A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Ribosomal proteins ofThermomyces lanuginosus — characterisation by two-dimensional gel electrophoresis and differential disassembly

One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungusThermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved fromTh. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800–36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000–48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH₄Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.     

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43_m. Cx43_m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all ³²P_i from Cx43_m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.     

Characterization of Calpain-Mediated Proteolysis of GluR1 Subunits of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate Receptors in Rat Brain

Abstract: Previous results have indicated that GluR1 subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [³H]AMPA or 6-cyano-7-[³H]nitroquinoxaline-2,3-dione ([³H]CNQX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 m M resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [³H]AMPA and [³H]CNQX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.     

Esophageal PCO2 as a monitor of perfusion failure during hemorrhagic shock

Sato, Yoji, Max Harry Weil, Wanchun Tang, Shijie Sun,Jianlin Xie, Joe Bisera, and Hidehiro Hosaka. EsophagealPCO₂ as a monitor of perfusionfailure during hemorrhagic shock. J. Appl.Physiol. 82(2): 558-562, 1997.Measurement ofgastric wall PCO₂(Pg_CO2) bytonometric method has emerged as an attractive option for estimatingvisceral perfusion during circulatory shock. However, gastric acidsecretion obfuscates the tonometric measurement. We, therefore,investigated the option of measuringPCO₂ in the esophagus to minimizethese restraints. Hemorrhagic shock was induced in five Sprague-Dawleyrats, and five rats served as sham controls.Pg_CO2 wasmeasured with an ion-sensitive field effect transistor that wassurgically implanted into the gastric wall. Esophageal luminalPCO₂(Pe_CO2) wasmeasured by a second ion-sensitive field effect transistor sensor.During hemorrhagic shock, mean aortic pressure declined from 150 to 50 mmHg. Gastric blood flow decreased from 58 to 12 ml ^· min^{1 ·} 100 g¹ (21% of preshock) andesophageal blood flow from 44 to 7 ml ^· min^{1 ·} 100 g¹ (16% of preshock).Pg_CO2simultaneously increased from 47 to 116 Torr andPe_CO2 from 47 to 127 Torr. The increases inPg_CO2 werehighly correlated with increases inPe_CO2(r = 0.90). Esophageal tonometry may,therefore, serve as a practical alternative to gastric tonometry.

     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

phbB,phbC基因克隆,序列分析及植物表达载体的构建

利用聚合酶链式反应技术,从真养产碱杆菌Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ Ｈ１６染色体ＤＮＡ中的主增并克隆了调控聚－β－羧基丁酸生物合成的两个关键酶基因;依赖ＮＡＤＰＨ的乙酰乙酰ＣｏＡ还原酶基因和ＰＨＢ合成酶基因。限制性内切酶图谱和核苷酸序列分析证实了克隆结果,并表明所克隆的基因与国外报道的有很高的同源性。     

脱落酸对线粒体Na^＋—K^＋ATPase活性的影响

在提取大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ）黄化子叶的线粒体时,于清洗和悬浮介质中加入ＡＢＡ,发现４０ μｍ ｏｌ／Ｌ（±） ＡＢＡ 对线粒体膜结合Ｎａ＋ －Ｋ＋ ＡＴＰａｓｅ 活性及线粒体吸氧速率有明显的促进作用。ＡＢＡ 可使１６℃与２７℃培养的大豆子叶线粒体膜结合Ｎａ＋ －Ｋ＋ ＡＴＰａｓｅ Ａｒｒｈｅｎｉｕｓ图的折点温度分别下降６．３℃和５．９℃,并将该酶的底物动力学曲线由正协同曲线转变为非协同曲线。表明ＡＢＡ 可降低线粒体的膜相变温度