耐药性癫癎患者术后脑组织中Tau蛋白的表达

目的研究Tau蛋白在耐药性癫癎患者脑组织中的表达,探讨其在耐药性癫癎发病机制中的作用。方法采用免疫组化法检测48例耐药性癫癎患者海马及颞叶皮层中Tau蛋白的表达,同步观察海马苔藓纤维芽生,并与对照组进行比较。结果耐药性癫癎组和对照组脑组织中总Tau蛋白表达无明显差异,而磷酸化Tau蛋白在耐药性癫癎患者海马CA3区(0·0450±0·0115)及齿状回内分子层(0·0463±0·0120)表达增多,同步伴有海马苔藓纤维芽生(耐药性癫癎组:3·18±0·35,对照组:0·61±0·19)。结论磷酸化Tau蛋白表达增强可能是耐药性癫癎患者海马突触重建的重要机制,改变磷酸化Tau蛋白的表达有可能改善耐药性癫癎患者的预后。     

耐药性癫(癎)患者颞叶中细胞周期依赖性蛋白激酶5的表达

目的 研究细胞周期依赖性蛋白激酶5(cyclin dependent kinases 5,CDK5)在耐药性癫(癎)患者颞叶中的表达,探索其在耐药性癫(癎)发病机制中的作用.方法 收集耐药性癫(癎)患者术后脑组织,用荧光定量PCR、免疫组化和Western blot 3种检测方法从基因和蛋白水平分别测定CDK5在耐药性癫(癎)患者颞叶中的表达,并与对照组进行比较.结果 荧光定量PCR发现CDK5 mRAN比对照组明显增加,免疫组化检测显示这种基因的蛋白表达产物主要分布在神经元轴突和胶质细胞中,Western blot检测在相对分子质量35 000处有一蛋白条带,并且可见实验组(颞叶和海马中分别为1.4293±0.1839和2.0733±0.4738)高于对照组(颞叶和海马中分别为0.9680±0.4147和1.403±0.6163,P＜0.05).结论 CDK5在耐药性癫(癎)患者颞叶中表达增强,提示他们可能参与了耐药性癫(癎)的形成.     

颞叶癫痫主穹窿蛋白表达及其作用研究

目的 探讨主穹窿蛋白(major vault protein,MVP)在颞叶癫痫大鼠模型脑组织的表达及其与难治性癫癎耐药是否相关.方法 用氯化锂-匹鲁卡品制作颞叶癫癎模型,并将它分为药物有效组和耐药组;用免疫组化和Western blot法检测MVP表达.结果 MVP主要表达在海马和皮质区的小胶质细胞,血管内皮细胞,血管周围的星形胶质细胞也有表达,神经元中少见表达;耐药组MVP表达较药物有效组和对照组明显增高,具有统计学意义(P<0.05).结论 耐药大鼠模型中MVP过量表达,提示MVP可能与难治性癫癎的耐药性有关.     

颞叶癫(癎)大鼠模型内嗅皮质和齿状回内Sema3A及其受体Np1的表达变化

目的 探讨颞叶癫(癎)大鼠脑内Sema3A及其受体Np1的表达变化在癫(癎)发病中的作用.方法 SD大鼠制成颞叶癫(癎)模型,Neo-Timm染色证实苔藓纤维出芽,免疫组化和原位杂交技术对致(癎)后不同时间点内嗅皮质和齿状回的Sema3A mRNA、Np1 mRNA和蛋白表达进行分析.结果 与对照组比较,颞叶癫(癎)大鼠海马齿状回内分子层存在苔藓纤维出芽(7 d:0.70±0.42,15 d:1.50±0.52,30 d:2.20±0.41,60 d:2.50±0.51,P＜0.05);在匹罗卡品致(癎)后7 d,实验组内嗅皮质区Sema3A mRNA的表达(0.3006±0.0675)明显低于对照组(0.4562±0.0457,P＜0.01);齿状回内Np1mRNA(0.2337±0.0358)及蛋白(0.2706±0.0389)的表达亦明显低于对照组(P＜0.01).结论 内嗅皮质区Sema3A mRNA、齿状回内Np1 mRNA和蛋白的表达下调,可能参与了苔藓纤维的出芽机制.     

颞叶癫癎主穹窿蛋白表达及其作用研究

目的探讨主穹窿蛋白（major vault protein，MVP）在颞叶癫癎大鼠模型脑组织的表达及其与难治性癫癎耐药是否相关。方法用氯化锂-匹鲁卡品制作颞叶癫癎模型，并将它分为药物有效组和耐药组；用免疫组化和Westernblot法检测MVP表达。结果MVP主要表达在海马和皮质区的小胶质细胞，血管内皮细胞，血管周围的星形胶质细胞也有表达，神经元中少见表达；耐药组MVP表达较药物有效组和对照组明显增高，具有统计学意义（P〈0．05）。结论耐药大鼠模型中MVP过量表达，提示MVP可能与难治性癫癎的耐药性有关。     

神经营养因子-3在颞叶癫(癎)后的表达与海马苔藓纤维发芽的关系

目的 探讨神经营养因子-3(NT-3)在颞叶癫(癎)大鼠海马内表达与苔藓纤维发芽(MFS)的可能关系.方法 大鼠侧脑室注射红藻氨酸(KA)建寺颞叶癫(癎)模型后,侧脑室注射NT-3反义寡核苷酸(ASODN)及正义寡核苷酸(SODN);应用免疫组化法观察海马NT-3表达;应用Timm银染方法,光镜和电镜观察海马MFS,并与空白对照组及脂质体对照组进行比较.结果 致(癎)后大鼠海马齿状回NT-3表达下降;海马苔藓纤维明显粗乱,侧支发芽;齿状回内分子层可见银标记突触末端,主要为轴-树型非对称突触,其中ASODN组海马NT-3蛋白水平降低及MFS程度更明显.结论 KA致痢和NT-3 ASODN均能降低大鼠海马齿状回NT-3表达,增加MFS程度.提示NT-3可能通过对MFS及突触重组作用来影响颞叶癫(癎)的发生和发展.     

肌球蛋白轻链激酶和Rho激酶在兔脑血管平滑肌细胞迁移中的作用

目的 探讨肌球蛋白轻链激酶(MLCK)和Rho激酶在颅内血管平滑肌细胞(VSMC)迁移中的作用.方法 体外培养免大脑中动脉VSMC.构建携带反义MLCK cDNA的腺病毒载体并转染VSMC以降低MLCK表达.应用Rh0激酶特异性抑制剂Y-27652来抑制Rho激酶的活性.免疫印迹法测定细胞内MLCK和gho激酶的表达水平;改良Boyden小室法检测血管紧张素11(ATII)诱导的VSMC迁移;甘油凝胶电泳和免疫印迹法检测肌球蛋白轻链(MLC)的磷酸化水平.结果 反义MLCK cDNA转染后,MLCK表达量较LacZ基因转染组下降(84.6+5.8)%(P＜0.01),但ATII刺激引起的MLC磷酸化和VSMC迁移未受明显影响(P均＞0.05).Y-27682预处理反义MLCK cDNA转染后的VSMC可明显降低基础水平以及ATII刺激引起的MLC磷酸化,并部分阻断VSMC迁移(P均＜0.05).结论 颅内、外动脉VSMC迁移的分子机制不同,在ATII诱导的颅VSMC迁移中MLCK作用有限,而Pho激酶可能通过非钙依赖型MLC磷酸化途径发挥重要作用.     

颞叶癫大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化

目的 研究颞叶癫(癎)大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化.方法 给SD大鼠腹腔注射匹罗卡品、氯化锂制作颞叶癫(癎)模型.用免疫组化法和原位杂交技术对致(癎)后不同时间点大鼠海马CA1区、CA3区、齿状回的Sema3F mRNA、Np2 mRNA和蛋白表达进行检测,并与正常对照组比较.结果 颞叶癫(癎)大鼠致(癎)后7 d、15 d,海马CA1区、CA3区Sema3F mRNA、Np2 mRNA和蛋白的表达明显低于正常对照组(P＜0.05～0.01), 致(癎)后30 d、60 d表达与正常对照组差异无统计学意义;而齿状回Sema3F mRNA、Np2 mRNA和蛋白的表达与正常对照组的差异无统计学意义.结论 颞叶癫(癎)大鼠海马CA1区、CA3区Sema3F、Np2表达在致(癎)后早期明显下调,而在慢性期恢复正常.     

匹罗卡品癫(癎)模型中蛋白质netrin-1的表达与苔藓纤维出芽的动态变化

目的:探讨神经诱向因子蛋白质netrin-1在癫(癎)持续状态后海马苔藓纤维出芽(MFS)中的作用.方法:氯化锂-匹罗卡品建立大鼠颞叶癫(癎)(TLE)模型,采用Timm染色和免疫组化的方法分别检测MFS和netrin-1在大鼠海马组织中的表达.结果:TLE组大鼠在模型形成的第2周和第4周,海马齿状回内netrin-1的表达较正常对照组明显增加,并可见到MFS,穿越齿状回颗粒细胞层到达内分子层,并形成一条致密的层状带.结论:癫(癎)状态后在海马齿状回netrin-1的表达上调,证明其可能参与了癫(癎)后MFS过程.     

癫(癎)幼鼠海马和颞叶皮质区缝隙连接蛋白43和突触体素表达的变化

目的观察神经元缝隙连接蛋白43(Cx43)和突触体素(synaptophysin P38)在戊四氮(PTZ)点燃癫癎幼鼠海马及颞叶皮质区中的表达,探讨两者与癫癎的关系及其在癫形成中的作用。方法将50只21日龄Wistar大鼠分为对照组和实验组。实验组采用PTZ点燃癫癎幼鼠,按点燃进程分为Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级及Ⅴ级发作组。采用免疫组化和图像分析技术,观察海马及颞叶皮质区Cx43和P38表达的变化。结果应用PTZ点燃后,实验各组幼鼠海马及颞叶皮质区Cx43和P38的表达明显高于对照组(P<0.01),且随发作级别的增高,幼鼠海马及颞叶皮质各区Cx43和P38的表达均增加。但各组间海马区和颞叶皮质区Cx43和P38的表达情况的比较差异无统计学意义(P>0.05)。结论Cx43和P38的表达水平与癫癎的发生发展有密切关系,为研究小儿癫癎的病因及发病机制提供依据。     

Increased serum myosin light chain 3 level in neuromuscular diseases

A radioimmunoassay for human skeletal muscle myosin light chain 3 (MLC-3) was developed. The serum level of MLC-3 was evaluated in 143 patients suffering from neuromuscular diseases. Increased MLC-3 level was observed in muscular dystrophies. There were significant positive correlations between serum levels of MLC-3 and creatine kinase (CK) in Duchenne and limb-girdle type muscular dystrophy, but the regression lines were different. Patients with neurogenic amyotrophy, especially amyotrophic lateral sclerosis, also showed elevated MLC-3 levels with or without high CK, and the frequency of increase in MLC-3 was greater than that of CK. The results of the present study suggest that circulating MLC might be a useful marker for muscle breakdown not merely in myopathies but in neurogenic disorders.     

Clinical significance of serum cardiac myosin light chain I in patients with muscular dystrophy

We examined serum cardiac myosin light chain I (LCI), serum creatine kinase (CK) levels and left ventricular function in patients with muscular dystrophy and secondary cardiac involvement. LCI levels were determined by a two-site immunoradiometric assay method in 25 patients with muscular dystrophy and 10 normal subjects. This study included 15 patients with Duchenne muscular dystrophy (DMD), 8 patients with Fukuyama type congenital muscular dystrophy (FCMD) and 2 sisters with non-Fukuyama type congenital muscular dystrophy (nFCMD). We measured the value of left ventricular fractional shortening (FS) using echocardiography. All patients with DMD and FCMD showed moderate or severe skeletal muscle weakness. The mean values of LCI were significantly higher in patients with DMD (11.0 +/- 8.3 ng/ml, p less than 0.01) and in patients with FCMD (1.6 +/- 1.4 ng/ml, p less than 0.05) than in normal subjects (0.3 +/- 0.2 ng/ml). In patients with DMD, LCI level correlated closely with CK level (r = 0.81, p less than 0.01) but not with FS (r = 0.35, n.s.). In patients with FCMD, LCI level correlated significantly with CK level (r = 0.75, p less than 0.05) but not with FS (r = 0.44, n.s.). Close correlation between LCI and CK levels was thought to result from the cross reaction between cardiac LCI and myosin light chains of skeletal muscle in the assay method we used. Two siblings with nFCMD showed mild skeletal muscle weakness. A 22-year-old sister with mild left ventricular dysfunction (FS = 0.41) showed high level of CK (4794/U/L) and mild elevation of LCI (7.3 ngml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution of calcium- and calmodulin-dependent myosin light chain phosphorylating activity in rat cerebral cortex

The subcellular distribution of Ca2+- and calmodulin-dependent myosin light chain phosphorylating activity in rat cerebral cortex was studied. The activity showed a high degree of association to nerve endings (either crude or purified by discontinuous sucrose density gradient centrifugation). After osmotic shock of the nerve endings, the activity was largely membrane associated and could not be released from membranes by freeze-thawing, dilution, 75 mM NaCl or 4 mM EDTA. The association of Ca2+/calmodulin-dependent myosin light chain phosphorylating activity with synaptosomal membranes suggests a role for calcium-dependent myosin phosphorylation in events relating to neurotransmission.     

Clinical significance of serum cardiac myosin light chain I in patients with Duchenne muscular dystrophy]

The cardiac myosin light chain I (LCI) is one of the cardiac muscle structural proteins. A sensitive immunoradiometric assay kit for LCI by using LCI monoclonal antibodies is developed. We estimated LCI in the patients with Duchenne muscular dystrophy (DMD) and Kugelberg-Welander disease (KW). The results suggested that LCI has close relationships with the functional disturbances of skeletal muscles, especially disturbances of pulmonary ventilation. Therefore we studied properties and localizations of LCI in the skeletal muscles by Western blotting and immunohistochemical methods. In Western blotting method LCI monoclonal antibodies have a band of 27 KD proteins of skeletal muscles. LCI has also found to be localized in type 1 fibers in frozen sections of biopsied of human skeletal muscles. LCI was measured from 47 patients with DMD and 8 patients with KW. The average serum LCI levels in the patients with DMD were 11.79 ng/dl and its levels in the patients with KW were in the normal range (under 2.5 ng/dl). Among 12 patients receiving negative pressure chest respirator, the levels of LCI were also under 2.5 ng/dl. Serum LCI decreased with increasing age and reduced physical activity. The levels of LCI has obvious positive correlations with CK and myoglobin. These results suggested that the measurements of serum LCI are useful as one of the markers of disease severity and the determination of suitable time of using respirator.     

Studies on myosin light chain phosphorylation in intact platelets, utilizing a cell-penetrating thiol protease inhibitor

By employing a cell penetrating thiol protease inhibitor, EST: ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)buty lcarbamoyl]- 2-oxiranecarboxylate, the role of calpain, a major thiol protease in platelets, on 20K protein (myosin light chain) phosphorylation was examined in intact human platelets. EST dose-dependently inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Phosphopeptides mapping revealed the phosphorylation by these agonists was rendered only by the action of myosin light chain kinase (MLCK). However, in TPA (12-O-tetradecanoylphorbol-13-acetate) stimulated platelets, EST did not inhibit 20K phosphorylation which was mediated by the action of C-kinase. [Ca2+]i determined by the use of quin-2 was elevated after the stimulation of thrombin, ionomycin or collagen but not TPA. Thus, it was suggested that calpain enhances MLCK activity on 20K phosphorylation in intact platelets following the stimulation by the agonist which elevates [Ca2+]i.     

Albumin causes increased myosin light chain kinase expression in astrocytes via p38 mitogen-activated protein kinase

Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton, leading to disruption of vascular barrier integrity in multiple organs, including the blood-brain barrier (BBB), after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia, and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase of MLCK in astrocytes and the signaling pathways involved in this increase. After midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MLCK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK, or rho kinase, also prevented this increase. These results are further evidence of a role of MLCK in the mechanisms of BBB compromise following TBI and identify astrocytes as a cell type, in addition to endothelium in the BBB, that expresses MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB.     

Effects of lithium and valproate on agonist-induced platelet intracellular calcium mobilization: relevance to myosin light chain kinase

Serotonin (5-HT)- or thrombin-stimulated platelet intracellular calcium (Ca) mobilization has been reported to be enhanced in patients with bipolar disorders. However, the mechanism of this enhancement is unknown. As a preliminary study, the authors examined the effects of a myosin light chain kinase (MLCK) inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), and two drugs that are mainstays of treatment for bipolar disorder, lithium and valproate, on 5-HT- or thrombin-induced Ca increase in the platelets of normal subjects. When preincubated with 30 microM ML-9, Ca responses to both agonists were enhanced. Valproate showed a dose-dependent attenuation of agonist-induced intracellular Ca rise, both in the absence and presence of ML-9. Although lithium alone had no significant effect on the Ca increase, a high concentration of lithium significantly decreased Ca mobilization only in the presence of ML-9. These results suggest that the enhanced Ca response observed in bipolar disorder might be relevant to decreased function of MLCK and that the mechanism of action of lithium may include a compensatory effect on MLCK modulation.     

NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells.

The relationship between phosphorylation of myosin light chain (MLC) and neurite outgrowth induced by nerve growth factor (NGF) was studied in PC12 cells. Inhibitors of Rho kinase, HA-1077 or Y-27632 also induced neurite outgrowth. As already reported botulinum exoenzyme C3 which inactivates Rho protein also induced neurite outgrowth. Calyeulin A, an inhibitor of phosphatase counteracted both NGF- and C3- induced neurite outgrowth. Treatments of both NGF and C3 resulted in significant and transient decrease in phosphorylated MLC. These results suggest that NGF induces neurite outgrowth of PC12 by a transient decrease in phosphorylated MLC which is brought about by activation of MLC phosphatase via inhibition of Rho-Rho kinase pathway.