Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.     

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree

The active principle in a methanolic extract of the laboratory-grown cyanobacterium, Fischerella sp. isolated from Neem (Azadirachta indica) tree bark was active against Mycobacterium tuberculosis, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli as well as three multi-drug resistant E. coli strains in in vitro assays. Based on MS, UV, IR ¹H NMR analyses the active principle is proposed to be hapalindole T having the empirical formula C₂₁H₂₃N₂ClSO and a molecular weight of 386 with the melting point range 179–182 ^°C. The estimated production of Hapalindole T from the cyanobacterium is 1.25 mg g⁻¹ lyophilized biomass. It is suggested that cyanobacteria colonizing specialized niches such as tree bark could be an antibacterial drug resource.     

Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice

Ability of Mycobacterium tuberculosis to survive under oxidative stress in vivo is an important aspect of pathogenesis. Rv3303c gene from M. tuberculosis encodes an NAD(P)H quinone reductase. These enzymes have been shown to manage oxidative stress in other pathogenic bacteria. We have hypothesized that Rv3303c protein will remove reactive oxygen species released by the host and hence reduce oxidative stress to M. tuberculosis. rv3303c was PCR cloned and the purified recombinant enzyme reduced superoxide generator menadione. Antisense and sense RNA constructs of rv3303c were electroporated in M. tuberculosis H37Rv. The transformants were characterized by difference in expression of specific mRNA and protein. Antisense transformants were markedly reduced in virulence as compared to sense transformants as judged by several parameters such as weight and survival of infected mice, growth in vivo, colonization and histopathology of lungs. In the presence of menadione, the sense transformant was more resistant to killing in vitro than the antisense transformant. It may be concluded that the rv3303c gene contributes to virulence of M. tuberculosis in vivo and this might be mediated in part by increased resistance to reactive oxygen intermediates thereby enhancing intracellular growth and colonization.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

Phylogenetic relationships in the cricket tribe Xenogryllini (Orthoptera,Gryllidae, Eneopterinae) and description of the Indian genus Indigryllus gen. nov.

The subfamily Eneopterinae is known greatly for its diversified acoustic modalities and disjunct distribution. Within Eneopterinae, tribe Lebinthini is the most studied group, due to its highest species diversity (ca. 150 species in 12 genera), endemic distribution on the islands of Southeast Asia and of the South West Pacific, males’ ability to produce high‐frequency calling songs, and evolution of females’ vibrational response. To investigate the distribution pattern and diversification of acoustic and behavioral attributes in a larger frame, clear understanding of phylogenetic relationships within other tribes of Eneopterinae is vital. In this study, we focus on the tribe Xenogryllini, sister group of Lebinthini. Xenogryllini, as opposed to Lebinthini, is known by fewer species (11 species in two genera), distributed widely in continental Asia and Africa, and for producing low‐frequency calling songs. We describe a new genus Indigryllus with a new species of the tribe Xenogryllini, discovered from the southwest of India. We used eight molecular genetic markers to reconstruct the phylogenetic relationships. The resultant phylogenetic tree is used to compare and discuss distribution patterns and acoustic modalities between Lebinthini and Xenogryllini.     

Size- and Age-Dependent Neurotoxicity of Engineered Metal Nanoparticles in Rats

Earlier we showed that chronic administration of engineered nanoparticles (NPs) from metals, e.g., Cu, Ag, or Al (50–60 nm, 50 mg/kg, i.p. daily for 1 week) alter blood–brain barrier (BBB) disruption and induce brain pathology in adult rats (age 18 to 22 weeks). However, effects of size-dependent neurotoxicity of NPs in vivo are still largely unknown. In present investigation, we examined the effects of different size ranges of the above-engineered NPs on brain pathology in rats. Furthermore, the fact that age is also an important factor in brain pathology was also investigated in our rat model. Our results showed that small-sized NPs induced the most pronounced BBB breakdown (EBA +480 to 680 %; radioiodine +850 to 1025 %), brain edema formation (+4 to 6 %) and neuronal injuries (+30 to 40 %), glial fibrillary acidic protein upregulation (+40 to 56 % increase), and myelin vesiculation (+30 to 35 % damage) in young animals as compared to controls. Interestingly, the oldest animals (30 to 35 weeks of age) also showed massive brain pathology as compared to young adults (18 to 20 weeks old). The Ag and Cu exhibited greater brain damage compared with Al NPs in all age groups regardless of their size. This suggests that apart from the size, the composition of NPs is also important in neurotoxicity. The very young and elderly age groups exhibited greater neurotoxicity to NPs suggests that children and elderly are more vulnerable to NPs-induced brain damage. The NPs-induced brain damage correlated well with the upregulation of neuronal nitric oxide synthase activity in the brain indicating that NPs-induced neurotoxicity may be mediated via increased production of nitric oxide, not reported earlier.