Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Development of immunoassay methods by use of liposomes

In order to develop liposomes for use in an immunoassay system, the preparation of immune liposomes and their characterization have been investigated. Liposomes have potential use in extremely sensitive analytical immunoassays, in addition to serving as an attractive drug delivery system. This liposome immunoassay system is based on membrane immunochemistry and an enzymatic reaction. An intense yellow color, easily detectable with the naked eye, was produced quite rapidly by the lysis of bovine serum albumin (BSA)-labeled, alkaline phosphatase-entrapped liposomes in the presence of anti-BSA rabbit serum and active complement under alkaline conditions. Sensitive detection is possible because of the antigen-antibody complex reaction, which leads to liposome lysis and an enzymatic reaction. The liposome immunoassay method offers a rapid, simple, and sensitive testing procedure which can quantitatively and qualitatively determine the presence or absence of antigenic materials and antibodies.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.     

季节、环境温度与黄鼠冬眠的关系

观察了达乌尔黄鼠在实验室内冬眠的一般情况。常温黄鼠的体温有着规律性的年周期,与环境温度的年周期变动不完全呈依从关系。出眠初期(4月下旬),动物体温高而稳定。4月至6月常温黄鼠的平均体温(皮温)为35.6℃,波动菹围32—37.5℃。随着体重达到顶峰,体温逐渐降低。8月份部分黄鼠出现低于32℃的低常体温,表明部分黄鼠自8月盛夏开始冬眠。但就整个种群而言,北京地区实验室内黄鼠冬眠季自9月下半月开始。3月底止,共6.5个月。秋季室温下降,动物入眠趋势增长。浅低体温(31.9—15℃)的比数逐渐升高。9月至12月,低体温(低于31.9℃)的百分比从47％增至84.8％,反映了动物从浅冬眠向深冬眠过渡。1月至2月份,低体温占85％以上,深低体温(低于15℃)占绝对优势。标志着动物种群的深眠月份。秋季动物从常温期向冬眠期转化的界线是不清的,而春季从冬眠期向常温相转化的界限却比较明显。     

记辽宁东部新鳞齿鱼属一新种

本文记述了产自辽宁东部红庙子盆地下桦皮甸子组的新鳞齿鱼属—新种——Neolepidotes liaodongensis sp. nov..根据新材料,将 Neolepidotes 与 Lepidotes 等属作了补充比较,增订了新鳞齿鱼属的特征.     

E.p.r.-spectroscopic studies on the molybdenum-iron site of nitrogenase from Clostridium pasteurianum.

The e.p.r. spectroscopy of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum was re-investigated. The sharpness of the delta Ms = +/- 3 g'z peak from the +/- 3/2 Kramer's doublet enables the observation and quantification of incompletely resolved hyperfine splittings from the stable magnetic nuclei 95Mo and 57Fe in samples enriched in these isotopes. No couplings to 1H or 17O could be discerned by examination of spectra from samples exchanged into 2H2O and H2(17)O respectively. Simulation of the spectrum from 95Mo-enriched samples yields a hyperfine coupling of 2.9 MHz, and indicates that the earlier electron-nuclear-double-resonance-derived estimate of 8.1 +/- 0.2 MHz is substantially in error.     

Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma

A cell line was established from an in situ squamous cell carcinoma of the skin (Bowen's disease), and its in vitro karyotypic evolution was cytogenetically analyzed. Initially, considerable genetic heterogeneity was evident. Nine cytogenetically abnormal clones, eight of which were apparently unrelated, were found among the 83 metaphases analyzed from the primary culture and the first passage. With increasing time in culture this complexity was reduced, so that a single clone dominated passages 7-11. The clone that emerged from this genetic convergence had a t(12;17)(p13;q21) as the sole abnormality. Our findings indicate that the cytogenetic multiclonality that has been repeatedly detected in short-term cultures of squamous cell carcinomas is not caused by the in vitro conditions. Instead, the principles of Darwinian selection apply: the altered, but stable, selection pressure facing a newly established and initially multiclonal cell line will lead to a reduction of genetic heterogeneity until the one clone that now has the proliferative advantage outgrows the other subpopulations.