不同底物与固氮酶及其钼铁蛋白相互作用的荧光光谱分析

 本文研究了不同底物(N_2,H_2,N_2O,NaN_3,C_2H_2)对棕色固氮菌固氮酶及其钼铁蛋白荧光光谱的影响。结果表明,上述底物均能络合在钼铁蛋白及固氮酶上,但络合程度不同,从而为固氮酶系统有多个不同的底物络合中心,底物络合中心在钼铁蛋白分子上,铁蛋白对钼铁蛋白有变构作用,提供了光谱学证据。     

固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

Regulation of intracellular calcium in epithelial cells

The role of [Ca2+]i as a second messenger in non-excitable cells has been appreciated for almost 3 decades. The advent of fluorescent Ca2+ indicators has allowed the monitoring of Ca2+ signalling in suspensions of these cells. Agonist mediated changes in [Ca2+]i usually show an initial Ca2+ transient followed by a maintained increase. The former has been shown to be due to Ca2+ release from one or more intracellular stores, the latter due to activation of receptor operated Ca2+ entry (ROCE). More recently it has been recognized that many cells show distinct maintained oscillatory behavior when examined by single cell optical methods. It is proposed here that these oscillations are the consequence of IP3 and Ca2+ stimulation of Ca2+ release and ligand activation of ROCE followed by Ca2+ inhibition of Ca2+ and ROCE as Ca2+ pumps are activated. These oscillations allow more exact regulation of a pump/leak controlled second messenger such as [Ca2+]i.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.     

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.     

神经敏感性的降低是棉铃虫对拟除虫菊酯抗药性的重要机制

用扫描电子显微镜和光学显微镜的染色观察,研究了棉铃虫Helicoverpa armigeraHubner 3龄幼虫腹部神经一肌肉的分布,表明其腹纵肌细胞适合于作电生理细胞内记录。用电生理细胞内微电极记录法研究了棉铃虫三个品系：对照品系、功夫菊酯抗性品系(Cy-R,抗性指数13.4)和氰戊菊酯抗性品系(Fn-R,抗性指数37.9)的3龄幼虫神经一肌肉材料对相应杀虫剂的神经敏感性。结果表明：用10^-6mol/L的功夫菊酯处理Cy-R,在30min内有40%的个体对药剂无反应,另外60%的个体产生反应所需的时间是对照品系的3.5倍。用10^-6mol/L的氰戊菊酯处理Fn-R,在30min内有47%的个体对药剂无反应,另外53%的个体产生反应所需的时间是对照品系的4.2倍。这些结果说明神经敏感性降低是棉铃虫对拟除虫菊酯抗药性的重要机制。     

内源植物激素与光敏核不育水稻农垦58S育性的关系

采用气相色谱－质谱联用选择离子检测（ＧＣ－ＭＳ－ＳＩＭ）技术,定性、定量分析了光敏核不育水稻农垦５８Ｓ（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ． ｓｕｂｓｐ． ｊａｐｏｎｉｃａ）及对照品种“农垦５８”在长（ＬＤ）、短（ＳＤ）日照处理下,不同发育时期顶端全展叶片中内源ＩＡＡ、ＡＢＡ 和生殖器官中内源ＩＡＡ、ＡＢＡ、ＧＡ１、ＧＡ４ 的含量变化。实验结果表明：在ＬＤ 处理下,农垦５８Ｓ雌雄蕊形成期的叶片和花粉母细胞形成期的幼穗中ＩＡＡ 相继发生亏缺;农垦５８Ｓ－ＬＤ花粉母细胞形成期、单核期和扬花期,生殖器官中内源ＡＢＡ 均低于农垦５８Ｓ－ＳＤ 的含量,“农垦５８”则与此相反;扬花期不育花药中内源ＧＡ１ 和ＧＡ４ 含量剧减。据此认为：生殖器官中早期ＩＡＡ 的亏损、ＡＢＡ 含量剧减伴随着抗逆性能的减弱及ＧＡＳ的不足共同导致了农垦５８Ｓ的雄性败育