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。     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

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)     

甘蔗叶不同部位ATP酶活性细胞化学定位

甘蔗叶片,叶鞘和肥厚带韧皮部 ATP 酶活性定位于筛管、伴胞的质膜、内质网和某些伴胞细胞基质、小囊泡和发育成熟的液泡上;叶片韧皮部薄壁细胞、厚壁细胞和厚壁通道细胞质膜及小囊泡中亦显示有 ATP 水解产物;维管束鞘细咆与厚壁细胞或厚壁通道细胞所构成的细胞间隙上也存在有 ATP 酶活性反应产物沉淀。甘蔗叶片大、中、小三种维管束,从小维管束到大维管束,面向细胞间隙的细胞表面上的 ATP 酶活性逐渐增强,而维管束鞘细胞质膜上的 ATP 酶活性则趋于减弱;同一维管束内则以韧皮部细胞的 ATP 酶活性最强。维管束鞘细胞与叶肉细胞之间存在很多的胞间连丝,并表现出高的 ATP 酶活性。讨论了 ATP 酶活性的分布状态与叶肉细胞的光合产物向韧皮部运输的关系。     

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.     

短尾猴（Macaca arctoides）和猕猴跟骨的功能形态研究

本文从形态描述和统计入手,对短尾猴(macaca arctoides)和猕猴的跟骨进行了比较研究。结果表明,所研究的跟骨变量无论数值大小还是几何图形结构都存在一定差异。特别是跟骨最大宽、跟长、后距骨连结面长、跟骨高度及相对跟长存在显著性差异水平。猕猴跟骨变量间的相关关系比短尾猴的表现得更为紧密。据其形态与功能的关系,我们认为:与猕猴相较,短尾猴更适应于地栖生活。这似乎与短尾猴具更大的体重有关。     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.