Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.     

DNA translocation by the restriction enzyme from E. coli K

The restriction endonuclease Eco K binds to a host specificity site and then proceeds to cleave the DNA at sites that may to several thousand bases away. It does this by translocating the DNA past the enzyme in an ATP-dependent reaction that results in the formation of highly twisted loop intermediates. DNA cleavage can occur on either side of the host specificity site.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

Metabolism of prostaglandins in spontaneously hypertensive rats: NAD"-dependent 15-hydroxyprostaglandin dehydrogenase activity is decreased in kidney and increased in lung.

Renal, pulmonary and gastric NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase activities were determined in both spontaneously hypertensive and normotensive rats at 6 and 12 weeks of age. Renal enzyme activity in hypertensive rats was only 30–40% of that present in normotensive controls at both ages. In contract, pulmonary enzyme activity in hypertensive animals was twice as active as that in normal controls. There was no significant difference in gastric enzyme activity. NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase activity, the enzyme responsible for the conversion of vasoinactive PGF metabolites to PGE metabolites, also failed to show any difference in two types of rat kidneys. The results indicate that, in hypertension, prostaglandin inactivation is impaired in kidney but is facilitated in lung.     

Preparation of new ω-aldehydroalkyl 1-thio-d-glycopyranosides,and their coupling to bovine serum albumin by reductive alkylation

Synthesis of two ω-aldehydoalkyl 1-thioglycosides of d-glucopyranose and of d-galactopyranose is described. 3-Oxopropyl and 2-oxoethyl 1-thioglycosides were prepared by treating a tetra-O-acetyl-1-thioaldose with either acrolein or 2-bromoacetaldehyde, followed by O-deacetylation under mild conditions. These ω-aldehydoalkyl 1-thioglycosides were successfully attached to bovine serum albumin (BSA) by reductive alkylation as described previously. With the 3-oxopropyl 1-thioglycosides, much higher levels of sugar attachment (e.g., ～80 mol of sugar per mol of BSA) were attained than hitherto possible with any sugar derivative tested.     

应用气-质联用仪测定三种螺旋藻中的甾醇成分

应用GLC/MS联用仪对室内培养的钝顶螺旋藻(Spirulina platensis (Nordstedt) Geitler)、极大螺旋藻(S.maxima (Stechell & Gardiner) Geitler)和盐泽螺旋藻(S.subsalsa Oerst)的甾醇成分进行了测定。从钝顶螺旋藻和盐泽螺旋藻中共分出11个相同的甾醇组分:胆甾醇、胆甾烷醇、芸苔甾醇、麦角甾醇、海绵甾醇、菜子甾醇、豆甾醇、24-乙基-Δ~(5,7,22)-胆甾醇、β-谷甾醇、异岩藻甾醇和4α,23,24-三甲基Δ~(5,22)-胆甾醇;从极大螺旋藻中只分离出8个甾醇组分。其中胆甾醇含量最高。4α,23,24-三甲基-Δ~(5,22)-胆甾醇为蓝藻中首次报导。     

人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。     

用固定化方法研究超氧化物歧化酶的活性与结构的关系

 采用吸附、包埋、共价交联等方法固定化超氧化物歧化酶(SOD)所得固定化酶活力回收都不高,表明酶的催化反应速率受超氧阴离子自由基(O_2~-)扩散速率的控制。用海藻酸钠包埋SOD,固定化酶不表现活力,破固定化后所得的糊状物却有很高的活力。用戊二醛交联所得的固定化酶活力回收率也很低,表明ε-NH_3~+的正电荷是酶活力所必需。     

根霉葡萄糖淀粉酶的复性复活动力学研究

本文利用荧光、紫外差光谱研究了根霉葡萄糖淀粉酶在盐酸胍变性后的复性、复活动力学。结果表明,该酶在小于4mol/L盐酸胍中变性是可逆的,其复性过程遵循一级反应方程。酶复活过程是由两个一级反应组成的复合反应,构象变化速度与复活过程中较快的反应速度相差无几,这可能是在Trp及Tyr微区的构象变化基本完成之后,酶活力恢复还没有完成造成的。     

Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch"

The family of mammalian extracellular matrix metalloproteases (MMPs) are secreted by cells in an inactive (latent) proenzyme form. A highly conserved amino acid sequence, PRCGVPDV, is found near the COOH-terminal end of the pro-domain of these MMPs and believed to act as an "autoinhibitor." Recent studies (Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and Wart, H. E. V. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 364-368) indicate the Cys of this sequence ligands to the active-site zinc keeping the proenzyme in an inactive state, and mutational analysis (Sanchez-Lopez, R., Nicholson, R., Gesnel, M. C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899) suggests that the conserved residues surrounding this Cys are required for latency. We have constructed 16 new site-directed mutations of the PRCGVPDV autoinhibitor region of the MMP transin (rat stromelysin) and tested whether these mutant enzymes are produced in a latent or activated form. We find that the conserved Arg as well as the Cys are essential for maintaining latency. The Cys cannot be replaced by other zinc-liganding amino acids, and the Arg cannot be replaced by Lys. Residues immediately surrounding the Cys are sensitive to even conservative amino acid substitutions. We show that a synthetic peptide PRCGVPDV is capable of acting as a weak inhibitor of transin and that replacement of the Cys with a Ser abolishes inhibition by the peptide. A review of the current knowledge of MMP substrate specificity in combination with these new results suggests that the PRCGVPDV sequence does not inhibit activity by mimicking the known substrates of the protease.