Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness

Genomic copies of three Xanthomonas oryzae pv. oryzae avirulence (avr) genes, avrXa7, avrXal0, and avrxa5, and four homologous genes, aB3.5, aB3.6, aB4.3, and aB4.5, were mutagenized individually or in combination to study the roles of avr genes in one component of pathogen fitness, i.e., aggressiveness or the amount of disease X. oryzae pv. oryzae causes in susceptible rice lines. These X. oryzae pv. oryzae genes are members of the highly related Xanthomonas avrBs3 gene family. Compared to the wild-type strain, X. oryzae pv. oryzae strains with mutations in avrXa7, avrxa5, and the four homologous genes caused shorter lesions on rice line IR24, which contains no resistance genes relevant to the wild-type strain. The contribution of each gene to lesion length varied, with avrXa7 contributing the most and avrXal0 showing no measurable effect on aggressiveness. The functional, plasmidborne copies of avrXa7, aB4.5, and avrxa5 restored aggressiveness only to strains with mutations in avrXa7, aB4.5, and avrxa5, respectively. Mutations in avrXa7 were not complemented by plasmids carrying any other avr gene family members. These data indicate that some, but not all, avr family members contribute to pathogen aggressiveness and that the contributions are quantitatively different. Furthermore, despite their sequence similarity, the aggressiveness functions of these gene family members are not interchangeable. The results suggest that selection and pyramiding resistance genes can be guided by the degree of fitness penalty that is empirically determined in avr gene mutations.     

Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization

Redistribution of postsynaptic AMPA- (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. We show that AMPA receptors underwent clathrin-dependent endocytosis, which was accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreased AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation-(LFS-) induced homosynaptic CA1 LTD were found to be mutually occlusive and were both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS.     

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

Purification and characterization of a novel inhibitor of the proliferation of hepatic stellate cells

An inhibitor of the proliferation of hepatic stellate cells (HSC) was purified from rat liver by a combination of gel filtration and ion exchange chromatography. The molecular mass of this non-arginase growth inhibitory factor (NAGIF) was determined to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The proliferation of HSC was inhibited by NAGIF with a 50% inhibitory dose of 5 nmol/liter. The inhibitory activity of NAGIF was not limited to HSC but also affected the growth of bovine endothelial cells and 3T6 fibroblasts. However, the growth of B16 mouse melanoma was not inhibited by NAGIF. The NH(2)-terminal sequence of NAGIF, AEPVEPWS, is identical to an internal sequence of rat Zn-alpha(2)-glycoprotein. Although the action mode of this inhibitor remains to be investigated, it seems very likely that NAGIF is involved in the negative control mechanism of HSC growth.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

Effects of lithium and insulin on glycogen synthesis in L6 myocytes: additive effects on inactivation of glycogen synthase kinase-3

In insulin-sensitive L6 myocytes, insulin stimulated glycogen synthesis in a dose-dependent manner and lithium further stimulated glycogen synthesis at all insulin concentrations. Lithium alone at 20 mM stimulated glycogen synthesis to the degree similar to the maximal insulin response. Effects of lithium and insulin were fully additive for both glycogen synthesis and glycogen synthase activity. In L6 myocytes, insulin increased phosphorylation of Akt1 and glycogen synthase kinase-3 alpha and beta (GSK-3 alpha and beta), resulting in its activation and inactivation, respectively. Unlike insulin, lithium directly inhibited GSK-3 (both alpha and beta) without affecting phosphorylation of GSK-3. Moreover, lithium in vitro could further inhibit enzyme activity of GSK-3 (both alpha and beta) that was isolated from insulin-stimulated cells (thus already phosphorylated and inactivated by insulin). In summary, insulin increases glycogen synthesis by the Akt1/GSK-3/glycogen synthase pathway, but lithium increases glycogen synthesis by direct inhibition of GSK-3 in L6 myocytes. Inhibitory effects of lithium and insulin on GSK-3 (both alpha and beta) were additive, which may account, at least in part, for their additive effects on glycogen synthase activity and glycogen synthesis in L6 myocytes.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.