用免疫荧光技术研究细胞周期中核基质的分布变化

本文利用含有抗核基质自发抗体的硬皮人血清,以小鼠艾氏腹水癌细胞为材料,用间接免疫荧光染色的方法,追踪了对应核基质抗原在细胞周期中分布的变化。结果显示,在末期和间期之间存在一个核基质抗原从细胞质向细胞核内转移的过程。由于这一过程是通过核膜进行的,从而提示核基质结构可能有解聚和再聚合的行为。用酶化学结合间接免疫荧光染色的方法,初步研究了抗原的化学性质。染色形态的比较研究显示所用血清中可能含有不同于以前发现的、抗新的核基质抗原的自发抗体。     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

改变乙型肝炎病毒核心抗原基因3′端序列对其在原核细胞中表达的影响

乙型肝炎病毒(HBV)的核心抗原基因(C基因)编码185个氨基酸残基,在原核细胞或痘苗病毒系统中能表达并装配成27nm大小的核心抗原(HBcAg)多聚体颗粒。已证实HBV C基因3′端编码近40个氨基酸的碱基序列,不是表达形成HBcAg颗粒所必需的。用外源基因替换这部分序列,已表达出表面带有外源基因产物的杂合颗粒,它具有很好的免疫原性,成为新型的基因工程多决定簇颗粒载体疫苗。但我们的实验中发现,用另外的外源基因替换3′端序列能显著影响HBV C基因在大肠杆菌中的表达,不同组成的外源基因其影响程度有所不同。     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N-linked carbohydrate-independent mechanism of three lysosomal integral membrane proteins

The biosynthesis, glycosylation, movement through the Golgi system, transport to lysosomes, and turnover of three lysosomal integral membrane proteins (LIMPSs) have been studied in normal rat kidney cells using specific anti-LIMP monoclonal antibodies. Immunoelectron microscopy studies revealed the presence of LIMPs in secondary lysosomes, Golgi cisterna, and coated and uncoated vesicles located in the trans-Golgi cisterna, area. Pulse-chase experiments recorded LIMP precursors of 27 (LIMP I), 72 (LIMP II), and 86 kDa (LIMP III) and mature LIMPs of 35-50 (LIMP I), 74 (LIMP II), and 90-100 kDa (LIMP III). Time course studies on the acquisition of endoglycosidase H resistance by LIMPs indicated that all three LIMPs moved from the site of their synthesis in the endoplasmic reticulum to the medial Golgi within 30-60 min after their synthesis. All three LIMPs were fully glycosylated before leaving the Golgi system, the process during which LIMP I was retained in the trans side of the organelle. LIMP I reached the lysosomes with a halftime of 2 h and LIMPs II and III with half-times of 1 h after their synthesis by a mechanism that was independent of N-linked carbohydrates. LIMPs free of N-linked carbohydrates displayed much shorter half-lives than fully glycosylated LIMPs, suggesting an important role of the sugars in protecting LIMPs against proteolytic degradation. Double immunofluorescence microscopy experiments showed that LIMP I, LIMP II, and LIMP III are localized in the same lysosomes.     

Fluorescence studies of chicken liver fatty acid synthase. Segmental flexibility and distance measurements

The 4'-phosphopantetheine of chicken liver fatty acid synthase was specifically labeled with the fluorescent substrate analog coenzyme A 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoate at low salt concentrations. A serine at the active site of the thioesterase was specifically labeled with the fluorescent compounds 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminopentylmethylphosphono fluoridate and/or pyrenebutyl methylphosphonofluoridate. Dynamic anisotropy measurements indicate the thioesterase has considerable segmental flexibility, whereas the fluorescent labeled 4'-phosphopantetheine does not display detectable local or segmental flexibility. Fluorescence resonance energy transfer measurements indicate that the distance between the fluorescent label at the end of the 4'-phosphopantetheine and NADPH bound to the beta-ketoacyl reductase or enoyl reductase site on the same polypeptide chain is essentially the same, approximately 38 A. The two types of reductases were distinguished by specifically blocking enoyl reductase with pyridoxal 5'-phosphate. No significant energy transfer occurs between sites on different polypeptide chains so that the distances must be greater than 55 A. The distance between the serine on the thioesterase and the 4'-phosphopantetheine on the same polypeptide is 48 A; again no interpolypeptide chain energy transfer was observed. The distance between the serines of the two thioesterases within a fatty acid synthase molecule is greater than 56 A. The monomeric enzyme obtained at 1 degree C does not have beta-ketoacyl synthase and reductase activities. Also fluorescent titrations indicate NADPH is not bound to beta-ketoacyl reductase in monomeric enzyme. The addition of potassium phosphate to the monomers at 1 degree C rapidly dimerizes the enzyme and restores the beta-ketoacyl reductase activity. The beta-ketoacyl synthase activity is slowly restored when the dimer is raised to room temperature. The results obtained suggest that relatively large conformational changes may be part of the catalytic cycle.     

辽宁海城小孤山遗址发掘简报

从小孤山晚更新世洞穴遗址出土了38种哺乳动物化石、上万件石制品以及一批制作精美的骨角制品和装饰品。石器工业在技术与类型上和华北旧石器关系密切。大致相同的骨针和穿孔兽牙于三十年代曾在周口店山顶洞遗址发现过,但是与欧洲马格德林鱼叉相似的角制鱼叉在中国旧石器文化中是头一次发现。     

用PFU法研究微型生物群集过程中数据的处理

根据MacArthur-Wilson的岛屿区系平衡模型S_t=S_(eq)(1-e~(GT)),可以从野外生态效应试验和室内毒性试验中,提出3个功能参数(S_(eq)、G、t_(90％))进行比较。本文提出两种计算方法:复合梯形法和最小二乘法,后者已在计算机上实现了BASIC计算程序。从数学理论上论证,最小二乘法误差较小,但如果实验布局合理,两种计算方法能得到十分一致的结果。实验模型是否符合理论模型,可以用统计学上的拟合差异度检验法来检验。     

Dual action of anti-sporozoite antibodies in vitro

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.