大熊猫基因指纹探针F2ZGP96060801的研制及比较实验分析

利用ＡＢＩ－３９４型ＤＮＡ合成仪合成的寡核苷酸５－「Ａ（Ｘ）ｎ－ｘＴＣＣＡＣ」ｎ－３,经高效液相色谱仪纯化后,制备成了命名为为Ｆ２ＺＧＰ９６０６０８０１的大熊猫基因指纹探针,用同位素标记法标记Ｆ２ＺＧＰ９６０６０８０１,ＬＺＦ－Ｉ、朋５、３３．６和（ＣＡＣ）６／ＧＴＧ）５等５种基因指纹探针,比较检测了大熊猫随机个体的被毛、大熊猫３雄配Ｉ雌所产１个双胞胎计６只个体的福尔马林固定的肝组织和粪便中的胃肠     

大鼠酰胺化酶的信号肽引导的昆虫细胞表达外泌系统

将大鼠酰胺化酶的信号肽及前导肽编码序列引入昆虫核多角体病毒转移表达载体,构建ＰＡＢＣｈＧＲＦ（Ｇｌｙ）、ＰＡＢＣＩＧＦＩ融合基因的昆虫细胞分泌表达质粒ｐＢａｃＰＡＧ２、ｐＢａｃＰＡＩ,并与经修饰的银纹夜蛾核多角体病毒ＢａｃＰＡＫ６线性化ＤＮＡ共转染秋粘虫细胞Ｓｆ２１,通过同源重组、筛选和鉴定,得到它们的重组病毒ＢａｃＰＡＧ、ＢａｃＰＡＩ。将重组病毒感染Ｓｆ２１细胞,ＰＡＢＣｈＧＲＦ（Ｇｌｙ）和ＰＡＢＣＩＧＦＩ均得到有效外泌表达,表达产物通过ＩｇＧＳｅｐｈａｒｏｓｅ柱可获得快速纯化。     

重组I型cAMP依赖的蛋白激酶调节亚基的表达,纯化和鉴定

Ｉ型蛋白激酶的调节亚基（ＲＩ）具有两个ｃＡＭＰ结合位点,对ｃＡＭＰ具有很高亲和力和特异性,我们从人神经细胞中克隆人ＲＩ亚基ｃＤＮＡ片段（编码氨基酸残基９３－３８１）并将其亚克隆至ｐＥＴ３０ａ原核表达载体,实验表明该表达质粒在大肠杆菌ＢＬ２１中,在ＩＰＴＧ诱导下,表达产生大量带聚组氨酸标记的重组ＲＩ亚基。这些蛋白质以可溶性蛋白形式存在,经组氨酸结合金属螯合树脂亲和柱层析纯化后,每０．１升培养菌可制备     

Hib在调理过程中补体成分C_3的外膜蛋白结合位点

Ｈｉｂ在调理过程中补体成分Ｃ_３的外膜蛋白结合位点用从正常人血清（ＮＨＳ）分离出的不同浓度的ＩｇＧ抗体调理Ｉ（１２５）标记的ｂ型流感嗜血杆菌（Ｈｉｂ）菌体,分离纯化出ＩｇＧ－外膜蛋白（ＯＭＰ）复合物。用ＳＤＳ－ＰＡＧＥ和放射免疫沉淀试验进行分析,结果...     

青海高原不同海拔地带生长的珠芽蓼光合特性的比较

对生长于青海高原不同海拔的珠芽蓼（Ｐｏｌｉｇｏｎｕｍｖｉｖｉｐａｒｕｍ）叶片的光合特性进行比较研究发现：随海拔升高叶绿素含量有降低趋势,叶绿素ａ／ｂ比值及类胡萝卜素含量有增大趋势。不同海拔的珠芽蓼叶片和叶绿体的Ｆｖ／Ｆｏ、Ｆｖ／Ｆｍ和Ｒｆｄ值随海拔升高而增大,表明随海拔升高,其潜在光合活力增加。低温荧光测定显示,随海拔升高叶绿体中ＰＳＩ和ＰＳＩ的相对荧光产量比值（ＰＳＩ／ＰＳＩＩ）减小。叶绿体蛋白质ＳＤＳ－ＰＡＧＥ结果表明,在２８－３２ＫＤ附近电泳谱带的变化显著。     

PAF对肺内小动脉平滑肌细胞TXA_2，PGI_2乃Ca~（2＋）－ATPase的影响

本工作采用分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭＣｓ），观察了外源性血小板活化因子（ｐｌａｔｅｌｅｔａｃｔｉｖａｔｉｎｇｆａｃｔｏｒ，ＰＡＦ）、ＢＮ５２０２１（ＰＡＦ受体拮抗剂）、吲哚美辛、维拉帕米对ＰＡＳＭＣｓ产生血栓素Ａ_２（ＴｘＡ_２）、前列环素（ＰＧＩ_２）及对细胞膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的影响。结果表明：（１）基础状态下ＰＡＳＭＣｓ存在花生四烯酸（ＡＡ）代谢。（２）外源性ＰＡＦ通过受体后途径激活环加氧酶促进ＡＡ代谢致ＴＸＡ_２及ＰＧＩ－２增加，ＴＸＡ_２／ＰＧＩ_２比值无明显变化。（３）外源性ＰＡＦ能直接抑制Ｃａ~（２＋）－ＡＴＰａｓｅ活力。（４）维拉帕米可逆转ＰＡＦ抑制ＰＡＳＭＣｓ膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的效应。     

9—蒽羧酸对豚鼠心室动作电位和L型Ca电流的影响

目的：研究９－蒽羧酶（９－ＡＣ）对豚鼠以肌动作电位（ＡＰ）和Ｌ型Ｃａ电流（Ｌｃａ）的影响。方法：电流钳配合制霉菌素膜等孔方法记录心室肌动作电位,用全细胞式膜片钳（Ｗｈｏｌｅ ｃｅｌｌ ｒｅｃｏｒｄｉｎｇ）技术记录Ｌｃａ。结果：在低ＣＩ＾－状态下,β肾上腺素能受体激动剂异丙肾上腺素（ＩＳＯ）可使动作电位时程（ＡＰＤ）明显延长。９－ＡＣ单独使用时对ＡＰ无作用,但在ＩＳＯ的作用下,蛋白磷酸酶抑制剂９－Ａ     

迷路损伤增强大鼠前庭内侧核生长相关蛋白mRNA水平

前庭代偿是研究神经可塑性的一个理想模型。生长相关蛋白（ＧＡＰ－４３）在神经再生和突触重组中起重要作用。用ＤＩＧ标记的ＧＡＰ－４３ ｃＤＮＡ片段作探针进行原位杂交,检测了大鼠迷路损伤５、１２、２０和３０ｄ后前庭内侧核ＧＡＰ－ｍＲＮＡ表达的变化。结果表明,迷路损毁后两侧前庭内侧核ＧＡＰ－４３ｍＲＮＡ的水平以不同的幅度和时程明显升高。这一结果表示,ＧＡＰ－４３ｍＲＮＡ水平的提高可能与前庭代偿中突触重组和     

5-HT和NA增强大鼠骶髓后连合核神经元甘氨酸门控Cl~-通道电流由蛋白激酶介导

用制霉菌素穿孔膜片钳方法研究５－ＨＴ和ＮＡ对急性分离的大鼠骶髓后连合核神经元甘氨酸门控氯离子通道电流（ＩＧｌｙ）的调控作用及其胞内机制。发现：（１）５－ＨＴ激活与非胰岛激活蛋白（ＩＡＰ）敏感型Ｇ蛋白偶联的５－ＨＴ２受体亚型,激活磷脂酶Ｃ（ＰＬＣ）,增加甘油二酯（ＤＡＧ）的生成。ＤＡＧ增强不依赖Ｃａ２＋的新型ＰＫＣ（ｎＰＫＣ）的活性,从而增强ＩＧｌｙ;（２）ＮＡ激活与ＩＡＰ敏感型Ｇ蛋白偶联的α２受体,抑制腺苷酸环化酶（ＡＣ）,减少ｃＡＭＰ的生成,使ＰＫＡ活性降低,从而增强ＩＧｌｙ。     

应用荧光原位杂交和RFLP标记检测多小穗小麦新种质10—A中的黑麦染 …

利用ＡＰＡＧＥ,荧光原位杂交技术和ＲＦＬＰ标记,对导入黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）多小穗等性状创制的小麦新种质１０－Ａ进行了分子标记检测,ＡＰＡＧＥ分析发现,１０－Ａ与其他１ＲＳ／１ＢＬ易位系一样,含有１ＲＳ的醇溶蛋白标记位点Ｇｌｄ１Ｂ３。以黑麦基因组总ＤＮＡ作探针,用中国春（Ｔｒｉｔｉｃｕｍａｅｓｔｉｕｍｃｖ．ＣｈｉｎｅｓｅＳｐｒｉｎｇ）基因组ＤＮＡ作封阻,与１０－Ａ根尖细胞有丝分裂染     

Activation of endogenous phosphorylase kinase in liver glycogen pellet by cAMP-dependent protein kinase

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.     

The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein beta subunit, GPB1, was cloned and disrupted. gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATalpha and MATa cells. The functions of GPB1 are distinct from those of the Galpha protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence. gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show that MATa cells stimulate monokaryotic fruiting of MATalpha cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gbeta subunit GPB1 and the Galpha subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

A spectrophotometric study of vitamin B2 and its coenzyme forms binding to muscle glycogen phosphorylase b

The vitamin B2 and its coenzyme forms binding to glycogen phosphorylase b from rabbit skeletal muscle has been studied by the spectrophotometric method. The spectral properties of riboflavin, FMN and FAD bound to muscle glycogen phosphorylase b were found to be identical at the wavelengths of 300 to 500 nm. According to data on spectrophotometric titration of muscle glycogen phosphorylase b by FMN, each subunit of the enzyme contains one flavin-binding site.     

Interaction of muscle glycogen phosphorylase B with methotrexate, folic and folinic acids

The interaction of rabbit skeletal muscle glycogen phosphorylase b with methotrexate, folic and folinic acids has been studied. Microscopic dissociation constant for the glycogen phosphorylase b--methotrexate complex determined by analytical ultracentrifugation is 0.43 mM. A subunit of glycogen phosphorylase b is shown to have two sites for methotrexate binding. AMP and FMN diminish the affinity of glycogen phosphorylase b to methotrexate, whereas glycogen does not influence the methotrexate binding to the enzyme. Methotrexate, folic and folinic acids are found to be inhibitors of the muscle glycogen phosphorylase b. The inhibition is reversible and characterized by positive kinetic cooperativity (the Hill coefficient exceeds one unity). The value of the pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: folic acid (0.65 mM), methotrexate (1.01 mM), folinic acid (3.7 mM). The antagonism between methotrexate, folic and folinic acids, on the one hand, and AMP and FMN, on the other, is revealed for their combined action.     

Insulin stimulates dephosphorylation of phosphorylase in rat epitrochlearis muscles

We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Pi to label cellular phosphoproteins, before being treated with hormones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control and insulin on phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the beta subunit of phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase alpha subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in phosphorylase and phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.     

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.     

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.     

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5)

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.