地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。     

Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.     

Changes of intracellular Na+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na⁺ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na⁺concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na⁺ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na⁺ , K⁺ -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Absence of pfr destruction in the modulation of phenylalanine ammonia-lyase synthesis of mustard cotyledons

The relationship between the amount of active phytochrome (Pfr) produced by 5-minute light pulses and the rate of subsequent enzyme accumulation (phenylalanine ammonia-lyase, EC 4.3.1.5) of mustard (Sinapis alba L.) cotyledons was investigated. The response rapidly adjusts to changes of the Pfr level produced by light pulses of different wavelengths. Regardless of total phytochrome levels in the cotyledons, response adjustments to new photostationary states (_λ) are correlated with _α values. On the other hand, the kinetics of enzyme accumulation shows no influence of Pfr destruction as determined spectrophotometrically (τ_½ = 45 min) in the same organ (see Schäfer et al. 1973 Photochem Photobiol 18: 331-334). It is concluded that the phytochrome molecules involved in the regulation of this response by light pulses comprise a small fraction of the total phytochrome of the cotyledons. In contrast to bulk phytochrome this fraction appears to be not subject to Pfr destruction.     

水稻OsPR10A的表达特征及其在干旱胁迫应答过程中的功能

利用免疫印迹(WB)分析了水稻(Oryza sativa) OsPR10A在其不同生长时期、不同组织部位及多种非生物逆境胁迫下的表达特征, 发现OsPR10A在干旱、盐胁迫以及茉莉酸甲酯(MeJA)和脱落酸(ABA)诱导下表达量明显升高, 表明该蛋白可能在干旱和盐胁迫应答过程中发挥作用。为证明这一推测, 我们构建了OsPR10A超表达载体, 经农杆菌介导转化水稻, 获得超表达OsPR10A的纯合株系。田间表型观察表明, 转基因株系株高变矮、穗长变短、结实率降低。用20% PEG6000在水稻种子萌发过程中进行干旱处理, 结果显示, OsPR10A超表达株系的根长和芽长均显著高于野生型, 证明超表达OsPR10A可增强水稻萌发期耐旱性。该研究有助于增进人们对水稻OsPR10A功能的了解。     

检测病毒气溶胶存活的方法学研究

以钝齿棒状杆菌噬菌体B271血清型为病毒等小颗粒生物粒子的模拟剂,建立了一种适合这类小颗粒生物粒子气溶胶存活研究的方法。本文从该噬菌体耐气溶胶化特性、气溶胶粒谱、用气溶胶示踪剂求算物理衰亡的方法和气溶胶采样回收技术等方面探讨了病毒气溶胶存活研究中的几个关键技术问题,为病毒气溶胶存活研究提供了参考。     

眼底图象定量分析

本文介绍了基于数学形态学的图象分析系统在生物医学,特别是眼底图方面日益广泛的应用.提出了运用图象分析系统取得眼底照片上一些参数的方法.主要参数包括:动静脉管径及其比例,无灌注区的面积等.这些参数不仅用于眼科方面的诊断和治疗,对于分析和诊断全身性疾病,如高血压、动脉硬化、糖尿病、静脉阻塞等都具有重要价值