Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.     

Parvalbumin isoforms differentially accelerate cardiac myocyte relaxation kinetics in an animal model of diastolic dysfunction

The cytosolic Ca(2+)/Mg(2+)-binding protein alpha-parvalbumin (alpha-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, alpha-Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg(2+) affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring alpha/beta-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with beta-Parv having an estimated 16% greater Mg(2+) affinity and approximately 200% greater Ca(2+) affinity than alpha-Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by beta-Parv than by alpha-Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 +/- 2 vs. 49 +/- 2 (SE) ms (P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with alpha- or beta-Parv adenoviral vectors. Upon Parv gene transfer, beta-Parv caused significantly faster relaxation than alpha-Parv (P < 0.05), even though estimated [beta-Parv] was approximately 10% of [alpha-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.     

Sustained biological effects of porcine interleukin 5 delivered to pigs as recombinant protein or via a DNA vector

The ability of cytokines to act as natural immunotherapeutics to enhance the health and the disease resistance of animals is of particular interest to the intensive livestock industries. Antibiotics have been used for such purposes over a long period of time, however, there is growing concern that this practice will enhance the development of antibiotic resistance in a range of bacterial pathogens. In several species, interleukin 5 (IL-5) is known to enhance B cell activity and to increase the numbers of eosinophils in blood and tissues. In this report, IL-5 was delivered to pigs, either as a recombinant protein or via a DNA delivery vector and was shown to elevate eosinophils in blood over a sustained period. Interleukin 3, a potent haemopoietic factor, did not synergise with IL-5 when both cytokines were given together, but did prime the pigs for a stronger response to IL-5. These results demonstrate that IL-5 can readily be delivered to commercial pigs to elicit a significant biological effect.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Single-cell Analysis of G-protein Signal Transduction

The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways.     

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.     

Phenotypic hypersusceptibility to multiple protease inhibitors and low replicative capacity in patients who are chronically infected with human immunodeficiency virus type 1

Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naive patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change 相似文献   

Synthesis and SAR of diazepine and thiazepine TACE and MMP inhibitors

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-alpha release. Oral activity in the mouse LPS model of TNF-alpha release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20.