高产融合子Q4413的形态学与生理生化特征的研究

本文通过对枯草芽孢杆菌BR151衍生株与北京棒杆菌1134衍生株的赖氨酸高产融合子Q4413株的形态学、生理生化特性等方面的研究,揭示了融合子与双亲株在这些方面的差异,为Q4413株确系双亲株的重组子或新的融合子增加了佐证.     

由大肠杆菌中提取重组猪生长激素的研究

 本文通过研究提出了一种由大肠杆菌细胞中提取重组猪生长激素的方法。提取回收率为52.4％,纯度为94.1％,具有生物学活性。     

双拷贝人α型心钠素基因的组建及大肠杆菌分泌型克隆与表达

将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。     

固定化葡萄糖异构酶最适pH值的调节

用多孔强碱性三乙醇胺基聚苯乙烯树脂作为载体,用CNBr与载体上多羟基作用共价偶联葡萄糖异构酶(GI)。最适偶联条件表明:CNBr量增多,蛋白载量增加,但比活下降。固定化葡萄糖异构酶(IGI)最适反应温度比天然酶提高15℃。并系统地研究了影响IGI活力-pH的曲线的各种因素:用具有不同平均孔径的载体(R=137A,185A,230A,365A)固定化GI,在低离子强度条件下(0.0064mol/L),测定其最适pH值分别7.76,7.56,7.50,8.20。选择平均孔径为230A且具有不同数量三乙醇胺基的载体(0.94,1.05,1.13,1.37mmol/g干胶)分别固定化GI,其最适pH值分别为7.70,7.50,7.46,7.36。     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.     

Exogenous C1q reconstitutes a secondary deficiency of C5-deficient AKR mouse macrophages for FcR-dependent cellular cytotoxicity and phagocytosis

Studies originally designed to assess the putative role of endogenous C5 in macrophage activation for antibody-dependent cellular cytotoxicity (ADCC) yielded unanticipated results. Resident and inflammatory peritoneal macrophages from C5-deficient AKR mice were found to have significantly lower capacity for FcR-dependent ADCC activation and phagocytosis of IgG-opsonized SRBC targets than did C5-competent C3HeB/FeJ (C3H) mice. Reconstitution of the ADCC response of AKR macrophages was accomplished initially with C5-sufficient C3H mouse serum, which suggested that endogenous C5 may be required for ADCC activation. However, further investigation largely eliminated C5 involvement in that a heat-labile component of C5-deficient AKR serum was shown to be active in the reconstitution of ADCC activation of AKR macrophages. Macrophages from AKR mice were found to have significantly lower levels of C1q mRNA synthesis, endogenous C1q levels, and C1q secretion than did C3H mouse macrophages as determined by Northern blot, Western blot, and presynthetic radiolabeling analysis, respectively. The addition of purified exogenous C1q to IgG-opsonized SRBC targets fully reconstituted ADCC activation for AKR inflammatory peritoneal macrophages to levels of normally FcR-responsive C3H macrophages. Similarly, exogenous C1q augmented FcR-dependent phagocytosis of AKR macrophages but had no effect on macrophages from responsive C3H mice. Our results indicate that AKR mice have a deficiency for FcR-dependent cellular cytotoxicity and phagocytosis that is related to their low potential for C1q synthesis and secretion rather than to their established genetic deficiency for C5 synthesis. We tentatively conclude that endogenous C1q is required as an accessory molecule for macrophage FcR-dependent effector functions and that C5 is not a prerequisite for ADCC activation.     

Functional analysis of the 3′-terminal sequence of the maize controlling element (Ac) by internal replacement and deletion mutagenesis

Using deletion analysis of the Ac transposable element, we have shown that replacement of internal sequences from base pairs 181–3559 does not abolish transposition. We have done sequential deletion analysis of the 3'-end of the Ac element and found that deletion of the major transposase binding sites (AAACGG) abolishes transposition. But, surprisingly, we found a 3'-terminal deletion of the transposase binding sites which also contained a 71-bp internal sequence between base pairs 3559 and 3630 retained transposition ability. This 71-bp internal sequence did not have a transposase (ORFa) binding motif. These data suggest that two different domains may be involved in the minimal sequence necessary for transposition. Finally, we have identified functional prokaryotic promoter sequences and ARS sequences within the 5' and 3'-termini of Ac, but cannot ascribe any function to these sequences.