Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS

We investigated the mechanism of exercise-induced late cardioprotection against ischemia-reperfusion (I/R) injury. C57BL/6 mice received treadmill exercise (60 min/day) for 7 days at a work rate of 60-70% maximal oxygen uptake. Exercise transiently increased oxidative stress and activated endothelial isoform of nitric oxide synthase (eNOS) during exercise and increased expression of inducible isoform of NOS (iNOS) in the heart after 7 days of exercise. The mice were subjected to regional ischemia by 30 min of occlusion of the left coronary artery, followed by 2 h of reperfusion. Infarct size was significantly smaller in the exercised mice. Ablation of cardiac sympathetic nerve by topical application of phenol abolished oxidative stress, activation of eNOS, upregulation of iNOS, and cardioprotection mediated by exercise. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine during exercise also inhibited activation of eNOS, upregulation of iNOS, and cardioprotection. In eNOS(-/-) mice, exercise-induced oxidative stress was conserved, but upregulation of iNOS and cardioprotection was lost. Exercise did not confer cardioprotection when the iNOS selective inhibitor 1400W was administered just before coronary artery occlusion or when iNOS(-/-) mice were employed. These results suggest that exercise stimulates cardiac sympathetic nerves that provoke redox-sensitive activation of eNOS, leading to upregulation of iNOS, which acts as a mediator of late cardioprotection against I/R injury.     

Human herpesvirus 6 infection of CD4+ T-cell subsets

The immune system includes CD4+ regulatory T (T reg) cells that play a role in self-tolerance and demonstrate functional variations that govern immune responses. HHV-6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T-cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV-6 to specific T-cell populations such as CD4+CD25 high, which includes the majority of T reg cells, and CD4+CD25(-). These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV-6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real-time PCR. The effects of virus infection on cytokine production from these T-cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV-6 replication. Virus infection enhanced the production of both Th1- and Th2-type cytokines from CD4+CD25(-) T cells; however, only Th2-type cytokine release was augmented from viral-infected CD4+CD25 high T cells. Further, while virusinfected CD4+CD25 high T cells shift their antiviral immunity toward Th2 dominance by producing IL-10, the role of virus-infected CD4+CD25(-) T cells remains obscure.     

Optimized method for determining free L-cysteine in rat plasma by high-performance liquid chromatography with the 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole conversion reagent

The analytical method was optimized for L-cysteine (Cys) in rat plasma with co-existing L-cystine (Cyss). We observed that more than 100% Cyss in rat plasma was converted to Cys under typical conditions for the conversion with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Another conversion reagent, 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), was then employed, with which the reaction could be carried out at a low temperature without the use of a reducing reagent. Under the optimized conditions of 4 °C and pH 8.3, the conversion ratio of Cyss to Cys in rat plasma was as low as 5-7%. We determined the Cys concentration in plasma of the portal vein of rats that had been orally administered with Cys and Cyss by applying this method. The result indicated that Cys administration and also Cyss administration effectively increased the plasma Cys level. The method developed in this study is well suited for determining the thiol compounds in biological samples.     

Biological evaluation of 3'-O-alkylated analogs of salacinol, the role of hydrophobic alkyl group at 3' position in the side chain on the α-glucosidase inhibitory activity

Four analogs with 3′-O-alkyl groups (9a: CH₃, 9b: C₂H₅, 9c: C₁₃H₂₇ or 9d: CH₂Ph) instead of the 3′-O-sulfate anion in salacinol (1), a naturally occurring potent α-glucosidase inhibitor, were synthesized by the coupling reaction of 1,4-dideoxy-1,4-epithio-d-arabinitols (18a and 18b) with appropriate epoxides (10a-10d). These analogs showed equal or considerably higher inhibitory activity against rat small intestinal α-glucosidases than the original sulfate (1), and one of them (9d) was found more potent than currently used α-glucosidase inhibitors as antidiabetics. Thus, introduction of a hydrophobic moiety at the C3′ position of this new class of inhibitor was found beneficial for onset of stronger inhibition against these enzymes.     

Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

IKKε regulates cell elongation through recycling endosome shuttling

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Highlights? Recycling endosomes shuttle along the proximal-distal axis of growing bristles ? IKK? locally antagonizes Rab11 effector Nuf at the bristle tip by phosphorylation ? Nuf-endosome pathway is independent of actin cytoskeleton or DIAP1-caspase pathway ? Mammalian IKK-related kinases antagonize Rab11-FIP3 through phosphorylation     

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.     

Isoflurane induces second window of preconditioning through upregulation of inducible nitric oxide synthase in rat heart

The second window of preconditioning (SWOP) induced by inhalation of volatile anesthetics has been documented in the rat heart and is triggered by nitric oxide synthase (NOS), but involvement of NOS in the mediator phase of isoflurane-induced SWOP has not been demonstrated. We tested the hypothesis that isoflurane-induced SWOP is mediated through upregulation of inducible NOS (iNOS). Rats inhaled 0.75 minimum alveolar concentration (MAC) isoflurane, 1.5 MAC isoflurane, or O2 for 2 h. After 24, 48, 72, and 96 h, the isolated heart was perfused with buffer and subjected to 30 min of ischemia followed by 2 h of reperfusion. Inhalation of 0.75 and 1.5 MAC isoflurane significantly limited infarct size after ischemia-reperfusion 24-72 h after isoflurane inhalation. The maximum effect was obtained 48 h after inhalation of 1.5 MAC isoflurane. Postischemic left ventricular function was improved only 48 h after inhalation of 1.5 MAC isoflurane. iNOS expression and activity in the heart were increased 24-72 h after inhalation of 1.5 MAC isoflurane; this increase was less pronounced after inhalation of 0.75 MAC isoflurane. A selective iNOS inhibitor, 1400W (10 microM), abolished iNOS activation and cardioprotection induced 48 h after inhalation of 1.5 MAC isoflurane. These results suggest that isoflurane inhalation induces SWOP after 24-72 h through overexpression and activation of iNOS in the rat heart.     

Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.     

Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet potato plants

Adiponectin is a 30kDa protein exclusively produced and secreted from adipocytes and as a cytokine has been found to link obesity, insulin resistance, and type 2 diabetes. Production of biologically active adiponectin in large scale is desirable for pharmaceutical applications. Mouse adiponectin cDNA was used for developing transgenic sweet potato plants via Agrobacterium-mediated transformation. The presence of the transgene was verified by PCR and DNA gel blot analysis. Further investigated were five independent transgenic lines, all of which expressed high levels of adiponectin mRNA. Immuno blot analysis with a mouse adiponectin antiserum revealed that, in addition to a 29 kDa-protein which co-migrates with the adiponectin protein produced in Escherichia coli cells, a 31 kDa-protein was produced, indicative of a post-translational modification of the protein. The transgenic plants did not show obvious differences in growth rate and morphology in response to adiponectin production.