G蛋白耦联受体激酶结合蛋白1通过调节细胞外调节激酶1/2的活性促进成骨细胞迁移

目的探索G蛋白耦联受体激酶结合蛋白1(GITI)在成骨细胞迁移中的作用,并分析其机理。方法通过Western blot方法检测GIT1蛋白在鼠的成骨细胞内的表达；用免疫荧光染色方法确定：在血小板衍生生长因子(PDGF)不刺激和刺激的条件下,GIT1和细胞外调节激酶1/2(ERK1/2)在成骨细胞内的位置；用共同免疫沉淀的方法测定GIT1和ERK1/2相互结合,并且用免疫荧光双染的方法确定这两种蛋白相互结合的位置；用包含GIT1-RNA发夹结构的腺病毒感染成骨细胞后,用免疫荧光染色方法确定磷酸化ERK1/2(pERK1/2)在成骨细胞内的位置,用划痕愈合法检测在PDGF刺激下的迁移能力。结果在成骨细胞内,PDGF刺激导致了GIT1和ERK1/2的相互结合,并且这种结合发生在成骨细胞的局部粘附内。包含GIT1-RNA发夹结构的腺病毒明显抑制了pERK1/2招募至成骨细胞局部粘附内以及PDGF所刺激的成骨细胞的迁移。结论在PDGF刺激下,GIT1招募pERK1/2至成骨细胞的局部粘附内,从而促进成骨细胞的迁移。     

SB203580对肾缺血/再灌注损伤时细胞凋亡及p38MAPK影响的实验研究

目的　探讨不同时间及不同浓度p38MAPK特异性抑制剂SB2 0 35 80对肾缺血 /再灌注损伤过程中肾功能、细胞凋亡及 p38MAPK活性、表达量、p38MAPK底物的影响。 方法　 4 9只大鼠按缺血 /再灌注及给药时间的不同 ,随机分为 7组 ,每组7只大鼠按正交拉丁方表的顺序 ,经尾静脉注射相同体积、不同剂量的SB ,使其在大鼠体内达到不同的浓度。测定BUN和Scr;用TUNEL试剂盒检测细胞凋亡情况 ;Westernblot技术用于蛋白定性及半定量分析。结果　SB可显著减轻大鼠肾缺血 /再灌注损伤造成的Scr和BUN的升高、肾小管上皮细胞的凋亡及 p38MAPK的激活 ,但存在模型、剂量及给药时机的差异 (P<0 .0 5 ) ,缺血前 3h之前给药 ,使其体内血药浓度达到 5 μmol/L左右可取得较好的效果。 结论　SB可显著减轻大鼠肾缺血 /再灌注损伤 ,在缺血前 3h之前给药 ,同时使其血药浓度达到 5 μmol/L左右可取得最佳效果。     

应用抑制消减杂交和cDNA表达谱芯片筛选肝星形细胞持续活化的相关基因

目的：利用抑制消减杂交（suppression subtractive hybridization,SSH）和cDNA表达谱芯片筛选早期活化的肝星状细胞与大鼠正常肝脏组织、轻度肝纤维化组织、重度肝纤维化组织中差异表达的基因.方法：从SSH构建的大鼠轻度和重度肝纤维化中两个差异cDNA文库中挑选1 000条上调显著的基因,与正常大鼠4 136条基因克隆制作成一张芯片,筛选持续期活化的肝星状细胞相关基因.结果：获得与HSC持续活化相关的上调基因633条,下调基因715条,其中血清和糖皮质激素调节蛋白激酶（SGK）在正常大鼠基因克隆和2、8周SSH上调差异基因中均呈上调信号.结论：联合应用SSH和cDNA表达谱芯片是筛选和鉴定不同样本中差异表达基因的快速、经济和有效方法;SGK可能作为多种细胞信号传导通路和细胞磷酸化级联反应的一个功能性交汇点,参与了肝星状细胞的早期活化和信号传导.     

Protein kinase C in focal ischemic rat brain: dual autoradiographic analysis of [C]iodoantipyrine (IAP) and [H]phorbol-12,13-dibutyrate (PDBu)

Protein kinase C (PKC) activity was measured in rat brain with 2 h of middle cerebral artery (MCA) and common carotid artery (CCA) occlusion, using dual autoradiography of [¹⁴C]iodoantipyrine (IAP) and [³H]phorbol-12,13-dibutyrate (PDBu). In the ischemic brain, it required more than 120 min of incubation to obtain a plateau in PDBu binding. In contrast, the binding of PDBu in non-ischemic brain reached a plateau with incubation for 60 min. This delay of PDBu binding in the ischemic brain suggests that the affinity of this ligand is reduced due to a change in structure of the cell membrane caused by ischemia. PDBu binding in the ischemic brain increased significantly compared to the non-ischemic brain. This finding provides further evidence that excessive activation of PKC in the ischemic brain may play an important role in ischemic neuronal damage. ©1997 Elsevier Science B.V. All rights reserved.     

Protein kinase C agonist and antagonist effects in normal human epidermal keratinocytes

Abstract Several lines of evidence implicate protein kinase C (PKC) in the development of basal cell and squamous cell carcinomas, tumors which originate from epidermal keratinocytes. To examine PKC in a model relevant to human skin, we exposed normal human epidermal keratinocytes (NHEK) in serum-free media to a variety of PKC agonists and antagonists. NHEK PKC activity increased up to 10-fold within the 1st hour of exposure to tetradecanoyl phorbol acetate (TPA), and gradually returned to control values within 72 h. TPA-induced PKC activity was enhanced by pretreatment of cultures with protein and RNA synthesis inhibitors. TPA-induced growth arrest and differentiation was antagonized by staurosporine. Down-regulation by bryostatin pretreatment blocked TPA-stimulated differentiation. Our overall conclusion is that activation of PKC in cultured human keratinocytes is required for differentiation. These results are crucial to the analysis of compounds suspected of promoting or inhibiting epidermal tumors.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Amadori糖化白蛋白对牛视网膜血管二酯酰甘油--蛋白激酶C级联的影响

目的 :探讨Amadori糖化血清白蛋白对牛视网膜血管二酯酰甘油 (DAG)———蛋白激酶C(PKC)信号级联的影响及d-α-生育酚的干预作用。方法 :体外制备的Amadori糖化的牛血清白蛋白 (AGSA)分别在生理浓度葡萄糖 (5 .5mmol/L)和高浓度葡萄糖 (2 0mmol/L)培养液中作用于新鲜牛视网膜血管。采用薄层层析和放射自显影法测定DAG含量和PKC活性 ;并检测d -α-生育酚预处理后DAG、PKC改变。结果 :在生理葡萄糖浓度下 ,牛视网膜血管暴露于AGSA2 4h或 72h后细胞内DAG含量、PKC活性均较对照组显著增加 ,当AGSA和高葡萄糖同时作用于牛视网膜血管时 ,DAG -PKC级联显著激活 ,分别为正常葡萄糖组的 3.4 7倍和 4 .5 4倍 ;在正常血清培养液中 ,与 5 .5mmol/L的葡萄糖相比 ,2 0mmol/L高糖在 2 4h时并不刺激DAG含量增加 (P >0 .0 5 ) ,而 72h时则较对照组增加 4 5 % ,PKC活性较对照增加 5 6 % ,而d -α -生育酚可逆转上述生化改变。结论 :Amadori糖化血清白蛋白可在生理葡萄糖浓度下刺激DAG -PKC途径活化 ,从而影响血管一系列生理功能 ,而d -α-生育酚对血管生化改变有保护作用。     

Pheophytin a, a low molecular weight compound found in the marine brown alga Sargassum fulvellum, promotes the differentiation of PC12 cells

We identified and characterized a neurodifferentiation compound from the marine brown alga Sargassum fulvellum collected from the Japanese coastline. Several instrumental analyses revealed the compound to be pheophytin a. Pheophytin a did not itself promote neurite outgrowth of PC12 cells. However, when PC12 cells were treated with a low concentration of pheophytin a (3.9 microg/ml) in the presence of a low level of nerve growth factor (10 ng/ml), the compound produced neurite outgrowth similar to that produced by a high level of nerve growth factor (50 ng/ml). Pheophytin a also enhanced signal transduction in the mitogen-activated protein kinase signaling pathway, which is also induced by nerve growth factor. The effect of pheophytin a on neurite outgrowth of PC12 cells was completely blocked by U0126, a representative mitogen-activated protein kinase kinase inhibitor. These results suggest that pheophytin a enhances the neurodifferentiation of PC12 cells in the presence of a low level of nerve growth factor and that this effect is mediated by activation of a mitogen-activated protein kinase signaling pathway.