Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon

To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

Expansion of GAA trinucleotide repeats in mammals

We have previously shown that GAA trinucleotide repeats have undergone significant expansion in the human genome. Here we present the analysis of the length distribution of all 10 nonredundant trinucleotide repeat motifs in 20 complete eukaryotic genomes (6 mammalian, 2 nonmammalian vertebrates, 4 arthropods, 4 fungi, and 1 each of nematode, amoebozoa, alveolate, and plant), which showed that the abundance of large expansions of GAA trinucleotide repeats is specific to mammals. Analysis of human-chimpanzee-gorilla orthologs revealed that loci with large expansions are species-specific and have occurred after divergence from the common ancestor. PCR analysis of human controls revealed large expansions at multiple human (GAA)(30+) loci; nine loci showed expanded alleles containing >65 triplets, analogous to disease-causing expansions in Friedreich ataxia, including two that are in introns of genes of unknown function. The abundance of long GAA trinucleotide repeat tracts in mammalian genomes represents a significant mutation potential and source of interindividual variability.     

Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens

N-ethyl-N-nitrosourea (ENU) is a widely used mutagen in genotypic and phenotypic screens aimed at elucidating gene function. The high rate at which ENU induces point mutations raises the possibility that an observed phenotype may be to the result of another unidentified linked mutation. This article presents methods for estimating the probability of additional linked coding mutations (1) in a given region of DNA using both Poisson and Bayesian models and in (2) an F(1) animal exposed to ENU that has undergone b number of backcrosses. Applying these methods to the mouse data set of Quwailid et al., we estimate that the probability that a confounding mutation is linked to a cloned mutation when the candidate region is 5 Mb is very slim (p < 0.002). Where mutants are identified by genotypic methods, we show that backcrossing in the absence of marker-assisted selection is an inefficient means of eliminating linked confounding mutations.     

Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures

Dynamic deformational loading has been shown to significantly increase the development of material properties of chondrocyte-seeded agarose hydrogels, however little is known about the spatial development of the material properties within these constructs. In this study, a technique that combines video microscopy and optimized digital image correlation, was applied to assess the spatial development of material properties in tissue-engineered cartilage constructs cultured in free-swelling and dynamically-loaded conditions (3h/day, 5 days/week, and maintained in free-swelling conditions when not being loaded) over a 6-week period. Although homogeneous at day 0, both free-swelling and dynamically loaded samples progressively developed stiffer outer edges and a softer central region. The distribution of GAGs and collagens were shown to mimic this profile. These results indicate that although dynamic loading augments the development of bulk properties in these samples, possibly by overcoming some of the diffusion limitation and nutrient transport issues, the overall profile of construct properties in the axial direction remains qualitatively the same as in free-swelling culture conditions. Poisson's ratio of these constructs increased over time in culture with increased fixed charged density contributed by the GAGs, but this increase was significantly less in dynamically loaded samples by day 42. Polarized light microscopy of Picrosirius Red labeled samples, at an angle perpendicular to the direction of loading, suggests that these differences in Poisson's ratio may be due to improved organization of collagen network in the dynamically loaded samples.     

Cutting edge: deficiency in the E3 ubiquitin ligase Cbl-b results in a multifunctional defect in T cell TGF-beta sensitivity in vitro and in vivo

Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b-/- CD4+CD25- T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-beta. We have now asked whether the resistance noted in Cbl-b-/- T cells is restricted solely to TGF-beta's antiproliferative effects, whether the TGF-beta resistance has in vivo relevance, and whether a defect can be identified in the TGF-beta signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-beta-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b-/- mice show a significantly enhanced response to a tumor that is strictly TGF-beta regulated; and 3) Cbl-b-/- T effector cells have defective TGF-beta-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-beta signaling, and that its absence results in multifunctional TGF-beta-related defects that have important disease-related implications.