Residues Val254, His256, and Phe259 of the angiotensin II AT1 receptor are not involved in ligand binding but participate in signal transduction

The role of the external third of helix VI of the angiotensin II (AII) AT1 receptor for the interaction with its ligand and for the subsequent signal transduction was investigated by individually replacing residues 252-256 by Ala, and residues 259 or 261 by Tyr, and permanently transfecting the resulting mutants to Chinese hamster ovary (CHO) cells. Binding experiments showed no great changes in affinity of any of the mutants for AII, [Sar1]-AII, or [Sar1, Leu8]-AII, but the affinity for the nonpeptide antagonist DuP753 was significantly decreased. The inositol phosphate response to AII was remarkably decreased in mutants V254A, H256A, and F259Y. These results indicate that AT1 residues Val254, His256, and Phe259 are not involved in ligand binding but participate in signal transduction. Based in these results and in others from the literature, it is suggested that, in addition to the His256 imidazole ring, the Phe259 aromatic ring interacts with the AII's Phe8, thus contributing to the signal-triggering mechanism.     

Selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection

A novel method to monitor specific peptidase activities in biological samples as complex as undiluted plasma/blood is described. The approach is based on the design of synthetic polypeptide substrates in which di- or triarginine sequences are linked to each other via one or more other amino acids recognized specifically by the peptidase to be determined. Detection of chymotrypsin and renin activities using synthetic substrates P4 (F-R-R-R-F-V-R-R-F-NH2) and P5 (R-R-R-L-L-R-R-L-L-R-R-R), respectively, serves to demonstrate the principles of this new assay system. A polyion-sensitive membrane electrode, prepared by doping polymer films with dinonylnaphthalene-sulfonate (DNNS), is shown to exhibit significant nonequilibrium electromotive force (EMF) responses toward these and other polycationic substrates at microgram/milliliter levels under physiological conditions. The same electrode, however, exhibits much smaller total EMF response toward the shorter fragments of the synthetic peptides generated by peptidase activity; hence, the addition of peptidase to a solution containing the synthetic substrate yields a change in electrode EMF response, the rate of which is proportional to the activity of peptidase present. Other synthetic polycationic peptides as well as natural polycationic peptides (e.g., protamine) that lack specific cleavage sites for chymotrypsin and renin, yet are detected by the DNNS-based membrane electrode, do not elicit any significant change in EMF response in the presence of the peptidases, confirming the feasibility and utility of the proposed bioanalytical method.     

低能重离子及其碰撞产生的级联原子在生物材料中的直接作用范围

用Ｍｏｎｔｅ Ｃａｒｌｏ法模拟计算了３０ｋｅＶ和２００ｋｅＶ的Ｎ＾＋与１１０ｋｅＶ的Ｆｅ＾＋在模拟细胞中的射程分布和径迹结构，并将１１０ｋｅＶＦｅ＾＋模拟计算的结果与ＲＳＢ测量的结果相比较，发现计算与测量的结果吻合较好，离子的作用范围小于１μｍ。计算和实验的结果都说明能量相当低的重离子不大可能直接作用引起麦胚深层生物效应。     

用复合烷基锂引发丁二烯聚合

对以环己烷为溶剂、四氢呋喃为微观结构调节剂、复合烷基锂为引发剂的丁二烯聚合反应过程进行了研究，考察了不同引发温度、不同配比的复合烷基锂对聚合反应的影响，并与以正丁基锂为引发剂的丁二烯聚合反应过程进行了比较。     

Initial results of an in vitro fertilization and embryo transfer program in a private health institution

Between October 1994 and the end of February 1995 115 patients were included in our programme. In these patients we monitored 141 cycles, performed 128 punctures and 100 ET. In 14 instances (9.2%) the cycle was abolished. By puncture of 1468 follicles 1370 oocytes were obtained which corresponded to a recovery rate of 93.3%. On average by aspiration 11.9 oocytes per patient were obtained. From the total number of acquired oocytes 54.6% were fertilized and 43.5% divided. Of 596 embryos 290 were transferred and the remainder was vitrified. A total of 17 clinical pregnancies were achieved, incl. 5 which terminated by abortion, the remainder proceeds. The authors are aware that they present a relatively small group during a short period of time, and thus the first results of the centre cannot be presented as a current standard. Despite this the results permit some optimism for the future and indicate a certain amount of standardization of the authors' clinical and laboratory work.     

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.     

Changes in mitochondrial calcium concentration during the cardiac contraction cycle

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.     

真空熔炼高压气体雾化制粉技术及设备

介绍了气体雾化粉的品种、用途及工业化生产。还介绍了真空熔炼-高压气体雾化制粉所需的设备。     

汛期采金船的缆绳牵拉

本文介绍了采金船渡汛时的停泊方向及采金船在洪水中可能产生拖曳力的估算方法，为了防止采金船汛期发生溜船事故，建议采用缆绳牵拉固船的措施，以保障采金船的汛期安全。