A continuous fluorescent assay for measuring protease activity using natural protein substrate.

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.     

Temporal relationships betweenZea mays,ostrinia nubilalis (Lep.: Pyralidae) and endophyticBeauveria bassiana

The entomopathogenic fungus,Beauveria bassiana (Balsamo) Vuillemin, was applied to whorl-stage (V7) corn,Zea mays L., by foliar application of a granular formulation of corn grits containing conidia or by injection of a conidial suspension. All plants were infested with European corn borer larvae,Ostrinia nubilalis (Hübner), at the V7 (whorl), V12 (late-whorl), or V17 (pretassel) stage of plant development. Plants infested at whorl and late-whorl stages had significantly more European corn borer tunneling than did plants infested at the pretassel stage. The percentage of plants colonized byB. bassiana did not differ significantly among the whorl, late-whorl, and pretassel stages. As the plants matured,B. bassiana was isolated from different plant areas, with the pith more frequently colonized than the leaf collars. Foliar application ofB. bassiana provided immediate suppression ofO. nubilalis in those plants infested at whorl stage. The reduced efficacy ofB. bassiana at the intermediate plant stages relative to efficacy at harvest is discussed. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

时域—频域结合分析法—一种分析果蝇求爱歌的新方法

我们设计了一种时域-频域结合分析法,并用此方法分析了6个种群12种果蝇的求爱歌,发现如果将时域与频域的研究结合起来,对求爱歌进行频谱分析,可以定量地揭示出求爱歌的频域特性及其在时域上的细微变化。我们还对果蝇求爱歌的时域模式进行了初步的探讨,发现它们是在同一基本成分上进行调制而产生的,亲缘关系较近的种具有相近的调制方式。在对杂交后代的求爱歌的频谱分析中,我们还发现频谱上的某些特点是能够遗传的。这一新的研究方法为果蝇的进化遗传学和神经遗传的研究提供了一种新的手段。     

华东地区黑果蝇自然群体同工酶遗传多态的研究

我们用标准垂直板聚丙烯酰胺凝胶电泳和水平板琼脂糖凝胶电泳技术检测了黑果蝇(D.virilis)在合肥、芜湖、九江、南昌、福州、泉州和常州7个自然群体中Est-α、Est-β、Amy、Acph和α-Gpdh 5个座位的遗传变异,发现Est-α、Est-β和Amy 3个座位是高度多态的,Acph、α-Gpdh两个座位则是单态的。根据这5个座位等位基因的频率,我们计算了群体间的遗传距离。综合何朝珍报道的宁波、杭州、南京和洪泽4个群体的结果和我们的结果,我们作出系统树并发现泉州、福州两群体和其他群体在基因频率的分布和遗传距离方面有显著差异;分析显示这种差异与群体间地理隔离有关。     

A rationale for the design of an inhibitor of tyrosyl kinase

Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.     

In vivo inhibition of cholesterol uptake in rabbit aortas by 7-ketocholesterol.

The iv injection of 7-ketocholesterol into rabbits, made soluble by combining with bile salts, inhibited cholesterol uptake by the aorta. However, the inhibition was not as marked or as uniform as previously demonstrated in in vitro experiments. This difference may have been the result of lower plasma concentrations of 7-ketocholesterol in the injected animals. Gastric feeding of 7-ketocholesterol failed to inhibit aortic cholesterol uptake, probably because of inadequate plasma concentrations of the inhibitory steroid. The results suggest that the mechanism of 7-ketocholesterol on aortic cholesterol uptake is through competitive inhibition.     

Turnover of plasma membrane polypeptides in nonproliferating cultures of Chinese hamster ovary cells and human skin fibroblasts.

When cultures of Chinese hamster ovary cells were maintained in stationary phase on medium deficient in l-isoleucine (A) or low in serum (B), active protein turnover occurs. These cells can be acetylated with trace levels of radioactive acetic anhydride in order to incorporate label into all of the major species of polypeptides of the plasma membrane. Four days following acetylation with [³H]acetic anhydride and removal from medium A containing l-[¹⁴C]leucine, the specific ³H and ¹⁴C radioactivities of the plasma membrane proteins had fallen 15- and 7-fold respectively. The lower value obtained with the radioactive leucine is probably due to reutilization of this amino acid. The ³H and ¹⁴C radioactivity profiles for the polypeptides separated by discontinuous gel electrophoresis, however, showed little qualitative change over the course of the experiment, suggesting that differential rates of protein turnover were not occurring. These results were confirmed in experiments with cells using both the above culture conditions in which two acetylations were carried out, one with ³H at time zero and the other with contrasting ³C label up to 96 h later. Two methods for plasma membrane isolation and a number of electrophoretic conditions were employed. Again, however, the radioactivity profiles along the gels coincided almost exactly, even though the ³H specific radioactivity had fallen several fold. Similar results have been obtained with confluent human skin fibroblasts. We suggest that the major proteins in the plasma membranes of cultured mammalian cells do not show markedly heterogeneous rates of turnover. In particular, larger species of polypeptides do not appear to have shorter half-lives than smaller ones.     

基于能值分析的环太湖城市群生态经济系统可持续发展研究

采用能值理论与分析方法,研究2015年环太湖城市群生态经济系统的能值流量,并构建多维度的生态经济系统指标体系,综合评价环太湖城市群可持续发展程度和健康水平。研究结果表明：（1）可更新资源利用不足,在能值利用总量中占比最大值仅为7.95%;（2）不可更新资源消耗过度,在能值利用总量中占比最小值达到56.95%;（3）（除苏州市外）可更新资源产品中肉类与水产品占比最小值已达63.08%,急需对传统的农业结构进行调整;（4）不可更新资源产品均以水泥和钢铁为主,占比最小值已达88.02%,应该加快传统的工业转型升级;（5）输入能值占比最大值仅为36.27%,商品进出口呈现贸易顺差,对外经济开放仍有发展空间;（6）废弃物与可更新能值比率最大值仅为12.8%,废弃物能值比最小值仅为0.19%,废弃物排放和利用水平有待提高;（7）湖州市可持续指标ESI和能值可持续发展指标EISD为1.59和2.99,在环太湖城市群中更具可持续发展潜力;（8）苏州市健康能值指数EUEHI和改进的健康能值指数EUEHI''为8.22和1.65,在环太湖城市群中健康水平较低。据此结果提出了有助于环太湖城市群经济社会发展与自然生态环境和谐共赢的可持续发展对策。