转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

山西朔县种子植物区系及其生态经济意义

朔县(现朔州市朔城区)植物种属相对丰富,具有维管束植物938种,隶属于420属,105科。其植物区系主要起源于古北大陆;种子植物属的地理成分复杂多样,以温带成分占绝对优势,具有典型的温带草原性质;与相邻地区植物区系的比较分析表明,联系最为密切的是蒙古草原植物区系,其次是黄土高原植物区系。该县植物资源利用应采取的对策是:既要合理开发利用,又要加强物种的保护。     

甲肝病毒在人血白细胞中体外连续传代和在分类白细胞中增殖的研究

1985年我们采用间接免疫荧光法(IF法)检测出甲肝患者外周血白细胞中有甲肝抗原(HAAg)存在,继而又将HAAg阳性白细胞直接种入PLC/PRF/5细胞,分离到两株甲肝病毒(HAV)NJ—3株和H—1株。为了弄清白细胞所携带的病毒究竟仅为吸附吞饮,抑或能在其中复制增殖,我们将分离到的HAV用正常人血白细胞进行体外增殖试验,现将结果报告如下。     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

Ligation activity of FLP recombinase. The strand ligation activity of a site-specific recombinase using an activated DNA substrate.

The FLP protein of the 2-microns plasmid of yeast belongs to the integrase family of site-specific recombinases whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. We have made an activated DNA substrate and have shown that FLP can promote efficient strand ligation without forming a covalent intermediate with the DNA substrate. The strand ligation activity of FLP is independent of its ability to cleave DNA. Since site-specific recombinases are members of the larger class of topoisomerases, these findings may be generally applicable to other members of this class of enzymes.     

Physiological aspects of l-lysine production: effect of nutritional limitations on a producing strain of Corynebacterium glutamicum

The effect of nutritional limitations, such as phosphorus and carbon, on the production of l-lysine by Corynebacterium glutamicum was studied in continuous culture. We observed that phosphate-limited cultures at low growth rates were favourable to l-lysine production. l-Lysine was produced when a culture at low dilution rate (0.03 h^–1) was established. A dilution rate of about 0.04 h^–1 should be maintained in order to assure good productivity and an l-lysine yield of 0.53 g/g. Under carbon-limiting conditions the maintenance energy and growth yield of 0.03 g/g·g^–1·h^–1 and 0.41 g/g, respectively, have been obtained. Under these limiting conditions the l-lysine production was not favoured even at lower dilution rates.Correspondence to: N. Coello     

Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters.

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.     

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.