Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli.

Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes. The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta). Three domains are recognized. The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site. The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit. Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit. Treatment of the transhydrogenase of E. coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity. Analysis of inhibition revealed that several sites on the enzyme were involved. NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase. Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity. Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme. The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit. Reaction of domain 2 and domain 3 was prevented by NADPH. Modification of domain 3 is probably not associated with inhibition of enzyme activity. Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3. The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation. In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed. The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase.     

人粒细胞集落刺激因子受体膜外区在昆虫细胞中的表达

以胎盘组织提取的mRNA为模板,RTPCR扩增人粒细胞集落刺激因子(G-CSF)受体膜外区CRH的cDNA片段,克隆于供体质粒pF_ASTB_AC1,与杆状病毒表达载体Bacmid同源重组后,转染昆虫细胞SF9,获得重组杆状病毒并证明了CRH的高效表达。表达产物经G-CSF亲和层析进一步纯化,纯度可达90%以上。受体竞争性结合实验结果表明,该表达产物能特异性结合G-CSF,具有较高的亲和力(Kd=3.8nmol/L)。     

葡激酶N端突变体的分子设计,高效表达与分离纯化

同源模建人微小纤溶酶原（ｍｉｃｒｏｐｌａｓｍｉｎｏｇｅｎ ,ｍｐｌｇ）的三维结构,建立葡激酶（ｓｔａｐｈｙｌｏｋｉｎａｓｅ,Ｓａｋ）,ｍｐｌｇ计算机分子对接的结构模型,模型与已有的实验结果基本相符。为研究葡激酶Ｎ端结构与功能的关系,进一步改造葡激酶分子,根据复合物结构模型设计了Ｎ端缺失１５个氨基酸的葡激酶突变体。     

Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP.     

考古标本微磨痕初步研究

从周口店第1地点和马鞍山遗址选取了20件燧石制品,以微磨痕的实验研究为基础,以扫描电子显微镜 (简称电镜) 为主要手段,通过对比分析,尝试了不同遗址间考古标本的微磨痕分析。结果表明,周口店第1地点和马鞍山遗址的石制品的功能都具有多样性;“楔” 的功能见于马鞍山遗址,并为周口店第1地点 “使用石片较多” 的说法提供了微磨痕方面的新证据。     

花生种子的磁处理对其萌发及幼苗生长的影响

本试验着重研究了用交、直流磁场对花生种子进行处理后,在种子萌发及幼苗生长过程中产生的影响。结果表明,一定强度的交流或直流磁场对种子的萌发及幼苗生长有一定的促进作用。处理过的种子存放一年后,磁场对种子萌发的作用依然存在。     

Forms and Fates of Rhizobial Bacteria Present in the Infected Host Cells of Nodules

There were two forms of rhizobial bacteria present in infected host cells of nodules. One was bacteroids which were enclosed in peribacteroid membrane originated from the infected host cells. The other was rhizobia as vegetative cells. The infected host cells were occupied by most of the bacteroids and a certain number of the vegetative cells respectively. With the nodule senescence, there were two kinds of fate of the bacteria: The bacteroids degenerated togather with the infected host cells at the same time and further disintegrated completely, so it is not possible that the disintegrated bacteroids could be returned into soil to revive: the vegetative cells did not disintegrate and die when the infected host cells senesced, eventually could be turned back into soil. The vegetative cells may play an important role, on the one hand, in cycle between legume and soil, on the other hand, maintain rhizobia in natural balance of population ecosystem.     

Plantlet Regeneration from Leaf Protoplast cultures of Japanese Butterbur

Protoplasts were isolated by enzyme digestion from leaf of Japanese butterbur (Petasites japonicus). The enzyme incubation mixture consisted of 4% (W/V)cellulase RS, 2% (W/V) hemicellulase, 1% (W/V) pectinase-dissolved Y-23 and polygalacturonase in a solution of 0. 5 mol/L mannitol at pH 5.7 . In the basic medium of 1/4 MS inorganic salts and 1/2 MS vitamins supplemented with 2 mg/L NAA, 0. 2–0. 5 mg/L BA, 0. 5 mol/L mannitol and 10 g/L sucrose, the cells divided luxuriantly. Regenerated plantlets were formed from callus after bud induction and root initiation.     

Studies on the Chemical Constituents of the Spores from Ganoderma lucidum Leyss et Fr,Karst

Two and ten compounds were isplated from the water portion and the ethereal soluble lipophilic fraction of Ganoderma lucidum respectively. On the basis of their chemical properties and co-TLC and IR spectral analysis, the two compounds from the water portion were identified as choline (Ⅰ) and betains (Ⅱ), ten compounds from thereal soluble fraction as tetra- cosanoic acid, stearic acid, palmitic acid, ergosta-7, 22-diene-3β-ol, nonadecanoic acid, benenic acid, tetracosane, hentriacontane, ergosterol and β-sitosterol. In addition, there are two compounds not be purified as contaminated by a small amount of ergosta-7, 22-diene-3β-ol.     

The Isolation and Identification of Phytoecdysones from Dacrydium pierrei Hickel

Three crystals were isolated from the bark of Dacrydium pierrei Hickel and were identified by melting point spectral data (UV, IR, MS, NMR) and GC. Hplc. Crystal Ⅰ is β-ecdysone, Crystal Ⅱ is a jugasterone C. Crystal Ⅲ consists of ponasterone A (ⅢA) and a new phytoecdysone named dacryhainansterone (ⅢB). Total yield of three crystals is 0.4%.