2种菌株来源的左旋门冬酰胺酶的鉴别和效价测定研究

目的:探讨由大肠埃希菌(E.coliASI.357)和欧文菌(Erwinia carotovora)制备的左旋门冬酰胺酶的鉴别方法,以及效价测定的最适酶促反应条件。方法:采用高效液相色谱法及等电聚焦电泳法对2种菌株来源的左旋门冬酰胺酶进行鉴别;考察缓冲液的种类及pH值对左旋门冬酰胺酶效价测定的酶促反应的影响。结果:大肠埃希菌和欧文菌2种来源的左旋门冬酰胺酶的色谱图有差别,主峰保留时间分别为11.0、11.8min,等电点色带分别为4.65～5.1、7.1～8.20;效价测定的最适酶促反应条件分别是三羟甲基氨基甲烷盐酸缓冲液(pH=9.0)、0.2mol·L-1磷酸盐缓冲液(pH=8.0)。结论:2种菌株来源的左旋门冬酰胺酶的等电点有显著差别,效价测定的最适酶促反应条件亦不相同,应作为2个品种制定质量标准。     

吗啡成瘾和正常大鼠的前额叶皮质表达蛋白质组变化的初步研究

目的建立和比较吗啡成瘾与正常大鼠前额叶皮质(PFC)蛋白质双向电泳图谱,寻找和鉴定吗啡成瘾大鼠PFC中的差异蛋白表达谱。方法以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和吗啡成瘾大鼠的PFC蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinumv5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果通过对2-DE图谱蛋白斑点的匹配及对比分析,与吗啡成瘾相关的差异表达蛋白斑点为87个;经质谱鉴定出2个有意义的差异表达的蛋白斑点:Snap25亚型β-Snap25突触相关蛋白25、β-肌动蛋白。结论吗啡成瘾组与正常对照组大鼠PFC蛋白表达存在差异;初步鉴定了大鼠前额叶皮质中与吗啡成瘾相关的差异蛋白,其表达的变化可能通过多种途经影响PFC神经元功能,为我们研究阿片类物质依赖作用机制提出了新的思路和方向。     

Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells

Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, lambda, theta, and zeta are present in rat primary cultured cerebellar granule cells (CGCs) 6-14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.     

The blood platelet proteome is changed in UREMIC patients

Comparative analysis of the protein profile of blood platelets isolated from nondialyzed and hemodialyzed uremic patients and healthy controls has been performed. These preliminary results indicate markedly changed expression of several proteins in blood platelets from both groups of patients compared with controls, with dramatic changes in hemodialyzed patients in the over-expression of low molecular peptides with a very wide range of pI values.     

阿霉素对端粒序列及其相似物电泳迁移速度的影响

目的 观察阿霉素对端粒序列d(TTAGGG)不同重复次数以及与其具有相似结构的单核苷酸序列二级结构的影响,探讨阿霉素诱导端粒复重序列d(TTAGGG)n形成鸟嘌呤四联体的机制.方法 人工化学合成线性单核苷酸序列d(TTAGGG)、d (TTAGGG)2、d(TTAGGG)3、d(TTAGGG)4、d(TTAGGG)5、d(TTGGAG)4、d(TGTAGG)4、d(TAGGTG)、d(TTGAGG)4;用非变性聚丙烯酰胺凝胶电泳法观察不同浓度阿霉素(0、1.25、2.5、5.0μg/mL)对端粒重复序列d(TTAGGG)4电泳迁移速度的影响;观察5.0μg/mL阿霉素对d(TTAGGG)、d(TTAGGG)2、d(TTAGG)3、d(TTAGGG)4、d(TTAGGG)5电泳迁移速度的影响;观察5.0 μg/mL阿霉素对d(TTAGGG)4、d(TTGGAG)4 、d(TGTAGG)4、d(TAGGTG)4 、d(TTGAGG)4电泳迁移速度的影响.结果 随着阿霉素的浓度的提高,端粒重复序列d(TTAGGG)4电泳迁移速度也随之加快;阿霉素可使端粒重复序列d(TTAGGG)4、d(TTAGGG)5序列的二级结构发生改变,从而加快其电泳迁移的速度,对d(TTAGGG)、d(TTAGGG)2、d(TTAGGG)3的电泳迁移速度没有影响;阿霉素可对含有GGG碱基排列的端粒重复序列发生电泳迁移速度的加快,而对无GGG碱基排列的端粒序列类似物的电泳迁移没有影响.结论 在阿霉素存在条件下,端粒序列d(TTAGGG)n重复4次以上的寡核苷酸序列的电泳迁移速度加快,其二级结构的改变是迁移速度加快的主要原因,这种改变后形成的二级结构可能为G-四联体.     

On the distribution of thyroglobin and larger iodoproteins in single rat thyroid follicles

Summary The distribution of the large iodoproteins in the lumen of thyroid follicles was tested. Samples of the colloid in rat thyroid follicles were obtained in vivo by micropuncture of superficial follicles in the isthmus of the gland. One sample was collected per follicle and several follicles were punctured in each gland. The protein composition of the samples was analysed by microgel electrophoresis. The protein separation patterns were recorded by microdensitometry after staining of the proteins and the amount of protein in the fractions was estimated from the densitometric recordings.Thyroglobulin (TG) and the aggregates of TG-here designated the S-TG fractions—were present in all samples of colloid. It was observed that there was a significant positive co-variation between TG and S-TG when the colloid contained only TG, S-TG and a prealbumin fraction. When the albumin-like protein was present in the samples the co-variation was less significant. The results show that the relative amount of the aggregates of TG is constant in the albumin free colloid and that these aggregates most probably are randomly distributed in the follicle lumen. The results further indicate that the aggregates of TG are non-randomly distributed in the larger peripheral follicles when the colloid contains the albumin-like protein.This study was supported by grant No 4482 from the Swedish Medical Research Council     

Hydrolysis of ibuprofenoyl-CoA and other 2-APA-CoA esters by human acyl-CoA thioesterases-1 and -2 and their possible role in the chiral inversion of profens

Ibuprofen and related 2-arylpropanoic acid (2-APA) drugs are often given as a racemic mixture and the R-enantiomers undergo activation in vivo by metabolic chiral inversion. The chiral inversion pathway consists of conversion of the drug to the coenzyme A ester (by an acyl-CoA synthetase) followed by chiral inversion by α-methylacyl-CoA racemase (AMACR; P504S). The enzymes responsible for hydrolysis of the product S-2-APA-CoA ester to the active S-2-APA drug have not been identified. In this study, conversion of a variety of 2-APA-CoA esters by human acyl-CoA thioesterase-1 and -2 (ACOT-1 and -2) was investigated. Human recombinant ACOT-1 and -2 (ACOT-1 and -2) were both able to efficiently hydrolyse a variety of 2-APA-CoA substrates. Studies with the model substrates R- and S-2-methylmyristoyl-CoA showed that both enzymes were able to efficiently hydrolyse both of the epimeric substrates with (2R)- and (2S)- methyl groups. ACOT-1 is located in the cytosol and is able to hydrolyse 2-APA-CoA esters exported from the mitochondria and peroxisomes for inhibition of cyclo-oxygenase-1 and -2 in the endoplasmic reticulum. It is a prime candidate to be the enzyme responsible for the pharmacological action of chiral inverted drugs. ACOT-2 activity may be important in 2-APA toxicity effects and for the regulation of mitochondrial free coenzyme A levels. These results support the idea that 2-APA drugs undergo chiral inversion via a common pathway.     

毛细管电泳在地中海贫血筛查中的应用

目的：了解毛细管电泳在地中海贫血筛查中的应用.方法：采用全自动毛细管电泳仪对3 196份标本进行血红蛋白电泳分析,测定HbA2、HbF及异常血红蛋白含量.结果：3 196份标本中检出异常血红蛋白426例,其中HbA2偏高165例,占5.16％; HbA2偏低183例,占5.73％.其中316例同步做地贫基因检测,毛细管电泳法对比基因检测：β地贫、HbH病、HbCS符合率为100％,静止型和标准型a地贫符合率为78.26％.结论：应用毛细管电泳开展地中海贫血的筛查,对预防和降低重型地中海贫血患儿出生具有重要意义.     

急性非ST段抬高型心肌梗死的特点及超声新技术的应用进展

急性非ST段抬高型心肌梗死（NSTEAMI）是急性心肌梗死的一种。它与传统的ST段抬高型心肌梗死（STEAMI）不同,好发于老年人,常累及多支血管,引起不完全闭塞,并容易形成侧支循环。目前临床的误诊和漏诊率都较高。超声二维斑点追踪技术（STD及其衍生出来的自动功能成像技术（AFI）等超声新技术克服了角度依赖性,能够通过斑点追踪,获取评价室壁运动的数据并以牛眼图显示。本文综述了NSTEAMI的特点和超声新技术的应用进展．     

皮肤角质形成细胞中TRPV1受体激活后的蛋白质组差异分析

目的 研究皮肤角质形成细胞中辣椒素受体TRPV1激活后细胞中蛋白质组学的变化,并鉴定差异蛋白质。方法 皮肤角质形成细胞株HaCaT细胞经辣椒素(CAP)刺激24 h后,提取蛋白样品,采用双向凝胶电泳技术分离HaCaT细胞总蛋白,用PDQuest软件分析对照组和CAP刺激组蛋白组图谱的表达差异点,再运用串联飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白。结果 CAP刺激HaCaT细胞后,磷酸甘油醛异构酶、烯醇化酶、6-磷酸葡萄糖酸内酯酶和组织蛋白酶D蛋白表达水平增高,角蛋白14和60S酸性核糖体蛋白P2表达量降低,这些蛋白可能在TRPV1激活后影响细胞功能的过程中有重要作用。结论 利用蛋白质组学技术,研究皮肤角质形成细胞株HaCaT细胞中TRPV1受体激活后蛋白表达差异,为研究TRPV1受体的作用机制,以及为研发针对性药物,提供新的线索和方向。