The killing of Entamoeba histolytica by activated human eosinophils.

Unlike normal, opsonin aided human eosinophils, fMLP-activated human eosinophils are capable of destroying virulent E. histolytica. Opsonins are not required for this action, although they enhance the effect. Some activated eosinophils succumb in the action as well, probably victims of toxic products released by dying amebas. Activated eosinophils thus appear to resemble activated macrophages in their dealing with this parasite.     

高葡萄糖介导MDCK细胞蛋白激酶C活性变化

观察了高葡萄糖对MDCK细胞蛋白激酶C（PKC）信号转导通路的影响。结果表明MDCK细胞在高葡萄糖环境下,PKC从细胞浆到细胞膜移位活化,同时细胞Na ,K －ATP酶活性下降,此可为PKC抑制剂staurosporine所阻止。因此,高糖引起肾小管细胞PKC信号系统异常可能是糖尿病肾病发生的重要机制。     

The fine structure of Entamoeba histolytica processed by cryo-fixation and cryo-substitution.

Recently, the use of cryo-fixation followed by freeze-substitution has been shown to produce a better ultrastructural preservation of cellular components due to the rapidity of the fixation procedure and the lack of distortion during dehydration. When these techniques are applied to study the fine structure of axenically cultured trophozoites of E. histolytica, an improved preservation of cytoplasmic and nuclear components was found. The surface coat of the parasite appears much thicker and uniform than in conventionally treated samples. Also, the ground substance of the cytoplasm is packed with fibrogranular material, which is usually extracted by chemical fixation. The extremely fast fixation procedure allows the visualization of fusion and/or fission processes of cytoplasmic vacuoles and vesicles. Nuclear microtubules and cytoplasmic microfilaments are clearly identified. Low-temperature techniques allow not only a better preservation of the cell structure of E. histolytica parasites, but will also facilitate considerably future immunoelectronmicroscopical studies.     

Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors.The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2 -terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTDinduced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.     

凝血酶诱导人类胚胎血管平滑肌细胞明胶酶分泌及活化

目的：探讨凝酶对血管平滑肌细胞明胶酶合成及活化的影响。方法：采用不同浓度的凝血酶（0.5,5,25,50u/ml)与培养的人类胚胎血管平滑肌细胞（HVSMC）或其条件培养上清液共育，用SDS-PAGE明胶酶谱法测定上清液中明胶酶的活性。结果：浓度大于或等于5u/ml的凝血酶可以诱导HVSMC合成MMP-2，且呈剂量递增关系，0.5u/ml的凝血酶可激活proMMP-2，且随剂量增加而增强，剂量大于或等于25u/ml的凝血酶有诱导HVSMC合成MMP-9的作用，凝血酶与HVSMC条件培养液共育，证实凝血酶具有时间和剂量依赖性直接激活的作用。结论：凝血酶以剂量依赖性方式促进proMMP-2分泌，并对MMP-2有直接激活作用。提示局部凝血酶浓度升高可通过降解基质，促进血管成形术后再狭窄或使斑块稳定性下降。     

蛋白激酶C不同亚型对丙泊酚血管舒张作用的影响

目的 探讨丙泊酚的血管舒张作用与蛋白激酶C(PKC)不同亚型间的关系.方法 将SD大鼠胸主动脉环随机分为内皮完整组(n=36)和去内皮组(n=36),每组各分6个亚组:①10 nmol/L Go6976+1×10<'-6>mol/L去甲肾上腺素(NA)+丙泊酚处理组(n=6);②10 μmol/L Rottlerin+1×10<'-6>mol/L NA+丙泊酚处理组(n=6);③2 μmol/L PKCε-Pseudo(假底物)+1×10<'-6>mol/L NA+丙泊酚处理组(n=6);④2 μmol/L PKCθ-Pseudo+1×10<'-6>mol/L NA+丙泊酚处理组(n=6);⑤2 μmol/L PKCζ-Pseudo+1×10<'-6>mol/L NA+丙泊酚处理组(n=6);⑥1×10<'-6>mol/L NA+丙泊酚处理组(对照组,n=6).PKCα抑制剂Go6976,PKCδ抑制剂Rottlerin,PKζ、θ和ε假底物孵育血管环30 min后,加1×10<'-6>mol/L NA收缩血管环达峰值,每15 min加递增浓度的丙泊酚(1×10<'-6>、5×10<'-6>、1×10<'-5>、5×10<'-5>、1×10<'-4>mol/L),观察血管张力的变化.结果 内皮完整时,Go6976、PKCε假底物和PKCθ假底物减小丙泊酚引起的血管舒张幅度,与内皮完整的对照组相比差异有统计学意义(P<0.05);Rottlerin抑制丙泊酚的血管舒张效应,且在1×10<'-6>-5×10<'-5>mol/L丙泊酚作用时将舒张效应转为收缩(P<0.05).去内皮时,Rotftlerin、PKCε假底物和PKCθ假底物分别增大相同浓度丙泊酚作用下的血管舒张幅度,与去内皮对照组相比差异有统计学意义(P<0.05);Go6976和PKCζ假底物分别增大1×10<'-5>-1×10<'-4>mol/L丙泊酚作用下的血管舒张幅度(P<0.05).结论 PKCα、δ、ε、θ参与了内皮完整时丙泊酚引起的血管舒张.     

蛋白激酶C不同亚型对丙泊酚血管舒张作用的影响

目的探讨丙泊酚的血管舒张作用与蛋白激酶C(PKC)不同亚型间的关系。方法将SD大鼠胸主动脉环随机分为内皮完整组(n=36)和去内皮组(n=36),每组各分6个亚组:①10 nmol/L Go6976+1×10-6mol/L去甲肾上腺素(NA)+丙泊酚处理组(n=6);②10μmol/L Rottlerin+1×10-6mol/L NA+丙泊酚处理组(n=6);③2μmol/L PKCε-Pseudo(假底物)+1×10-6mol/L NA+丙泊酚处理组(n=6);④2μmol/L PKCθ-Pseudo+1×10-6mol/L NA+丙泊酚处理组(n=6);⑤2μmol/L PKCζ-Pseudo+1×10-6mol/L NA+丙泊酚处理组(n=6);⑥1×10-6mol/L NA+丙泊酚处理组(对照组,n=6)。PKCα抑制剂Go6976,PKCδ抑制剂Rottlerin,PKCζ、θ和ε假底物孵育血管环30 min后,加1×10-6mol/L NA收缩血管环达峰值,每15 min加递增浓度的丙泊酚(1×10-6、5×10-6、1×10-5、5×10-5、1×10-4mol/L),观察血管张力的变化。结果内...     

Signal transduction in Entamoeba histolytica induced by interaction with fibronectin: presence and activation of phosphokinase A and its possible relation to invasiveness

Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activates signaling pathways through G-protein-coupled receptors. Increments of adenylyl cyclase activity and cAMP produce a striking reorganization of actin into structures that apparently facilitate adhesive, locomotive, and secretory activities. The reorganization of actin is induced by phosphorylation of actin-associated proteins by diverse kinases activated during the signaling process. Although cAMP-dependent kinases have not yet been identified in this parasite, the activation of the adenylyl cyclase route and its effects on particular motility-related functions strongly suggest their presence. Phosphokinase A (PKA) was detected by phosphorylation of the specific substrate, kemptide, its further activation by cAMP, and its inhibition by H89. The catalytic subunit of the enzyme was identified by immunofluorescence microscopy and by immunoprecipitation. Adhesion and damage to cultured cells were monitored by FN-binding and cytotoxicity assays. A cAMP-dependent kinase activated by effectors and agonists of adenylyl cyclase and also during interaction of trophozoites with fibronectin (FN) was found. The enzyme is associated with small granules in the cytoplasm and upon activation, a fraction of its catalytic subunit with an Mr of 100 kDa was translocated to the nucleus, while another fraction was aggregated into big clusters. Activity and translocation were blocked by H89, a specific inhibitor of PKA. Trophozoites stimulated by dBcAMP or forskolin-formed lamellae and restructured actin, but no significant increase in their adhesion to FN was observed and only showed 10% stimulus in their capacity to damage target cells. Treatment with H89 decreased adhesion to 40% and caused 80% inhibition in cell damage. These amebas showed altered organization of the actin structures induced by dBcAMP or FN. Our results support previous suggestions concerning the participation of PKA in the response elicited by the interaction of E. histolytica trophozoites with ECM proteins. They also indicate that adhesion and secretion in conjunction with motile activities are related to invasion processes.     

蛋白激酶C激活与糖尿病血管并发症研究的新进展

蛋白激酶C(PKC)是一类由多种同工酶组成的丝氨酸／苏氨酸蛋白激酶家族。高血糖和游离脂肪酸(FFA)可影响信号传导通路，特别是激活二酰甘油(DG)-PKC途径，PKC的激活可引起胰岛素抵抗(IR)及糖尿病血管并发症。PKC-β选择性抑制剂(LY333531)和维生素E对糖尿病血管并发症治疗方面已显示出良好的前景。     

The regulatory effects of protein kinase C on the proliferation of cultured human low-passage meningioma cells

Protein kinase C ( PKC) is a phospholipid- de-pendent,serine/threonine protein kinase,and repre-sents an important part of intracellular signal trans-duction system.Recent studies have suggested thatPKC also took part in cell proliferation of brain tu-mors. Ithas been shown thatseveralgrowth factors,such as epidermal growth factor( EGF) and platelet-derived growth factor( PDGF) may involve PKC intheir cell signaling processes,and the abnormalactiv-ity of PKC may lead to the promotion o…     

The role of protein kinase C and its effect on GHRH in the regulation of hormone secretion by somatotrophinomas

In recent years, the most exciting advance inthe research of human pitllitary tumors is the discovery that 30 %--40 % of hfuman pituitary somatotrophinomas carry somatic missense single--basemutations within codons 201 and 227 of the genefor the a--subunit of the stimulatory GTP--bindingprotein, Gs (Gsa). These mutations, termed gsponcogenes, result in constitutive activity of adenylyl cyclase and the subsequent elevations in intracellular cAMP levels are thought to be responsiblefor tumor g…     

蛋白激酶C信号传导通路在缺氧性心肌细胞凋亡中的作用

目的　研究蛋白激酶C信号通路在心肌细胞缺氧性凋亡中的作用。方法　将培养的大鼠心肌细胞随机分成 8组 :(1 )对照组 ;(2 )缺氧 6h组 ;(3)缺氧 1 2h组 ;(4 )缺氧 2 4h组 ;(5 )PMA1 0nM组 ;(6 )PMA1 0 0nM组 ;(7)CHE1 μM组 ;(8)CHE1mM组。对照组常氧培养 2 4h。缺氧培养即将心肌细胞 95 %N2 ～ 5 %CO2 下 37℃缺氧培养。 (5 )和 (6 )组即于缺氧培养前 ,加入PKC激动剂PMA(Phorbol 1 2 myristate 1 3 acetata) ,使其终浓度分别为 1 0nM、1 0 0nM ,预处理 1 0min后再缺氧 2 4h。 (7)和 (8)组即于缺氧培养前 1 0min ,加入PKC抑制剂CHE(chelerythrine) ,使其终浓度分别为 1 μM、1mM ,然后再缺氧培养 2 4h。其余各组缺氧不同的时间。终止培养后分别检测细胞的凋亡率和活性。结果　单纯缺氧能引起心肌细胞的凋亡和坏死 ,以缺氧 2 4h最显著。PMA1 0nM预处理能明显减少心肌细胞缺氧性凋亡 (P <0 .0 5 ) ,而PMA1 0 0nM组和CHE1 μM组心肌细胞凋亡率明显增加 (P <0 .0 5 )。PMA和CHE对 2 4h缺氧诱导的心肌细胞坏死无明显影响。结论　蛋白激酶C信号传导通路参与了抗心肌细胞缺氧性凋亡的信号传导 ,但可能与抗缺氧性坏死的信号传导之间存在差异     

木犀草素对宫颈癌HeLa细胞增殖及蛋白激酶C活性的影响

目的 研究中药木犀草素对人宫颈癌HeLa细胞蛋白激酶C（PKC）及细胞增殖的影响,并探讨其抗肿瘤机制.方法 分别以不同浓度的木犀草素作用于HeLa细胞后,用MTT法检测HeLa细胞生长抑制率,同步采用Western blot法检测 HeLa细胞PKC活性水平.结果 木犀草素作用于HeLa细胞后,细胞生长抑制率与对照组相比明显增高（P〈0.05）,且呈时间和剂量依赖性.各组HeLa细胞PKC的表达均呈阳性,且随木犀草素浓度的升高,PKC的表达呈明显下降趋势.结论 木犀草素可能通过下调肿瘤细胞PKC活性,抑制肿瘤细胞增殖而达到抗肿瘤活性.     

蛋白激酶C在人骨肉瘤细胞多药耐药中的作用及机制

目的 研究蛋白激酶C(PKC)主要亚型在入骨肉瘤143B细胞中的表达情况及其与143B细胞耐药的关系。方法采用Western blot法检测PKC主要亚型α、β和γ磷酸化形式p-PKCα、p-PKCβ、p-PKCβγ和多药耐药蛋白-1(mutidrug resistance protein-1,MDR-1)在人骨肉瘤1...     

纤维蛋白原降解产物对大鼠主动脉血管细胞的作用及蛋白激酶C抑制剂的影响

目的：研究蛋白激酶C（PKC）抑制剂对纤维蛋白原降解产物（FFDP）引起的大鼠主动脉血管内皮细胞（RAEC）损伤的保护作用和平滑肌细胞（RASMC）增殖的抑制作用。方法：RAEC损伤以乳酸脱氢酶释放测定,RASMC增殖以结晶紫染色法测定。结果：FFDP能促进RAEC释放乳酸脱氢酶,诱导RASMC增殖;PKC抑制剂槲皮素、Ro 31-8220剂量依赖性地抑制FFDP对RAEC的损伤,并抑制FFDP诱导的RASMC增殖。结论：PKC抑制剂对FFDP引起的RAEC有保护作用,对RASMC增殖有抑制作用。     

视网膜下液促使视网膜色素上皮细胞膜上蛋白激酶C变化的机制分析

目的：研究视网膜下液（SRF）能否引起体外培养的视网膜色素上皮（RPE）细胞浆内蛋白激酶C（PKC）的激活和转位，探讨SRF与RPE细胞中PKC信号系统变化的关系。方法：实验对象为体外培养的RPE细胞；不同的刺激因素分别在不同的时间刺激RPE细胞，通过细胞裂解和离心获取细胞浆和细胞膜蛋白粗提液，用同位素^32P标记和液体闪烁计数法检测细胞浆和细胞膜PKC活性水平。结果：SRF和佛波酯（PMA）都可以激活RPE细胞浆中的PKC（c-PKC），并使其由胞浆向胞膜转位，但胞膜上PKC（m-PKC）活性峰值水平和出现的时间不同。结论：SRF可引起RPE细胞膜上PKC的活性发生变化，变化的幅度与增殖性玻璃体视网膜病变（PVR）的分级呈正相关。     

蛋白激酶C对氯化铵上调大鼠脑星形胶质细胞AQP-4表达的影响

目的：研究经氯化铵处理的大鼠脑星形胶质细胞水通道蛋白-4（AQP-4）的表达情况和蛋白激酶C（PKC）对AQP-4表达的调节作用.方法：取出生1～2d的SD大鼠大脑皮质,原代培养星形胶质细胞.将培养的细胞随机分为对照组,用等量培养基培养24h;NH4CL组,用终浓度为5mmol/L氯化铵培养6,12,24,48h;DMSO组：用10g/L二甲基亚砜（DMSO）预处理30min,余处理同NH4CL组;TPA组：蛋白激酶C激动剂TPA（1μmol/L）预处理30min,余处理同NH4CL组;RT组：蛋白激酶C拮抗剂Ro31-8220（1μmol/L）预处理30min后,余处理同TPA组.用光学显微镜观察细胞的形态学变化,用WesternBlot法检测各组细胞AQP-4的表达情况.结果：AQP-4表达量NH4CL组与DMSO组分别为（0.69±0.03）,（0.67±0.03）,与对照组相比较分别增加约90%,87%,细胞肿胀明显,差异显著（P〈0.05）;而NH4Cl组与DMSO组组间差异不显著;TPA组AQP-4表达量（0.40±0.02）与NH4CL组相比较,降低约41%（P〈0.05）;RT组AQP-4表达量（0.68±0.02）与TPA组相比较,明显增加,而与NH4CL组相比较差异无统计学意义.结论：经5mmol/L氯化铵处理的星形胶质细胞其AQP-4的表达明显增加,而PKC的激活能下调AQP-4的表达.     

脂多糖干预气道上皮细胞细胞因子及蛋白激酶C的表达

目的 探索脂多糖(lipolysaccharide,LPS)干预气道上皮细胞细胞因子的分泌及蛋白激酶C(protein kinase C,PKC)的表达.方法 培养A549细胞株,0、0.1、1.0、10.0、20.0、30.0、50.0、100.0 μg/mL的LPS干预A549细胞2、4、8、24、30、48 h以后收集细胞上清液及细胞质总蛋白,ELASA法检测A549细胞IL-1、IL-6、IL-8、IL-11的分泌,Western blot 法检测相应的PKC的表达.结果 不同浓度LPS干预A549细胞相同时间后细胞因子IL-1、IL-6、IL-8、IL-11的分泌量及PKC的表达量之间的差异均有统计学意义(P＜0.01);PKC的表达和IL-11的分泌之间具有相关性(P＜0.05).结论 LPS干预细胞因子IL-1、IL-6、IL-8、IL-11的分泌及PKC的表达,且LPS干预细胞因子IL-11的分泌可能和PKC信号通路有关.     

肿瘤坏死因子-α激活人肺微血管内皮细胞p38 丝裂原激活蛋白激酶信号通路

目的　建立体外人肺微血管内皮细胞 (HPMEC)培养技术 ,观察肿瘤坏死因子 α(TNF α)对HPMECp38丝裂原激活蛋白激酶 (MAPK)活性的影响。方法　 ( 1)建立体外人肺微血管内皮细胞培养模型 ;( 2 )采用Westernblot和免疫复合物激酶技术检测TNF α对HPMECp38MAPK的活化作用。结果　 ( 1)TNF α可迅速诱导HPMECp38MAPK的磷酸化和活化 ,在一定程度上呈时间、剂量依赖关系 ;( 2 )p38MAPK特异性阻断剂———SB2 0 35 80可显著抑制由TNF α诱导的p38MAPK活化。结论　TNF α激活p38MAPK信号通路 ,在介导肺微血管内皮细胞的细胞内信号转导过程中起着十分重要的作用     

激活细胞外信号调节激酶-丝裂原蛋白激酶信号通路对人结肠癌细胞增殖及相关基因的影响

目的探讨野生型RAF和MEK持续激活细胞外信号调节激酶．丝裂原蛋白激酶信号通路（ERK-MAPK）对人结肠癌细胞增殖及细胞周期相关基因的影响。方法培养人结肠癌细胞系SW1116,脂质体介导RAF、MEK基因和空质粒转染,G418筛选阳性克隆。流式细胞仪分析细胞周期,光学显微镜下观察细胞形态学变化,四甲基偶氮唑蓝（MTT）法检测细胞活力,生长曲线和软琼脂集落形成检测细胞增殖,实时定量PCR检测p21^WAF1和p16^INK4A及癌基因c-myc转录水平。结果建立稳定表达RAF或MEK的细胞系,RAF或MEK高表达均能明显降低G0／G1期细胞百分比,促进细胞周期由G1向S期进行,增加DNA合成的S期细胞百分比,细胞活力和增殖力升高3—4倍,细胞分裂增殖旺盛。转染RAF／MEK质粒后,细胞均呈现p21^WAF1和p16^INK4A转录水平降低,c-myc转录水平升高。结论ERK-MAPK信号通路激活可下凋细胞周期相关基因p21^WAF1和p16^INK4A表达,并上调c-myc表达,减少G0期时相,加速G1／S期转化,促进细胞增殖。