Relationship between cAMP and Ca2+ fluxes in human platelet membranes

The effect of cAMP (which involved a 23 kDa protein phosphorylation) has been studied on the Ca2+ uptake and Ca2+ release from a human platelet membrane vesicle fraction. It was tested in the presence of the catalytic subunit of the cAMP-dependent protein kinase (C Sub). The addition of C Sub increased the steady state level of the Ca2+ uptake into the membrane vesicles. The effect was enhanced when tested in the absence of Ca2+ precipitating agent. The response was proportional to the dose of C Sub. Moreover, the effect varied with the Ca2+ concentration. The effect of C Sub has been tested on the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. A phosphorylated state of the 23 kDa protein appeared to be necessary. Indeed, a phosphorylation inhibition prevented the IP3 effect and the addition of C Sub increased the percentage of released Ca2+ (without modification of the time course). However, the C Sub dose-dependent response was not linear. The effect of cAMP on the two functions (Ca2+ uptake and Ca2+ release) appears to be different. Therefore, these results led us to suggest a more complex role of cAMP in the regulation of platelet Ca2+ concentration.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Complete characterization of the human IGF-I nucleotide sequence isolated from a newly constructed adult liver cDNA library

A full-size cDNA sequence coding for insulin-like growth factor I (IGF-I) was isolated from a human liver library. For the construction of this bank, a new method was developed which anneals dG-tailed cDNA with a synthetic adaptor 5'-AATTCCCCCCCCCCC-3' followed by ligation into the EcoRI site of a lambda immunity-insertion vector. Based on the sequence analysis of the complete IGF-I messenger we concluded that the protein is synthesized as a precursor containing a signal peptide of 22 or 25 amino acid residues. In addition, the sequence extended in the 3'-direction and showed the presence of multiple polyadenylation sites in the IGF-I message.     

基于近红外漫反射光谱快速测定淫羊藿蛋白质含量

采用近红外漫反射光谱技术对淫羊藿(Epimedium)的蛋白质含量进行快速且无损检测。近红外漫反射光谱经二阶导数处理、标准多元离散校正及主成分分析聚类处理后, 采用改进最小二乘法回归得到的定标模型预测效果最佳, 定标决定系数、交互验证标准差及交互验证相关系数分别为0.923、0.554和0.717。近红外光谱分析法的测定结果与用凯氏定氮法所得结果无显著差异, 两种方法测定值的相关性较高(R²=0.933 9)。重复性实验表明, 近红外光谱分析法的相对标准偏差为0.937%。该研究首次采用近红外光谱分析法测定了8种淫羊藿的蛋白质含量。该方法简便、精确, 在淫羊藿资源开发利用和药材质量控制方面具有参考意义。     

外科手术在高血压脑出血治疗中的应用

高血压脑出血是当今社会一种发病率及死亡率都非常高的神经科疾病,对于它的治疗,则是神经内外科的重点难点。目前治疗高血压脑出血的方法分为内科保守治疗和外科手术治疗两种,作者阅读了大量国内外临床相关文献,综述了高血压脑出血相关内外科治疗的方法,以及具体应用,望能为其相关研究提供有益的思路。     

In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage

The ability of frozen immature goat oocytes to undergo in vitro maturation (IVM) and fertilization (IVF) was investigated. Fully grown germinal vesicle stage (GV-stage) goat oocytes were submitted to different variables of cryopreservation: 1) exposure to propanediol before maturation but without freezing to detect the level of damage attributable to propanediol alone, 2) removal of cumulus cells to mimic damage attributable to osmotic stress during cryoprotectant exposure or freezing procedure, and 3) rapid freezing with propanediol. Maturation and fertilization rates were 82.1, 71, 65.3 and 23.7% and 71.2, 40, 58.4 and 23.1% for control, exposed, denuded and frozen oocytes, respectively. These results indicate that freezing sticto sensu (i.e., cooling and warming phases) have detrimental effects on IVM of GV-stage oocytes, whereas the reduced IVF rates of post-thaw matured oocytes are imputable to a cryoprotectant effect.     

From insulin receptor signalling to Glut 4 translocation abnormalities in obesity and insulin resistance

Insulin resistance is commonly associated with obesity in rodents. Using mice made obese with goldthioglucose (GTG-obese mice), we have shown that insulin resistance results from defects at the level of the receptor and from intracellular alterations in insulin signalling pathway, without major alteration in the number of the Glut 4 glucose transporter. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundly affected in response to insulin. This defect appears very early in the development of obesity, together with a marked decrease in IRS 1 tyrosine phosphorylation. In order to better understand the abnormalities in glucose transport in insulin resistance, we have studied the pathway leading from the insulin receptor kinase stimulation to the translocation of the Glut 4 containing vesicles. This stimulation involves the activation of PI 3-kinase, which in turns activates protein kinase B. We have then focussed at the mechanism of vesicle exocytosis, and more specifically at the role of the small GTPase Rab4 in this process. We have shown that Rab4 participates, first in the intracellular retention of the Glut 4 containing vesicles, second in the insulin signalling pathway leading to glucose transporter translocation.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.