A comparison of the effects of chromate,molybdate and cadmium oxide on respiration in the yeastSaccharomyces cerevisiae

Summary Growth ofSaccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by chromate than growth on fermentable medium. Chromate was selectively toxic against oxygen uptake in cells grown in non-fermentable medium and also inducedpetite mutations. CdO demonstrated similar but lesser effects on growth and respiration. However, molybdate had little toxicity to yeast non-fermentable growth and stimulated oxygen uptake in cells grown in fermentable and non-fermentable media. These results suggest that chromate, a carcinogen, may act more directly against the mitochondria ofS. cerevisiae than related chemical species, CdO and molybdate.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2

Tissue inhibitor of metalloproteinases-3 (TIMP3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). TIMP3, which encodes a potent angiogenesis inhibitor, is mutated in Sorsby fundus dystrophy, a macular degenerative disease with submacular choroidal neovascularization. In this study we demonstrate the ability of TIMP3 to inhibit vascular endothelial factor (VEGF)-mediated angiogenesis and identify the potential mechanism by which this occurs: TIMP3 blocks the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling and angiogenesis. This property seems to be independent of its MMP-inhibitory activity, indicating a new function for this molecule.     

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11(RK) protein devoid of methylated arginines. The Mre11(RK/RK) mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11(RK/RK) MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M(RK)RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M(RK)RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.     

Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10⁻⁵). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ∼5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ∼4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias.     

A novel bacterial community index to assess stream ecological health

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Fixation of oligosaccharides to a surface may increase the susceptibility to human parainfluenza virus 1, 2, or 3 hemagglutinin-neuraminidase

The hemagglutinin-neuraminidase (HN) protein of human parainfluenza viruses (hPIVs) both binds (H) and cleaves (N) oligosaccharides that contain N-acetylneuraminic acid (Neu5Ac). H is thought to correspond to receptor binding and N to receptor-destroying activity. At present, N′s role in infection remains unclear: does it destroy only receptors, or are there other targets? We previously demonstrated that hPIV1 and 3 HNs bind to oligosaccharides containing the motif Neu5Acα2-3Galβ1-4GlcNAc (M. Amonsen, D. F. Smith, R. D. Cummings, and G. M. Air, J. Virol. 81:8341–8345, 2007). In the present study, we tested the binding specificity of hPIV2 on the Consortium for Functional Glycomics'' glycan array and found that hPIV2 binds to oligosaccharides containing the same motif. We determined the specificities of N on red blood cells, soluble small-molecule and glycoprotein substrates, and the glycan array and compared them to the specificities of H. hPIV2 and -3, but not hPIV1, cleaved their ligands on red blood cells. hPIV1, -2, and -3 cleaved their NeuAcα2-3 ligands on the glycan array; hPIV2 and -3 also cleaved NeuAcα2-6 ligands bound by influenza A virus. While all three HNs exhibited similar affinities for all cleavable soluble substrates, their activities were 5- to 10-fold higher on small molecules than on glycoproteins. In addition, some soluble glycoproteins were not cleaved, despite containing oligosaccharides that were cleaved on the glycan array. We conclude that the susceptibility of an oligosaccharide substrate to N increases when the substrate is fixed to a surface. These findings suggest that HN may undergo a conformational change that activates N upon receptor binding at a cell surface.     

Worldwide experience with biosimilar development

Limited access for high-quality biologics due to cost of treatment constitutes an unmet medical need in the US and other regions of the world. The term “biosimilar” is used to designate a follow-on biologic that meets extremely high standards for comparability or similarity to the originator biologic drug that is approved for use in the same indications. Use of biosimilar products has already decreased the cost of treatment in many regions of the world and now a regulatory pathway for approval of these products has been established in the US. The Food and Drug Administration (FDA) led the world with the regulatory concept of comparability and the European Medicines Agency (EMA) was the first to apply this to biosimilars. Patents on the more complex biologics, especially monoclonal antibodies, are now beginning to expire and biosimilar versions of these important medicines are in development. The new Biologics Price Competition and Innovation Act (BPCIA) allows the FDA to approve biosimilars and allows the FDA to lead on the formal designation of interchangeability of biosimilars with their reference products. The FDA''s approval of biosimilars is critical to facilitating patient access to high-quality biologic medicines and will allow society to afford the truly innovative molecules currently in the global biopharmaceutical industry''s pipeline.Key words: monoclonal antibodies (mAbs), biosimilars, recombinant biopharmaceuticals     

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca)

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.