在吲哚乙酸不同位点偶联载体蛋白对其抗体特异性的影响

分别选择吲哚乙醇分子上的Ｃ１位羧基和吲哚环上的Ｎ位作为偶联载体蛋白的位点,用混合酸酐法和甲醛搭桥法分别合成了两种免疫原ＩＡＡ—ＣＯ—ＮＨ一ＨＳＡ和ＩＡＡ－Ｎ－ＢＳＡ,并进而制得了对吲哚乙酸侧链识别能力不同的两种多克隆抗体,分别可特异识别甲酯化ＩＡＡ和游离态ＩＡＡ;用碳化二亚胺法和甲醛搭桥法分别合成ＩＡＡ—ＣＯ—ＮＨ－ＢＳＡｂＩＡＡ—Ｎ—ＯＶＡ两种复合物,以之为包被物,建立了两种ＩＡＡＥＬＩＳＡ。其灵敏度分别为０．３５ｐｍｏｌ和１．８０ｐｐｍｏｌ;检测范围分别为０．７８～８００ｐｍｏｌ和１．９５～２０００ｐｍｏｌ;批内变异系数分别为４．４５％和４．７９％;批间变异系数分别为１．１５％和１．５０％。笔者用这两种ＥＬＩＳＡ检测了兰花气生根和桑树苗样品中ＩＡＡ的含量,发现两种检测结果相当一致。     

LDL受体基因cDNA的RT－PCR分离、克隆及其在RFLP中的应用

利用ＲＴ－ＰＣＲ技术分离低密度脂蛋白受体基因ｃＤＮＡ,将长为２６０５ｂｐ的ｃＤＮＡ插入质粒ｐＪＮ６,并经全序列测定证实,该序列与已报道序列相比,存在两个变异碱基,即第７５４位Ｃ→Ｔ,和１６５４位的Ｇ→Ａ,这两个变异碱基并不改变编码的氨基酸,利用ＬＤＬ受体基因ｃＤＮＡ中的ＣｌａⅠ片段作为探针,检测到ＬＤＬ受体基因上外显子８的ＳｔｕⅠ位点是个限制性片段长度多态性位点。所克隆的ｃＤＮＡ含有可译框架的全部密码,因此可作为基因表达材料。     

水稻巯基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂（ＣＰＩ）经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达７２μｇ时,即可产生明显的抑制作用。     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.     

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43_m. Cx43_m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all ³²P_i from Cx43_m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.     

Esophageal PCO2 as a monitor of perfusion failure during hemorrhagic shock

Sato, Yoji, Max Harry Weil, Wanchun Tang, Shijie Sun,Jianlin Xie, Joe Bisera, and Hidehiro Hosaka. EsophagealPCO₂ as a monitor of perfusionfailure during hemorrhagic shock. J. Appl.Physiol. 82(2): 558-562, 1997.Measurement ofgastric wall PCO₂(Pg_CO2) bytonometric method has emerged as an attractive option for estimatingvisceral perfusion during circulatory shock. However, gastric acidsecretion obfuscates the tonometric measurement. We, therefore,investigated the option of measuringPCO₂ in the esophagus to minimizethese restraints. Hemorrhagic shock was induced in five Sprague-Dawleyrats, and five rats served as sham controls.Pg_CO2 wasmeasured with an ion-sensitive field effect transistor that wassurgically implanted into the gastric wall. Esophageal luminalPCO₂(Pe_CO2) wasmeasured by a second ion-sensitive field effect transistor sensor.During hemorrhagic shock, mean aortic pressure declined from 150 to 50 mmHg. Gastric blood flow decreased from 58 to 12 ml ^· min^{1 ·} 100 g¹ (21% of preshock) andesophageal blood flow from 44 to 7 ml ^· min^{1 ·} 100 g¹ (16% of preshock).Pg_CO2simultaneously increased from 47 to 116 Torr andPe_CO2 from 47 to 127 Torr. The increases inPg_CO2 werehighly correlated with increases inPe_CO2(r = 0.90). Esophageal tonometry may,therefore, serve as a practical alternative to gastric tonometry.

     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.