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。     

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.     

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.     

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。     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-activeδ-endotoxin gene of Bacillus thuringiensis aizawai 7.29

The nucleotide sequence of a 2711bp DNA segment which contains the N-terminal coding sequence and the 5' flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino-acid polypeptide corresponding to a carboxy-terminally truncated delta-endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran-active delta-endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N-terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran- and coleopteran-active delta-endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran-active genes. Deletions of the 3' region of the gene were carried out and the toxic fraction of the bta delta-endotoxin was identified with the N-terminal half of the molecule.     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.