黄精凝集素Ⅱ分子稳定性与生物学活性研究

黄精凝集素Ⅱ分子稳定性与生物学活性研究鲍锦库,曾仲奎,周红（四川大学生物系,成都,６１００６４）本文在黄精凝集素Ⅱ纯化及性质研究的基础上,应用多种变性条件,研究其分子特性,同时对分子的巯基和色氨酸进行修饰,研究该凝集素分子保持其生物学活性与这些基团的...     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

The allometry of algal respiration

For 30 years, study after study has shown that respiration ratesincrease as {small tilde}0.75 of body size for organisms rangingfrom protozoans to mammals. However, a number of studies suggestedthat the respiration-size relationship for algae may be a rareexception to this general rule. Algal respiration may be almostproportional to cell size, such that the slope of the respiration-sizerelationship is closer to unity. The present study examinedthe effect of cell size and taxon on phytoplankton respiration,using data collected from the literature. To this end, we collecteda data set of 178 observations of algal respiration and cellsize representing six divisions-chlorophytes. chrysophytes,cyanophytes, euglenophytes, pyrrophytes and rhodophytes. Therelationship between respiration (R, in p1 O₂ cell^–1 h^–1)and cell carbon content (C, in pg C cell^–1) is describedas R = 0.030C^{0 93} and the exponent is significantly >3/4.When we expressed cell size in terms of volume, the exponentdecreased to 0.88 but this is still significantly >3/4. Amongthe six divisions studied, chlorophytes, euglenophytes and rhodophytesseemed to differ significantly in their respiration-size relationshipfrom other taxa. However, euglenophytes and rhodophytes havesuch small size ranges that no meaningful relationships canbe developed for those groups alone. The chlorophyte respiration-sizerelationship has obvious patterns in its residuals which mayindicate that significant sources of error were not controlledin these heterogeneous data. Thus, for the present, the generalmodel seems most appropriate for the prediction of respirationrates of phytoplankton.     

Process monitoring of acetic acid in Escherichia coli cultivation using electrochemical detection in a flow injection system

A flow-injection system for determination of acetic acid in Escherichia coli cultivations with electrochemical detection based on immobilized acetate kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) was developed to cope with the problems related to measurements under process conditions such as interferences from pyruvate, drift of electrode baseline and making the cultivation medium and reagents compatible. The results obtained by flow injection analysis were compared with those obtained with an enzymatic test kit.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

罗汉果双受精过程的细胞学观察

罗汉果（Ｓｉｒａｉｔｉａｇｒｏｓｖｅｎｏｒｉ（Ｓｗｉｎｇｌｅ）Ｃ．Ｊｅｍｅｙ）双受精过程属有丝分裂前配子融合类型,授粉后２４～４８ｈ,花粉管进入胚囊,穿过一个助细胞,放出两个精子。雌雄核融合和雄核与次生核融合同时发生在授粉后６２～７２,雄核与次生核融合速度快于配子融合,７２ｈ后即可见到初生胚乳核分裂。合子中的雌雄核仁在授粉后第５～６ｄ融合,授粉后８～９ｄ合成分裂形成二细胞胚。在双受精过程中,多次观察到有多条花粉管进入胚囊和多精入极核现象。原胚期有附加花粉管从珠孔进入。     

磷脂模型膜的时间分辨纳秒荧光光谱的研究

叙述了用时间分辨率纳秒荧光技术研究大豆磷脂或合成磷脂模型膜（脂质体）的物理状态变化与荧光寿命以及时间分辨各向异性测量参数变化相关性的实验结果。用荧光探剂ＭＣ５４０标记模型膜的结果表明,荧光寿命（τ）的变化与脂质／探剂比例、磷脂组成、磷脂的极性头部、脂肪酸酰链长度等有关。用ＤＰＨ（１,６－ｄｉｐｈｅｎｙｌ－１,３,５－ｈｅｘａｔｒｉｅｎｅ）标记磷脂模型膜的时间分辨各向异性分析结果表明,序参数（Ｓ）、