry1 and cry2 family genes demonstrated 100% mortality against first-instar larvae of the Bihar hairy caterpillar (lepidopteran pest). Our study of the ecological and molecular diversity among newly identified B. thuringiensis isolates suggests that these could be useful in planning new strategies for integrated pest management in sustainable agricultural systems.