NAC拮抗As_2O_3对外周血淋巴细胞的毒性作用

目的探讨N-乙酰-L-半胱氨酸(N acetylcysteine,NAC)拮抗As2O3对人淋巴细胞的毒性作用及机制。方法从正常人外周血中分离淋巴细胞,台盼蓝染色检测细胞活性;FITC-Annexin-V/PI染色检测细胞凋亡;电镜观察形态学改变;流式细胞术观察细胞内ROS水平及Bcl-2表达水平。结果 As2O3抑制人外周血淋巴细胞的增殖,细胞呈现典型的凋亡细胞特征性形态学改变,FITC-Annexin-V/PI染色发现凋亡细胞增多,同时,细胞内ROS水平增高和Bcl-2蛋白的表达水平下降。NAC与As2O3联合作用于淋巴细胞,细胞增殖抑制和凋亡均明显降低,细胞内ROS水平明显降低,Bcl-2蛋白表达水平回升。结论 As2O3对人淋巴细胞有明显的毒性效应,NAC可通过清除细胞内ROS和促进Bcl-2蛋白的表达而拮抗As2O3对淋巴细胞的毒性。     

阿米洛利对青霉素致痫小鼠行为及前皮层谷氨酸含量的影响

阿米洛利是经典的Na^＋／H^＋交换体（NHE）抑制剂，能抑制细胞内H^＋外排，降低细胞内pH；而细胞内酸化能抑制痫性放电，防止神经元损伤。青霉素为经典的致痫剂，本文以青霉素致痫模型评价了阿米洛利的抗癫痫作用，并利用微透析结合高效液相-电化学检测（HPLC-ECD）考察了其对小鼠前皮层谷氨酸（Glu）含量的影响。     

海洛因对人 T淋巴细胞线粒体产能代谢的影响

目的：研究不同海洛因吸食年限者T淋巴细胞线粒体功能,探讨海洛因对人 T淋巴细胞线粒体产能代谢的影响。方法采集不同海洛因吸食年限人员外周血,提取T淋巴细胞;MTT法检测T淋巴细胞增殖活性;流式细胞仪检测T淋巴细胞凋亡情况;荧光分光光度法检测细胞内ROS、三磷酸腺苷（ATP）含量。结果随着海洛因吸食年限的增加,T淋巴细胞增殖活性逐渐下降,细胞凋亡逐渐增加,与此同时,细胞内ROS含量增高、细胞线粒体产能逐渐减少。结论长时间吸食海洛因可能导致机体损伤,其机制可能与海洛因对人 T 淋巴细胞线粒体功能的影响有关。     

前胡丙素对高血压大鼠血管肥厚、细胞内钙、胶原及一氧化氮的影响

目的 研究前胡丙素对自发性高血压大鼠SHR及肾型高血压大鼠RHR的血管肥厚、细胞内钙、胶原、一氧化氮（NO）及血管反应性影响。方法 用显微测微仪测定血管中膜层，细胞大小，用Fura-2/AM为荧光指示剂，测定单细胞内[Ca^2 ]i，以测定羟脯氨酸含量反映胶原含量，用Griess法测定NO含量，以血管环观察收缩反应。结果 前胡丙素减轻血管中膜层增厚，维持细胞内[Ca^2 ]i稳态。减少胶原形成，增加SMCs释放NO。抑制血管环高反应状态。结论 前胡丙素抑制高血压血管肥厚，降低胶原含量及血管异常反应。     

扇贝多肽对UVB辐射小鼠胸腺淋巴细胞PI3K/Akt和ASK1-JNK信号通路的影响

目的 研究扇贝多肽(polypeptide from Chlamys farreri,PCF)对紫外线B(UVB)辐射损伤小鼠胸腺淋巴细胞后P13K/Akt和ASK1-JNK信号通路的影响.方法 UVB辐射小鼠胸腺淋巴细胞,用比色法测定胸腺淋巴细胞活性氧(reactive oxygen species,ROS)水平;western-blot检测Akt的活性,预先加入或不加入P13K/Akt通路特异性抑制剂LY294002检测细胞凋亡信号调节激酶Ⅰ(apoptosis signal regulating kinase-1,ASK1)、JNK的活性、线粒体膜电位(ACM)和DNA ladder.结果 PCF能激活AKT的活性,抑制UVB对小鼠胸腺淋巴细胞ASK1凋亡通路的活化.结论 PCF通过降低细胞内活性氧的含量,提高AKT的活性,引起ASK1的降解,导致ASK1-JNK诱导的细胞凋亡抑制.     

温度通过cAMP及cGMP对人淋巴细胞E受体的影响

作者用放射免疫测定及E玫瑰花环测定法检测了不同温度,不同保存时间对淋巴细胞E受体及细胞内cAMP相cGMP的影响,结果表明,随着温度的降低及保存时间的延长,E玫瑰花环的抑制率逐渐增加(P<0.05及0.01)。淋巴细胞内的cAMP含量升高,cGMP含量稍下降,cAMP/cGMP比值增大(P<0.05及0.01)。并对温度、淋巴细胞E受体及环核苷酸三者之间关系进行探讨。     

腹膜炎的解毒和免疫矫正综合治疗

本文对48例腹膜炎病人(20例为泛发性,28例为限局性)的T—淋巴细胞的含量,茶碱拮抗性和茶碱敏感性T—淋巴细胞,白细胞的趋化活性和淋巴细胞内ATP含量进行了研究。结果证明:术后早期阶段免疫系统的状况可以预测炎症过程的严重程度和后果。T—淋巴细胞含量的剧烈减少和在白细胞趋化活性持续下降的基础上淋巴细胞内ATP量的增加,在疾病的预测中是必须的指标。用次氯酸钠液间接电解氧化作为综合性加强治疗,除了有解毒作用外,还能持续影响淋巴细胞的代谢过程并激活细胞的免疫作用。用胸腺肽和白细胞介素作复合免疫矫正治疗,具有高潜力的免疫生物学作用。并可用于矫正泛发性腹膜炎病人的严重的继发性免疫缺欠。     

羊栖菜多糖对P388小鼠红细胞免疫促进作用的机制研究

研完了羊栖菜多糖(SFPS)对淋巴细胞白血病P368小鼠红细胞免疫促进作用的机制。结果表明，SFPS增强淋巴细胞白血病P368小鼠虹细胞免疲功能的机制与其降低P368小鼠红细胞膜过氧化脂质(LPO)含量，抑制红细胞膜蛋白与收缩蛋白变联高聚物(HMP)的形成，增加红细胞膜封闭度、唾液酸含量，增强红细胞膜超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、Na^ ，K^ -ATP酶活性有关。     

阿托伐他汀对金黄地鼠糖尿病模型巨噬细胞胆固醇外流的影响

目的：研究不同剂量阿托伐他汀对糖尿病动物模型体内巨噬细胞胆固醇含量的影响，进一步探讨他汀类药物抑制动脉粥样硬化斑块进展的机制。方法：建立糖尿病金黄地鼠动物模型，体外培养腹腔巨噬细胞，在体外与不同剂量阿托伐他汀孵育，利用酶法结合高效液相色谱法测定细胞内胆固醇及胆固醇脂含量。RT—PCR方法检测细胞ABCA1 mRNA表达量的不同。结果：在apoA1存在时，阿托伐他汀可以显著促进巨噬细胞内胆固醇外流，可能与药物增强细胞内胆固醇酯水解酶活性、促进巨噬细胞AB—CA1的表达有关。结论：阿托伐他汀促进ABCA1介导的细胞内胆固醇外流。     

外源性硫化氢抑制ox-LDL诱导血管内皮细胞组织因子的表达与降低ROS产生和抑制NF-κB活化有关

目的探讨外源性硫化氢抑制氧化型低密度脂蛋白(ox-LDL)诱导内皮细胞组织因子(TF)表达的机制。方法分别用不同浓度NaHS(25、50、100和200μmol·L-1)和50mg·L-1ox-LDL孵育人脐静脉内皮细胞(HUVECs),用RT-PCR和ELISA法检测HUVECs TF mRNA表达和蛋白含量,DCFH法测细胞内活性氧(ROS)的含量,Western blot测核蛋白转录因子(NF-κB)的活化。结果 ox-LDL明显诱导HUVECs TF mRNA的表达和蛋白含量增加;细胞内ROS水平升高,NF-κB活化。NaHS明显抑制了ox-LDL对TF mRNA和蛋白表达的诱导作用,同时降低ox-LDL诱导的细胞内ROS生成和NF-κB活化。此外,NF-κB抑制剂BAY 11-7082(10μmol·L-1)或抗氧化剂N-乙酰半胱氨酸(1 mmol·L-1)也能抑制ox-LDL诱导TF mRNA表达和蛋白含量的增加,同时降低细胞内ROS生成和NF-κB活化,该作用与200μmol·L-1NaHS的效应相似。结论 NaHS抑制ox-LDL诱导内皮细胞TF的表达与降低ROS产生和抑制NF-κB活化有关。     

环氧氯丙烷染毒大鼠肝和脑干组织中cAMP,cGMP和CaM含量的变化

应用放免技术和钙调泰(CaM)的酶联免疫测试法(ELISA)测定了大鼠EPI染毒后肝和脑干组织中cAMP、cGMP及CaM的含量变化。结果表明,随着染毒剂量的增加肝组织中cAMP、cGMP和CaM含量均呈上升趋势,其中,cAMP和CaM上升一致,cAMP/cGMP值升高;脑干组织中cAMP含量增加,cGMP和CaM无明显变化。本文还讨论了EPI染毒后脑干组织中cAMP、cGMP和DaM含量变化的可能机制和意义。     

Brazilein, an important immunosuppressive component from Caesalpinia sappan L

Caesalpinia sappan has been shown to have interesting immunosuppressive properties. Its heartwood has long been used in Chinese medicines for treating a variety of immune-mediated pathology and inflammatory disease. The purpose of this work was to evaluate the immunocompetence effects of brazilein on mice lymphocytes in vitro and in vivo. The results showed that brazilein and Caesalpinia sappan ethanol extract (SME) could distinctly inhibit the proliferation of T lymphocyte stimulated by Concanavalin A (Con A) and the proliferation of B lymphocyte stimulated by lipopolysaccharides (LPS), and brazilein could suppress mice humoral immune response by plaque forming cell (PFC) test. In addition, immune organs (thymus and spleen) in mice treated with brazilein were notably atrophied and weight loss in vivo (intraperitoneal injection, i.p.). In attempting to investigate the mechanisms of the immunosuppressive activity of brazilein, we discovered that brazilein can induce apoptosis in mice spleen lymphocytes by flow cytometry analysis and DNA fragmentation assay, which may be one of the pathways that brazilein inhibited immunocompetence of mice lymphocytes.     

Cytosolic and membrane-bound cyclic nucleotide phosphodiesterases from guinea pig cardiac ventricles.

Cyclic nucleotide phosphodiesterase (PDE) activities were characterized in the cytosolic and post-nuclear membrane preparations of guinea pig cardiac ventricles. The cytosolic PDE activities were stimulated 5-fold by calmodulin (CaM) on both substrates (1 microM) and 1.2-fold by cGMP (5 microM) on cAMP hydrolysis. Conversely, in the membrane preparation, CaM only stimulated PDE activities 1.2- to 1.4-fold, but cGMP induced a 3-fold increase of the hydrolysis of cAMP. In both the cytosolic and the membrane preparations, the hydrolysis of cAMP was inhibited by 100 microM of either the PDE III inhibitor SK&F 94120 (27% and 31% respectively) or the PDE IV inhibitor rolipram (14% and 23% respectively). Four peaks were resolved from the cytosolic preparation by chromatography. Peak A and peak B hydrolyzed both cAMP and cGMP and were stimulated respectively by CaM and cGMP. Peak C and peak D selectively hydrolyzed cAMP. Peak C had an apparent Km value for cAMP of 3.3 microM and was inhibited by PDE IV inhibitors. Peak D showed an apparent Km value for cAMP of 0.43 microM and was inhibited by cGMP and by cardiotonic inhibitors of PDE III. Similar potencies of these inhibitors were observed in the membrane preparation. These results suggest that in guinea pig cardiac ventricles: (1) PDE I (CaM-activated) is almost exclusively cytosolic; (2) PDE II (cGMP-stimulated), PDE III (cGMP-inhibited and cardiotonic-sensitive) and PDE IV (rolipram-sensitive) are present in cytosolic and membrane preparations; (3) PDE III and PDE IV differ in their apparent Km values for cAMP. The latter observation could explain the differential effects of PDE III and PDE IV inhibitors in the regulation of cardiac contraction.     

Immunosuppressive properties of chloroquinoxaline sulfonamide

Chloroquinoxaline sulfonamide (CQS), a sulfanilamide derivative with antitumor activity, was found to be toxic to lymphoid tissue during preclinical studies. The mechanism of this toxicity appears to involve profound inhibition of lymphocyte activation. Incubation of human peripheral blood mononuclear cells (PBMNCs) with CQS decreased cellular incorporation of thymidine and deoxyuridine in a dose-dependent manner. Analysis of cell cycle distribution by flow cytometry indicated that CQS blocked movement out of the G0/G1 phase. Drug-treated cells were smaller and expressed fewer receptors for interleukin-2 (IL-2) and transferrin than untreated mitogen-stimulated lymphocytes. These observations support the notion that CQS has cell cycle specificity in regulating lymphocyte proliferation. As little as 10 microM CQS markedly inhibited both human lymphocyte and murine CTLL cell replication in response to IL-2 containing growth factors. However, CQS did not block secretion of IL-2 into culture supernatant fractions by human PBMNCs. Finally, CQS inhibited in vitro production of immunoglobulins G and M by mitogen-stimulated lymphocytes, primarily by causing cytotoxicity. In all of these drug effects, CQS was approximately one to two logs more potent than the parent compound, sulfaquinoxaline (SQ). These studies indicate that CQS inhibits essential basic processes in human lymphocytes. This agent may find use as an immunosuppressive drug.     

Cytosolic and membrane-bound cyclic nucleotide phosphodiesterases from guinea pig cardiac ventricles

Cyclic nucleotide phosphodiesterase (PDE) activities were characterized in the cytosolic and post-nuclear membrane preparations of guinea pig cardiac ventricles. The cytosolic PDE activities were stimulated 5-fold by calmodulin (CaM) on both substrates (1 μM) and 1.2-fold by cGMP (5 μM) on cAMP hydrolysis. Conversely, in the membrane preparation, CaM only stimulated PDE activities 1.2- to 1.4-fold, but cGMP induced a 3-fold increase of the hydrolysis of cAMP. In both the cytosolic and the membrane preparations, the hydrolysis of cAMP was inhibited by 100 μM of either the PDE III inhibitor SK & F 94120 (27% and 31% respectively) or the PDE IV inhibitor rolipram (14% and 23% respectively). Four peaks were resolved from the cytosolic preparation by chromatography. Peak A and peak B hydrolyzed both cAMP and cGMP and were stimulated respectively by CaM and cGMP. Peak C and peak D selectively hydrolyzed cAMP. Peak C had an apparent K_m value for cAMP of 3.3 μM and was inhibited by PDE IV inhibitors. Peak D showed an apparent K_m value for cAMP of 0.43 μM and was inhibited by cGMP and by cardiotonic inhibitors of PDE III. Similar potencies of these inhibitors were observed in the membrane preparation. These results suggest that in guinea pig cardiac ventricles: (1) PDE I (CaM-activated) is almost exclusively cytosolic; (2) PDE II (cGMP-stimulated), PDE III (cGMP-inhibited and cardiotonic-sensitive) and PDE IV (rolipram-sensitive) are present in cytosolic and membrane preparations; (3) PDE III and PDE IV differ in their apparent K_m values for cAMP. The latter observation could explain the differential effects of PDE III and PDE IV inhibitors in the regulation of cardiac contraction.     

Effect of serum on isoproterenol-induced cyclic AMP accumulation in human lymphocytes

The effects of autologous serum on basal and isoproterenol (IPR) or prostaglandin E1 (PGE1) stimulated adenosine 3',5' cyclic monophosphate (cAMP) levels were investigated in human lymphocytes. For all blood donors, serum (25% (v/v)) lowered the basal cAMP content. In contrast, the responsiveness of the lymphocyte cAMP accumulation to (-)-IPR was increased. This effect was most clearly demonstrable in bicarbonate buffered incubation medium (40-50% increase of maximal response), but was also seen in phosphate buffered medium (10-20% increase). Serum did not alter the sensitivity of the lymphocytes to IPR. The response to PGE1, which was a considerable more effective stimulator of cAMP accumulation than IPR, was not affected in any consistent way by serum. The results indicate that serum influences the regulation of lymphocyte cAMP and that this effect may partly be exerted at the level of the beta-adrenoceptors.     

Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I

The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.     

Adenosine receptors of human leukocytes--II. Characterization of an inhibitory P-site

We have investigated the effects of 2',5'-dideoxyadenosine (DDA), 9'-beta-D-arabinofuranosyladenine (ARA), and 9-beta-D-xylofuranosyladenine (XFA), which have been classified as P-site adenosine agonists, on the cyclic adenosine 3',5'-monophosphate (cAMP) metabolism of human lymphocytes and polymorphonuclear leukocytes (PMNs). DDA (10(-5)-2 x 10(-4) M), ARA and XFA caused a dose-dependent decrease in cAMP content of human lymphocytes. In addition to decreasing lymphocyte cAMP levels, DDA, ARA, and XFA markedly inhibited the effects of many adenylate cyclase-stimulating agents including beta-adrenergic stimuli, prostaglandin E1 (PGE1), histamine, adenosine, forskolin and cholera toxin. Theophylline and 3-isobutyl-1-methylxanthine, which are known antagonists of adenosine A1/Ri and A2/Ra receptors, did not modify the inhibiting effects of DDA. Mn2+ (1 mM) increased the sensitivity to inhibition of adenylate cyclase agonists by DDA. We also search for the presence of adenosine P-sites in human PMNs. DDA caused a significant decrease of PMN cAMP levels only at the highest concentrations used (2 x 10(-4) M). In contrast, even low concentrations of DDA (10(-6)-10(-4) M) concentration-dependently blocked the stimulatory effect of PGE1 and forskolin on PMN cAMP accumulation. The results support the existence of a purine P-site that regulates cAMP metabolism of human lymphocytes and PMNs.     

Inhibition of a phosphodiesterase III in the lysis-sensitive target-induced elevation of cyclic AMP (cAMP) in human natural killer cells

Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.     

Evidence that FTY720 induces T cell apoptosis in vivo

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.