用免疫荧光单克隆抗体对脊髓灰质炎病毒抗原表位的分析

用间接免疫荧光与中和试验筛选出来的抗脊髓灰质炎2型与3型不同毒株的12个单克隆抗体,其中3个仅有免疫荧光活性,9个具有中和与免疫荧光活性。用免疫荧光活性的单克隆抗体进行试验,发现它们在识别特异性抗原表位方面与中和性单克隆抗体相似,显示出株特异的、几个毒株共同特异的或型特异的抗原表位。根据表位分布关系及特征,可以用来鉴别型内毒株的特征、毒株的抗原分析、抗原变异的研究以及疫苗相关病例的鉴别。所得结果与中和性单克抗隆抗体及T1-寡核苷酸指纹图谱分析一致。而免疫荧光单克隆抗体识别抗原表位的活性范围比中和性单克隆抗体更广。另外还发现某些兼有荧光与中和两活性的同一个单克隆抗体,用不同方法(IF与NT)进行试验时,与相同毒株出现不同表位反应,这点是值得引起注意需待进一步证实的重要问题。     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Effect of hyperlipidemic serum on lipid peroxidation, synthesis of prostacyclin and thromboxane by cultured endothelial cells: protective effect of antioxidants

An increased lipid peroxides and a decreased production of prostacyclin have been shown in advanced atherosclerotic lesions and plasma. Our purpose was to determine whether the similar findings could be observed in cultured endothelial cells, and whether antioxidants could protect the cell against peroxide injury. In these experiments we have used bovine aortic endothelial cells in culture to address the issue of hyperlipidemia-induced arterial damage. Results of the present study showed that different concentration of hyperlipidemic sera from atherogenic rabbits induced a time- and dose-dependent alteration in the production of prostacyclin and levels of lipid peroxides in endothelial cells. Endothelial cells incubated with hyperlipidemic serum increased prostacyclin generation significantly during the initial stages and then continuously decreased. When endothelial cells were incubated for 36 h, TXA2 generation was also impaired and at the same time the cellular lipid peroxides content increased. There was a positive correlation between the concentration of hyperlipidemic serum and lipid peroxides and an inverse correlation with prostacyclin synthesis. The medium supplemented with antioxidant selenium or vitamin E showed a significant decrease in lipid peroxides and an increase in prostacyclin synthesis. These results suggest that both hyperlipidemic serum and lipid peroxides injury endothelial cells and inactivate prostacyclin synthetase, resulting in a decrease of prostacyclin production, while antioxidants have a protective effect. We conclude that the increase in lipid peroxides in association with hyperlipidemia results in alteration of prostacyclin synthesis that may play an important role in the pathogenesis of atherosclerosis.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Apolipoprotein E localization in rat hepatocytes by immunogold labeling of cryothin sections

The distribution of apolipoprotein (apo) E in rat hepatocytes was investigated with an affinity-purified polyclonal antibody raised against apoE isolated from hepatogeneous very low density lipoproteins (VLDL). The distribution of this antibody was visualized with colloidal gold complexed to anti-rabbit IgG. By epipolarization microscopy, apoE was found uniformly along the basolateral surfaces of all hepatic parenchymal cells, showing a striking intensity along the sinusoidal front. Punctate deposits of colloidal gold appeared to be randomly distributed within all hepatocytes. Widely scattered Kupffer cells also stained for apoE. Electron microscopic examination of immunogold-labeled cryothin sections showed that hepatocytic microvilli projecting into the space of Disse consistently contained clusters of immunogold. The gold particles were variably associated with evident lipoprotein particles, raising the possibility that apoE alone may bind to receptors or other macromolecules at the surface of hepatocytes. Endosomes near the sinusoidal front and multivesicular bodies in the Golgi/biliary area labeled intensely for apoE, consistent with a high content of apoE associated with triglyceride-rich lipoprotein remnants contained within these organelles. Some but not all nascent VLDL particles within putative forming Golgi secretory vesicles were labeled, but many other Golgi vesicles and cisternae that lacked evident VLDL particles were also labeled. These results suggest that at least some apoE associates with nascent VLDL in forming Golgi secretory vesicles. Unexpectedly, the matrix of all hepatocytic peroxisomes was heavily labeled. Immunoblots with the affinity-purified anti-rat apoE IgG against proteins from highly purified peroxisomes isolated from rat hepatocytes revealed a protein with an apparent molecular mass of 34.5 kDa, similar to that of rat apoE in rat blood plasma. In addition, gold was sometimes found in the area either adjacent to peroxisomes or between multivesicular bodies and the bile canaliculus not evidently associated with a membranous compartment. These observations suggest that apoE may participate in interorganellar cholesterol transport within hepatocytes.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.     

轴浆转运在神经再生中的作用

夹伤坐骨神经阻断标记蛋白在轴浆中的快、慢转运。3天后转运再现。第14天的转运距离与对照相似,说明再生神经的转运动能基本恢复。用快、慢转运测出的14天平均再生速度分别为1.77±0.14与1.96±0.07mm/d,比对照神经的正常生长速度快6.3—7倍,提示再生需要更多的转运物质。进一步发现再生神经中某些标记蛋白(慢转运波W1)的转运速度为10.25±0.66mm/d,约比对照快1倍,因此这些标记蛋白可能包含适应再生需要而加速转运的结构和功能物质。