Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity

Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin.     

睾丸间质细胞—研究自体吞噬的一种正常细胞模型

In the present study, we tried to estimate, in a semiquantitative way, the relative frequency of the autophagic activity in various cell types under physiological condition. The results indicated that the highest activity appeared to be in the Leydig cells of rat testes. Autophagosomes were frequently observed in electron microscope photographs of Leydig cells, which provide a good model to study the autophagocytosis in normal cells. The autophagic process in Leydig cells was observed with the electron microscope in preparations treated to show CMPase activity. The mode of formation of autophagosomes in Leydig cells can be divided into three steps. Step 1, flattened membranous elements expand to enclose a small cytoplasmic territory to form pre-autophagosome. Step 2, The double membrane profile of the pre-autophagosome then completely encloses the cytoplasmic territory to form early autophagosome in which structurally normal organelles are contained. Step 3, the transformation of an early autophagosome into a late one is accompanied by the loss of one of the two delimiting membranes, the partial disintegration of the enclosed content and simultaneous acquisition of acid phosphatase activity. The enzymatic reactivity is acquired following a close association with the lysosomes. The late autophagosome then reaches the cell surface and appear to exocytose their residual content.     

A procedure for the construction of linear restriction fragment maps of a 50- to 100-kb DNA.

A simple and effective procedure for the construction of linear restriction fragment maps was developed. Using a two-enzyme digestion, two-dimensional (2-D) electrophoresis procedure, all the restriction fragments in a 50- to 100-kb DNA can be individually resolved and displayed on a 2-D plane. This 2-D gel pattern, with appropriate markers, provides a fixed set of x, y coordinates for each fragment obtained from the single and double digestion as well as the relationship between the two steps. A matrix is constructed from the 2-D pattern. The vertical column shows all the singly digested individual fragments and their sizes obtained from each restriction enzyme treatment, and the dividing horizontal row shows all the doubly digested DNA fragments and their sizes after treatment with two enzymes. The order of arrangement is always from the smallest to the largest fragments. Using this matrix, two linear DNA restriction maps for these two enzymes can be simultaneously constructed in a self-reconfirming manner. As examples for this procedure, we describe the construction of two linear restriction fragment maps, a combination of EcoRI and BamHI digestion as well as a combination of EcoRI and HindIII digestion of lambda-phage DNA.     

Stable Defined Substrate for Turbidimetric Assay of Endoxylanases

A stable xylan suspension was prepared and characterized. Hydrolysis of the particles converts them into soluble fragments, thereby lowering the turbidity of the suspension. The small volume of the assay mixture, the short incubation time required, and the simplicity of the procedure permit the rapid analysis of many samples. Furthermore, the procedure can be used to assay xylanase activities in the presence of other reducing materials and is also useful for monitoring low-level xylanase activities.     

Intervention trial with selenium for the prevention of lung cancer among tin miners in Yunnan,China

This pilot study evaluated the feasibility and effectiveness of conducting a double-blind clinical trial for the prevention of lung cancer with selenium (Se) in Yunnan Tin Corporation, the People's Republic of China, where the incidence rates of lung cancer are extraordinarily high among the miners. Forty healthy miners were randomized to either 300 μg of Se in high Se malt cakes or an identical placebo of malt cakes daily for one year. Subjects consumed their usual daily diet. The low Se concentrations in plasma (0.05±0.008 μg/mL) and hair (0.442±0.085 μg/g) reflected their low dietary Se intake in the control subjects. In Se-supplemented group, the Se status was increased by 178% for serum and 194.8% for hair. The serum GSHpx activity was increased by 155.7%, whereas the lipid peroxide level was reduced by 74.5% compared to the placebo. The results of UDS assay indicated that the lymphocyte DNA damage induced by ultraviolet irradiation and carcinogen 3,4-benzpyrene could be protected by Se supplementation. Se-supplementation did not affect the liver function test (SGPT), as well as the concentrations of hemoglobin, albumin, and cholesterol. Thus, daily intake of 300 μg Se in form of Se-malt as a chemopreventive measure is safe and effective to humans with low Se status.     

应用纯化的重组Epstein-Barr病毒早期抗原建立检测鼻咽癌病人血清IgA/EA抗体的ELISA方法

利用凝胶和离子交换柱(Mono Q)两次层析,将大肠杆菌表达的EB病毒早期抗原P138片段多肽纯化。以此P138为抗原,增加鼠抗人IgA单克隆抗体以扩大IgA的反应,建立了三步ELISA法。用本法检查了100例鼻咽癌病人和63例正常人血清中抗EB病毒IgA/EA抗体,病人血清的阳性检出率为86％,正常人有3例阳性(4.7％)。此结果表明,三步ELISA法较常用的间接ELISA法(阳性检出率为71％)敏感。     

外源脂肪酸及胆固醇对莱氏衣原体膜糖脂含量的影响

 <正> 单葡萄糖甘油二酯(MGDG)和双葡萄糖甘油二酯(DGDG)是莱氏衣原体膜上主要的极性脂。糖脂的生理功能过去很少研究,至今仍不清楚。近年来实验结果表明,MGDG在膜上容易形成六角形Ⅱ结构,而DGDG则形成脂双层结构。六角形Ⅱ结构的出现与生物膜的生理功能的关系是当前瞩目的研究内容。本文首次研究了外源脂肪酸和胆固酵对莱氏衣原体AIH089菌株膜上糖脂含量的影响。