Digitonin effects on photoreceptor adenylate cyclase

Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.     

Modulation of adenylate cyclase activity in liver and fat cell membranes by insulin

Insulin depresses both the activity of adenylate cyclase stimulated by glucagon, epinephrine, and sodium fluoride in liver cell membranes and the activity of adenylate cyclase stimulated by epinephrine and adrenocorticotropin in particulate preparations from homogenates of isolated fat cells. Significant inhibition is detected with very low concentrations (10(-11) molar) of insulin but not with unphysiologically high (10(-9)molar) concentrations of the hormone. These direct effects of insulin on an enzymatic system in broken-cell -preparations suggest a fundamental role of adenylate cyclase activity and of cyclic adenosine monophosphate in the mechanism of action of insulin.     

Somatomedin: inhibiton of adenylate cyclase activity in subcellular membranes of various tissues

Somatomedin in concentrations between 3 and 20 units per milliliter significantly inhibits the basal activity of adenylate cyclase in crude membrane preparations obtained from homogenates of fat cells, liver, and spleen lymphocytes of the rat, and from chondrocytes and cartilage of chick embryos. The enzyme activity measured in the presence of stimulating hormones (epinephrine, prostaglandin PGE(1), parathyroid hormone) is also inhibited in these preparations by somatomedin. These observations may be relevant in a general way to the mechanism of action of growth-prmoting substances and to the processes which normally regulate cell growth.     

Message transmission: receptor controlled adenylate cyclase system

The adenylate cyclase system is composed of an activating hormone or neurotransmitter (H), its receptor (R), the guanosine triphosphate (GTP) binding protein (Gs), and the catalytic unit (C). The activation of the receptor R involves a transient change in conformation, from a loose binding of the neurotransmitter H to an extremely tight interaction, termed locking. The system is regulated in the activation steps and also by three deactivation processes. A guanosine triphosphatase activity is built into the Gs protein so that the active GsGTP has only a limited lifetime during which it is able to activate C. In addition, the continued occupation of R by H causes desensitization of R. Finally, there are inhibitory receptors, such as alpha-adrenergic and opiate receptors, which inhibit the adenylate cyclase by way of a specific GTP binding protein (Gi). Yet to be determined are the conformational transformations of pure R on binding of an agonist or a partial agonist; the genes that code for the many different receptors that activate the adenylate cyclase, and the possibility that the G components interact with systems in the cell other than the adenylate cyclase.     

Phagocyte impotence caused by an invasive bacterial adenylate cyclase

For unknown reasons, humans infected with the bacterium Bordetella pertussis are exceptionally vulnerable to secondary infections. Bordetella species elaborate a soluble, heat-stable, and highly active adenylate cyclase. This enzyme is internalized by phagocytic cells and catalyzes the unregulated formation of adenosine 3',5'-monophosphate (cyclic AMP), thereby disrupting normal cellular function. This unusual phenomenon may explain Bordetella-induced aphylaxis and may prove to be useful for investigating a variety of cyclic AMP-governed processes.     

苯并(a)芘诱导对双齿围沙蚕腺苷酸环化酶(AC)基因及酶活性表达的影响

为探讨双齿围沙蚕Perinereis aibuhitensis对外源污染物的毒性响应,分析了不同苯并(a)芘[B(a)P]浓度(0.5、5、10、15μg/L)胁迫下双齿围沙蚕(体质量为1.5～2.5 g)腺苷酸环化酶(adenylate cyclase,AC)的基因表达和酶活性变化特征,利用RACE方法获得了沙蚕AC基因cDNA全长,并利用实时荧光定量PCR技术分析了苯并(a)芘诱导下沙蚕AC基因的变化。结果表明:双齿围沙蚕AC基因cDNA全长为4900 bp,包括5′非翻译区127 bp,3′非翻译区1536 bp,开放阅读框3237 bp,编码1078个氨基酸;该序列包含两个保守环化酶催化结构域,与其他动物氨基酸序列一致性为34%～47%;不同浓度的苯并(a)芘诱导会引起沙蚕AC基因表达量上升,0.5、10μg/L苯并(a)芘浓度组AC表达量在诱导第7天时达到最大,而5、50μg/L苯并(a)芘浓度组在诱导第14天时达到最大;利用ELISA试剂盒分析苯并(a)芘诱导下双齿围沙蚕AC酶活性变化,结果显示,在第4天时0.5、5、50μg/L浓度组AC酶活性上升到最高,之后随时间的延长酶活性降低,而10μg/L浓度组AC酶活性略低于空白对照组且在第7天时达到最高值。研究表明,苯并(a)芘会在一定程度上诱导沙蚕AC的基因表达及酶活性变化。     

Biotin enhances guanylate cyclase activity

Biotin and its analog, (+)-biotin-p-nitrophenyl ester enhanced guanylate cyclase activity two- to threefold in rat liver, kidney, colon, cerebellum, and heart. Dose-response relationships revealed that at concentrations as low as 1 micromolar, both biotin and its analog caused maximal augmentation of guanylate cyclase activity. These data suggest a role for the activation of guanylate cyclase in the mechanism of action of this vitamin.     

Bee venom enhances guanylate cyclase activity

Bee venom and phospholipase A2 extracted from bee venom enhanced guanylate cyclase (E.C. 4.6.1.2) activity two- to threefold in rat liver, lung, heart, kidney, ileum, and cerebellum. Dose-response relationships revealed that bee venom at concentrations as low as 1 microgram per milliliter and phospholipase A2 at 1 microunit per milliliter caused a maximal enhancement of guanylate cyclase.     

Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.     

Role of serotonergic input in the regulation of the beta-adrenergic receptor-coupled adenylate cyclase system

The action of desipramine on the norepinephrine-sensitive adenylate cyclase system and the density of beta-adrenergic receptors in rat cortex was studied after selective lesioning of serotonergic neurons with 5,7-dihydroxytryptamine. In animals with lesions desipramine failed to reduce the density of beta-adrenoceptors but decreased the response of adenosine 3',5'-monophosphate to isoproterenol and norepinephrine to the same degree as in animals without lesions. The results demonstrate a functional linkage between serotonergic and noradrenergic systems in the rat cortex, with beta-adrenergic receptors and neurohormonal sensitivity of the adenosine 3',5'-monophosphate-generating system being under separate regulatory control.     

Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.     

Adenyl cyclase activity in rat pineal gland: effects of chronic denervation and norepinephrine

Adenyl cyclase activity in the pineal gland of rats was determined by measuring the rate of formation of radioactive cyclic 3',5'-adenosine monophosphate from (14)C-labeled adenosine triphosphate. Norepinephrine added in vitro to pineal homogenates enhanced this activity, while denervation of the pineal gland by superior cervical ganglionectomy did not significantly reduce it. The enzyme in these denervated glands was more responsive to the stimulatory effects of norepinephrine.     

Esterase, malate, and lactate dehydrogenases activity in murine neuroblastoma

Mouse neuroblastoma tumors have only the fifth isozyme band (A(4)) of lactate dehydrogenase, whereas this band is missing in the brain which contained four other bands of lactate dehydrogenase. The alpha-esterase isozyme patterns of tumors, kidney, and brain are similar except that there is an additional slowest-moving form of esterase in all tumor tissues. The malate dehydrogenase pattern is not altered in any of the tissues.     

Phosphoribosylamidotransferase: regulation of activity in virus-induced murine leukemia by purine nucleotides

Infection with Friend leukemia virus causes a marked increase in the activity of splenic phosphoribosylamidotransferase in mice. Intraperitoneal injection of purine nucleotides and their free bases inhibits this enzyme. This is the first example of the control of phosphoribosylamidostransferase in vivo in the mammalian system as well as in virus-induced leukemia. Experiments in vitro support the findings in vivo.     

Inhibitors of DNA polymerases of murine leukemia viruses: activity of ethidium bromide

Ethidium bromide, compared on a molar basis, was a more effective inhibitor of the DNA polymerases of the Rauscher and Moloney murine leukemia viruses than either 4-N-demethylrifampicin or 4-N-benzyldemethylrifampicin. Daunomycin inhibited the polymerases weakly, and chromomycin A(3) inhibited almost not at all. 4-N-Benzyldemethylrifampicin was a more active inhibitor than the 4-N-demethyl congener.     

Demonstration of biological activity of a murine leukemia virus of New Zealand black mice

Although New Zealand Black mouse embryo and adult tissues show evidence of murine leukemia viral particles and antigens, efforts to demonstrate biological activity of a murine leukemia virus by standard methods have proved negative. Cocultivation of tissues of these mice with non-virus-yielding hamster cells transformed by Moloney sarcoma virus, however, has resulted in the rescue of a pseudotype sarcoma virus, presumably carrying the New Zealand Black mouse leukemia virus coat. This virus has an unusual host restriction, producing foci of cell alteration only in rat cells.     

小鼠Toll样受体9基因的克隆、表达及其活性的初步鉴定

从小鼠巨噬细胞中提取细胞总RNA,采用RT-PCR技术扩增小鼠Toll样受体9(mTLR9)cDNA,构建真核表达质粒p3XFLAG-CMV-7.1-mTLR9,转染293T细胞,利用Western Blotting检测蛋白表达,并用双荧光素酶报告基因系统检测mTLR9对其介导的信号通路下游转录因子NF-κB转录活性影响.结果表明,本试验成功克隆到小鼠TLR9cDNA,构建的重组体转染细胞后表达的蛋白分子质量与预计相符,并能激活下游转录因子NF-κB的转录活性.     

花生种子萌发过程中子叶内肽酶活性研究

探讨花生种子在萌发过程中子叶内肽酶的活性及其同工酶数量的变化,研究金属型蛋白酶抑制剂EDTA与不同温度对内肽酶的活性的影响。采用直立玻板法萌发花生种子,运用分光光度法测定内肽酶的活力,结果发现内肽酶活力在种子萌发6 d达到最大值;运用SDS-PAGE凝胶电泳法发现在种子萌发6 d出现5条内肽酶同工酶(其中2条是金属型内肽酶);在种子萌发8 d和10 d均出现7条内肽酶同工酶(其中4条是金属型内肽酶),这些内肽酶同工酶的最适温度约为40℃,EGTA对萌发6 d时存在的2种金属型内肽酶同工酶有明显抑制作用,但并不能完全抑制其活性。