Angiotensin II modulates nitric oxide-induced cardiac fibroblast apoptosis by activation of AKT/PKB

Previously we found that interleukin-1beta (IL-1beta)-activated inducible nitric oxide (NO) synthase (iNOS) expression and that NO production can trigger cardiac fibroblast (CFb) apoptosis. Here, we provide evidence that angiotensin II (ANG II) significantly attenuated IL-1beta-induced iNOS expression and NO production in CFbs while simultaneously decreasing apoptotic frequency. The anti-apoptotic effect of ANG II was abolished when cells were pretreated with the specific ANG II type 1 receptor (AT1) antagonist losartan, but not by the AT2 antagonist DP-123319. Furthermore, ANG II also protected CFbs from apoptosis induced by the NO donor diethylenetriamine NONOate and this effect was associated with phosphorylation of Akt/protein kinase B at Ser473. The effects of ANG II on Akt phosphorylation and NO donor-induced CFb apoptosis were abrogated when cells were preincubated with the specific phosphatidylinositol 3-kinase inhibitors wortmannin or LY-294002. These data demonstrate that ANG II protection of CFbs from IL-1beta-induced apoptosis is associated with downregulation of iNOS expression and requires an intact phosphatidylinositol 3-kinase-Akt survival signal pathway. The findings suggest that ANG II and NO may play a role in regulating the cell population size by their countervailing influences on cardiac fibroblast viability.     

β3受体激动剂对培养的大鼠心肌细胞搏动频率和细胞内环-磷酸腺苷水平的影响

目的：观察β3受体激动剂(BRL-37344)对培养的大鼠心肌细胞搏动频率和细胞内环-磷酸腺苷(cAMP)水平的影响,以探讨β3受体在心肌细胞中的作用。方法：分离培养乳鼠心肌细胞,随机分为八组：对照组、ISO组、Nadolol ISO组、BRL组、PTX BRL组、L-NAME BRL、Nadolol BRL组和Buprandol BRL组,观察心肌细胞搏动频率,并应用酶联免疫方法测定cAMP含量,逆转录多聚酶链反应(RT-PCR)方法测β3受体mRNA表达。结果：ISO(非选择性β受体激动剂)可显著增加心肌细胞搏动频率和升高cAMP水平,这种作用可被Nadolol(为β1,β2受体抑制剂)阻断。BRL-37344可显著降低心肌细胞搏动频率和cAMP含量,这种作用可被PTX(Gi蛋白抑制剂)和Bupranolol(非选择性β受体阻滞荆)完全阻断,同时可被L-NAME(一氧化氮合酶抑制剂)部分阻断,不受Badolol影响。RT-PCR方法测出心肌细胞中有β3受体mRNA表达。结论：心肌细胞中存在β3受体,它在心肌表现为负性变力作用,β3受体的效应不受β1,β2受体抑制剂影响。心脏β3受体信号途径中可能有Gi蛋白的参与,并且经过一氧化氮合酶途径发挥其作用。     

Molecular Motions of the Outer Ring of Charge of the Sodium Channel: Do They Couple to Slow Inactivation?

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain beta1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the alpha carbons of these residues of no more than 7 A. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.     

IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, thereby partially mimicking human disease. Using this model, we have demonstrated that mice deficient in gammadelta T cells are more susceptible to WN virus infection. TCRdelta(-/-) mice have elevated viral loads and greater dissemination of the pathogen to the CNS. In wild-type mice, gammadelta T cells expanded significantly during WN virus infection, produced IFN-gamma in ex vivo assays, and enhanced perforin expression by splenic T cells. Adoptive transfer of gammadelta T cells to TCRdelta(-/-) mice reduced the susceptibility of these mice to WN virus, and this effect was primarily due to IFN-gamma-producing gammadelta T cells. These data demonstrate a distinct role for gammadelta T cells in the control of and prevention of mortality from murine WN virus infection.     

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.     

The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase

Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.     

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.     

Novel interactions identified between micro -Conotoxin and the Na+ channel domain I P-loop: implications for toxin-pore binding geometry

micro -Conotoxins ( micro -CTX) are peptides that inhibit Na(+) flux by blocking the Na(+) channel pore. Toxin residue arginine 13 is critical for both high affinity binding and for complete block of the single channel current, prompting the simple conventional view that residue 13 (R13) leads toxin docking by entering the channel along the pore axis. To date, the strongest interactions identified are between micro -CTX and domain II (DII) or DIII pore residues of the rat skeletal muscle (Na(v)1.4) Na(+) channels, but little data is available for the role of the DI P-loop in micro -CTX binding due to the lack of critical determinants identified in this domain. Despite being an essential determinant of isoform-specific tetrodotoxin sensitivity, the DI-Y401C variant had little effect on micro -CTX block. Here we report that the charge-changing substitution Y401K dramatically reduced the micro -CTX affinity ( approximately 300-fold). Using mutant cycle analysis, we demonstrate that K401 couples strongly to R13 (DeltaDeltaG > 3.0 kcal/mol) but not R1, K11, or R14 (<1 kcal/mol). Unlike K401, however, a significant coupling was detected between toxin residue 14 and DI-E403K (DeltaDeltaG = 1.4 kcal/mol for the E403K-Q14D pair). This appears to underlie the ability of DI-E403K channels to discriminate between the GIIIA and GIIIB isoforms of micro -CTX (p < 0.05), whereas Y401K, DII-E758Q, and DIII-D1241K do not. We also identify five additional, novel toxin-channel interactions (>0.75 kcal/mol) in DII (E758-K16, D762-R13, D762-K16, E765-R13, E765-K16). Considered together, these new interactions suggest that the R13 side chain and the bulk of the bound toxin micro -CTX molecule may be significantly tilted with respect to pore axis.     

Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.