Extractive partition of L-aspartase and fumarase fromEscherichia coli using aqueous polyethylene glycol-ammonium sulfate systems

Summary Inorganic sulfate salts are used to form aqueous two-phase systems with polyethylene glycol (PEG) for enzyme purification. Two enzymes, L-aspartase and fumarase produced byEscherichia coli are efficiently separated into different phases in spite of the high degree of similarity in molecular weight and amino acid sequence between them. The ratio of L-aspartase to fumarase in the PEG-rich phase is more than sixty (60) times the ratio before extraction. A high degree of purification in a single extraction step can be achieved by careful selections of PEG molecular weight, pH, cation of the salts, and sodium chloride levels. Cations of sulfate-containing salts in the following order: NH ₄ ⁺ >Na⁺>Mg²⁺ tend to increase the partition of L-aspartase in the PEG-rich phase. The maximal degree of enzyme purification is obtained by using PEG 4000 and ammonium sulfate as a phase system at a stable pH for both enzymes.     

应用蛋白印迹法检测鼻咽癌病人血清中抗Epstein—Barr病毒IgA/EA抗体

曾毅等建立了一系列检测EB病毒IgA/VCA和IgA/EA抗体的鼻咽癌早期诊断方法,取得了满意的结果。为了进一步提高对鼻咽癌诊断更为特异的IgA/EA抗体的检出率,我们建立了检测EB病毒IgA/EA抗体的蛋白印迹法。方法敏感特异,结果令人满意。 本法中所用的两个质粒系由本实验室与西德Pettenkofer研究所Wolf教授的实验室合作构建。pUCARG1140和pUC9MBcE3.2质粒均为表达质粒,前者携带着来源于EB病毒Bam     

应用明胶颗粒凝集试验检测人免疫缺陷病病毒(HIV-1)抗体

明胶颗粒凝集试验是测定HIV-1抗体的新方法。本研究将明胶颗粒凝集试验与ELISA法、蛋白印迹法和间接免疫荧光试验做了比较,观察本方法的敏感性和特异性。共检测了195份来自法国和非洲象牙海岸的血清,凡是蛋白印迹法阳性的血清,明胶颗粒凝试验都是阳性。这表明本方法是特异和敏感的,方法简便,不需特殊仪器,省时,可用于HIV-1抗体的筛选,但多数蛋白印迹法可疑的血清,明胶颗粒试验均阴性。因此,对蛋白印迹法测出的可疑者应该用数种方法进行追踪检测。     

一种纯化细菌有机磷水解酶的简便方法

Cibacron blue T_3GA与溴化氰活化的Sepharose 4B偶联后,产生一种能有效地分离有机磷水解酶的吸附剂。用0.15mol/L MgCl_2溶液从黄杆菌P3—2细胞抽提出的粗酶液通过柱层析分离,即可得到纯化8倍、酶活性回收率为269.4％的纯酶制品。该酶制品用凝胶电泳测是均一的。     

樗蚕丝腺5SrRNA的核苷酸顺序

本文用凝胶直读法、末端鉴定法等相配合,测定了樗蚕(Philosamia cynthia)絲腺5SrRNA的核苷酸顺序:AGACAACGUCCAUACCACGUUGAAAACACCGGUUCUCGUCCGAUCACCGAAGUCAAGCAACGUCGGGCGCGGUCAGUACUUGGAUGGGUGACCGCCUGGGAACACCGCGUGCUGUUGGCUU比较了樗蚕、蓖麻蚕、柞蚕、家蚕、果蝇等5SrRNA结构差异,在分子水平上探讨了昆虫的分化。     

Expression of c-myc protooncogene in rat lens cells during development,maturation and reversal of galactose cataracts

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.     

Assignment of a novel protein tyrosine phosphatase gene (Hcph) to mouse chromosome 6.

Hematopoietic cell phosphatase (Hcph) was identified by amplification of conserved protein tyrosine phosphatase sequences from a myeloid cell line and is predominantly expressed in hematopoietic cells. Hcph is unique in containing two, tandemly repeated, src-homology 2 domains in the amino terminal region of the phosphatase. Using a genomic probe in interspecific backcross analysis, the murine Hcph gene maps to mouse Chromosome 6 and is tightly linked to the Tnfr-2 and Ly-4 genes.     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

Changes in pulmonary Cu−Zn contents in superior mesenteric artery occlusion shock of rabbit

Contents of Cu and Zn of into-pulmonary blood (IPB), out-pulmonary blood (OPB), Lung tissue, and supernatant and macrophages of Lung Lavage were determined in superior mesenteric artery occlusion (SMAO) shock of rabbits. Zn of pulmonary tissue was 11.42 +/- 0.60 and 14.52 +/- 1.78 (micrograms/g wet wt) in SMAO shock and control groups, respectively. Content of Zn was found to be lower, Cu was not changed, and Cu/Zn ratio increased in lung tissue in SMAO shock. Contents of Cu and Zn in other samples were not changed. The results suggest that lower Zn in lung tissue related to acute lung injury.     

Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products.

Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the homeotic products of the Antennapedia and bithorax complexes. Here the requirements for Pc group genes in controlling the abdA and AbdB products of the bithorax complex are described. Embryos containing mutations in the genes Polycomb (Pc), extra sex combs (esc), Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex comb extra (Sce), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho) were examined. In every case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior (A-P) axis, consistent with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. We suggest that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments.