上海地区汉族优秀游泳运动员ACE基因I/D多态性研究

目的:探讨上海地区汉族不同水平优秀游泳运动员ACE(血管紧张素转化酶)基因I/D多态性的分布特点。方法:采用PCR方法,对上海地区85名汉族优秀游泳运动员和90名汉族普通人的ACE基因I/D多态性进行检测。结果显示,上海地区汉族优秀游泳运动员的ACE基因的基因型和等位基因频率与上海和成都地区汉族普通人无明显差异(P>0.05);上海地区汉族游泳运动员和普通人以及成都地区汉族普通人的基因型和等位基因频率均与高加索人群存在高度显著性差异(P<0.0001),表现出明显的种族差异性。7名上海地区汉族国际健将ACE基因均为II型,运动水平越高的组别,II基因型和I等位基因频率越高,提示具有II基因型或I等位基因频率高的运动员经过多年运动训练,具有成为优秀运动员的可能,特别是II基因型的运动员可能性更大。     

血管紧张素转换酶2与糖尿病肾病

The identification of angiotensin-converting enzyme (ACE)2 opened new recognition of renin-angiotensin system (RAS). ACE2 degrades Ang Ⅱ to Ang (1-7), maintains homeostasis of RAS with ACE. Studies have revealed that ACE2 has important functions in diabetic nephropathy. It may be a target for drug and is used as gene therapy for diabetic nephropathy. Amplifying ACE2 activity may have a potential therapeutic role for diabetic nephropathy.     

Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

C端序列的删除对非出血重组蛇毒纤溶酶的影响

[目的]研究C端序列对非出血重组纤溶酶(rFⅡ)特异性、活性和热敏感性的影响.[方法]通过SOEing PCR方法构建删除C端序列的突变体,突变体和rFⅡ分别在P.pastoris中诱导表达.表达和纯化的蛋白用SDS-PAGE和Westeren blot进行鉴定.之后分析其生化特性.[结果]rFⅡ及突变体通过SDS-PAGE和Westeren blot得到证实.生化分析揭示:①突变体对显色底物N-(p-Tosyl)-Gly-Pro-Lys-pNA的催化效率(Kcat/Km)是rFⅡ的1.9倍;②突变体和rFⅡ对氧化的胰岛素B链拥有共同的优先裂解位点,可是在随后的裂解中开始展现差异;③突变体显示了更高的纤(原)活性;④热处理表明突变体相较r FⅡ对温度的增加更敏感;⑤通过圆二色谱测量,突变体的螺旋比例有所增加.[结论]删除的序列参与rFⅡ的特异性、活性和热敏感性,该序列可作为下一步优化的候选序列.     

青霉素酶电极的研制

探讨了青霉素酶电极的制备方法，以青霉素酶为敏感膜偶合玻璃电极制成青霉素酶电极，在０．１５￣１．５ｍ ｍｏｌ／Ｌ青霉素钠浓度范围内，该电极的动力学响应与青霉素钠浓度的对数呈良好的线性关系，相关系数为０．９９８８，对电极性能的影响因素如介质条件，温度、膜厚度等进行了考察，并试验了电极的有效活性期。测得平均回收率为９９．０％。     

A binding study of phospholipase A2 with lecithin,lysolecithin and their tetrahedral intermediates using molecular modeling

We used molecular modeling to examine the binding of 1, 2-dioctanoyl-sn-glycero-3-phosphocholine (a lecithin), 1-octanoyl-sn-glycero-3-phosphocholine (a lysolecithin) and their tetrahedral intermediates in the catalytic site of phospholipase A₂ (PLA₂). We performed energy minimization on each complex, computed the binding energy, determined the relative binding energy among the complexes and calculated the difference in inter- and intramolecular energies of the components in the complexes. We found that the calculated orientation of the sn-1 ester bond of lysolecithin in the active site is similar to that of the sn-2 ester bond in lecithin, thus permitting PLA₂ to hydrolyze lysolecithin using the same mechanism as it uses to hydrolyze lecithin. On the other hand, the binding of lecithin is energetically more favorable by 4.5 kcal/mol than the binding of lysolecithin to the enzyme, and the binding of the lecithin tetrahedral intermediate is also energetically more favorable by 19.7 kcal/mol than the binding of the lysolecithin tetrahedral intermediate to the enzyme, which explains why lecithin is a better substrate than lysolecithin in the catalytic site. These results indicate that the activation energy for the hydrolysis of lysolecithin is higher than that for lecithin, consistent with the observed slower rate for the hydrolysis of lysolecithin.     

Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells

The cellular and regional distribution of glutathione (GSH) and GSH-related enzyme systems involved in cellular defense against reactive oxygen species and electrophilic xenobiotics in the nervous system has been extensively studied. However, little is known about the subcellular distribution of GSH systems in brain tissue and cultured neural cells. The present study investigates the distribution of mitochondrial and cytosolic GSH and GSH-related enzymes in cultured cerebellar astrocytes and granule cells, and compares them with levels in the adult rat cerebellum. Cytosolic GSH levels and cytosolic activities of glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in astrocytes were 57, 153, 245, and 92% higher than those found in granule cells, respectively. In contrast, granule cells contained significantly higher mitochondrial GSH levels than astrocytes. Granule cells also demonstrated comparable mitochondria/cytosolic concentrations of GSH and GR, GPX and GST activities to those observed in the cerebellar tissue, whereas ratios in astrocytes were markedly lower. Although in vitro treatments with 100 μM ethacrynic acid depleted both cytosolic and mitochondrial GSH in cultured astrocytes and granule cells in a time-dependent fashion, cellular GSH in granule cells was more resistant to the GSH-depleting agent than astrocytes. These results suggest that although GSH and GSH-related enzymes are abundant in cytosolic compartments of astrocytes, mitochondrial pools are relatively small. Since brain mitochondria are sites of significant hydrogen peroxide generation, the mitochondrial localization of GSH and its associated enzymes in neural cells provide important defenses against toxic oxygen species in the nervous system. Differences in subcellular distribution of GSH systems in individual neural cell types may provide a basis for selective cellular and/or subcellular expression of neurotoxicity.     

一种新的五步蛇蛇毒类凝血酶Cdna的克隆和序列分析

目的：克隆出五步蛇的类凝血酶cDNA，为研究其结构和功能的关系。并为下一步基因表达开发抗血栓药物提供依据和基础。方法：从五步蛇蛇腺中提取了总RNA，通过反转录合成第一链cDNA，经PCR扩增出五步蛇毒腺中类凝血酶TLE1的cDNA片段，并将其连接到质粒载体扩增，然后进行DNA测序。结果：成功克隆出一种新的五步蛇类凝血酶的cDNA片段。此片段全长794bp，包括编码部分的全长为777bp。推导其编码的蛋白质序列为258个氨基酸，其中包括18个氨基酸的信号肽，6个氨基权的前肽和234个氨基酸的成熟氨基酸顺序。TLE1成熟肽与其它蛇毒类凝血酶相比，蛋白质一级结构具有较高的同源性。结论：从五步蛇中成功克隆出一 种新的类凝血酶cDNA，并确定了它的DNA顺序。     

海藻、甘草及其相伍用对小鼠肝药酶的影响

目的：研究海藻、甘草单煎液及其不同比例合煎，单煎后混合液以及单体A、B；C、D、E对小鼠肝药酶的影响。方法：取小鼠随机分组，分别ig给药，qd,连续4d后禁食20h，处死取肝脏，称重后制成匀浆，紫外分光光度计于450nm,490nm波长下测其光密度值，计算肝匀浆中细胞色素P－450的含量。结果：甘草、甘草与海藻合煎液及单体A，B，C，D，E均能显著提高小鼠肝匀浆中细胞色素P－450的含量，而海藻，甘草与海藻单煎后混合液未能提高小鼠肝心浆中的细胞色素P－450的含量。结论：甘草，甘草与海藻合煎液及单体，A，B，C，D，E均能显著提高小鼠肝匀浆中细胞色素P－450的含量，对肝药酶有诱导作用。     

VASOCONSTRICTOR RESPONSES TO COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM IN CYCLOSPORIN-INDUCED HYPERTENSION IN THE RAT

1. Since plasma renin activity is increased in cyclosporin A (CsA)-induced hypertension in the rat, the role of the vascular renin-angiotensin system (RAS) in CsA-induced hypertension was investigated in rat mesenteric resistance vessels. 2. Female Wistar rats received CsA (10 mg/kg per day, s.c.) or vehicle for 30 days. CsA treatment increased tail-cuff systolic blood pressure (CsA treated 135 ± 3 mmHg vs control 125 ± 1 mmHg, P<0.0001). 3. Mesenteric resistance arteries (200–300 μm) were isolated and mounted in a microvessel myograph. Concentration-response curves to tetradecapeptide renin substrate (10-¹¹-10^?6 mol/L), angiotensin I (10-^l1-10^?6 mol/L) and angiotensin II (10-¹²-10^?6 mol/L) showed no differences between CsA-treated and control groups. 4. Mesenteric vascular angiotensin-converting enzyme (ACE) characteristics were determined by radioligand binding. There were no differences in the content or affinity of ACE between CsA-treated and control rats. 5. These results suggest that the mesenteric vascular RAS does not play a major role in CsA-induced hypertension in the rat.