Acute Immobilization Stress and Intraventricular Injection of CRF Suppress Naloxone-Induced LH Release in Ovariectomized Estrogen-Primed Rats

The present study was undertaken to evaluate the role and possible interaction of the endogenous opioid peptide (EOP) and corticotropin-releasing factor (CRF) in the acute stress-induced suppression of gonadotropin secretion in ovariectomized estrogen-primed rats. An intravenous (i.v.) injection of naloxone (10 or 20  mg/kg), an EOP antagonist, significantly elevated serum luteinizing hormone (LH) levels within 10  min in non-stressed animals. The naloxone-induced LH release was completely eliminated when tested 30  min after the onset of acute immobilization. In a subsequent study, it was found that suppression of the naloxone-induced LH release occurred as early as 5  min after the stress onset, and was still evident 60  min after the end of a 30-min period of immobilization. The effect of naloxone was restored 3  h after liberation of the animal from the 30-min immobilization. An intraventricular (i.c.v.) injection of CRF (1 or 5  μg) also significantly suppressed, in a dose-related manner, the effect of a subsequent i.v. injection of naloxone. However, an i.c.v. injection of α -helical CRF(9-41) (25 or 50  μg), a CRF antagonist, prior to immobilization, could not interfere with the suppressive effect of stress on naloxone-induced LH release. These results suggest that both acute immobilization stress and CRF can inhibit the LH secretory activity without mediation by EOP neurons. However, the stress-related suppression may involve non-CRF mechanism(s).     

Development of the Qualeffo–31, an osteoporosis-specific quality-of-life questionnaire

Introduction Vertebral deformities are a common consequence of osteoporosis and are known to decrease quality of life. The Qualeffo–41 is a quality-of-life questionnaire especially developed for measuring quality of life in patients with vertebral deformities. It consists of 41 questions arranged in five domains: pain, physical function, social function, general health perception, and mental function. The objectives of this study were: (1) to develop a shorter version of the Qualeffo–41 by removing redundant questions; and (2) to investigate the scale characteristics, reliability, and validity of this shorter version. Methods The study was performed using data from the Qualeffo validation study and the Multiple Outcomes of Raloxifene Evaluation (MORE) study. The analyses were performed in patients with vertebral deformities (n=579). Factor analysis on polychoric correlations and an item response theory (IRT) model, i.e., the generalized partial credit model (GPCM), were used to create a shorter version of Qualeffo–41. Using GPCM, scoring weights were computed for all items. Results Three items were removed from the data set because of too many missing values. Factor analysis identified three instead of five domains: (1) pain, (2) physical function, and (3) mental function. Five items had factor loadings <0.4 and were not included in the GPCM. After excluding several items, the domains pain (four items), physical function (18 items), and mental function (nine items) showed a good, reasonable, and excellent fit, respectively. This indicates that the mental function domain and the pain domain are more unidimensional than the physical function domain. All three domains showed a very high correlation (r ≥0.95) with the corresponding domains of the Qualeffo–41. Conclusions Qualeffo–31 was developed, consisting of three domains with a reasonable to excellent fit to the GPCM. Although the fit to the GPCM supports the construct validity of the Qualeffo–31, validation in a new study should be performed before using it in practice.     

Inhibition of HIV-1 by modification of a host membrane protease

While it is clear that CD4 Is the receptor for the gp120 envelopeprotein of HIV-1, substantial evidence suggests that other hostcell proteins are required for successful membrane fusion. Studieswere initiated to examine the potential for a protein receptorwhich has an elastase-like character to participate in fusionof HIV-1 with permissive host cells. A synthetic elastase inhibitorwas shown to significantly reduce HIV-1 infectivity when presentduring, but not after, the initial contact between virus andcells. A human T cell elastase-like membrane component was purifiedand shown to be lipid-associated. By competitive Inhibition,the purified protein was shown to bind gp160 within the HIV-1fusion domain. The binding parameters of whole T cell membraneextract, with a hydrophobic pentapeptide representative of thefusion domain, suggested an elastase-like protein is the single,secondary T cell receptor for HIV-1 (K = 1x10³ M^–1). Thepentapeptide interacted with porcine and human (epithelial andpolymorphonuclear leukocyte), but not murine, elastase isoforms,suggesting its participation In the permissiveness of host cellsto infection.     

重组小鼠干细胞逆转录病毒载体高效基因转染CD41+、UT7、U937和MDA-MB-435细胞

目的:研究重组小鼠干细胞逆转录病毒载体介导基因转染,探索一条高效基因转染的途径,为重组小鼠干细胞逆转录病毒载体在基因转染中的应用提供理论依据和奠定实验基础。方法:①逆转录病毒载体的构建:EC1-4(repeats1-4ofcadherin-5extracellulardomains)基因克隆产物和mutant(Ser222A)MEK1基因克隆产物,Bg1Ⅱ和EcoRⅠ限制性内切核酸酶切割后,克隆进入逆转录病毒表达载体pMSCV。②CD41⁺细胞的获取和细胞培养:从脐带血分离的CD34⁺细胞通过TPO诱导表达CD41,FACS分离CD41⁺细胞。高糖DMEM培养液培养NIH3T3和MDA-MB-435细胞,U937细胞培养在RPMI-1640培养液,UT7细胞是细胞因子依赖性细胞株,Iscove'smodifiedDulbeco's培养液中加入GM-CSF。③测定病毒滴度:逆转录病毒载体转入包装细胞293,36h后收集病毒上清液,感染NIH3T3细胞,流式细胞仪测定病毒滴度。④Westernblot:基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,Westernblot检测基因产物的表达。结果:293细胞产生高滴度MEK1pMSCV病毒:3.1×10⁷,高滴度EC1-4pMSCV病毒:1.0×10⁸。用稀释8倍的病毒转染基因,重组逆转录病毒MEK1pMSCV转染白血病细胞株UT7和U973,GFP阳性细胞(转染阳性细胞)分别是60.73％、72.56％。重组逆转录病毒MEK1pMSCV转染原代培养细胞CD41⁺,GFP阳性细胞为30.57％。重组逆转录病毒EC1-4pMSCV转染人乳腺癌细胞株MDA-MB-435,GFP阳性细胞为97.54％。TPO作用CD41⁺和UT7细胞以及血清对U973细胞的作用,显示出外源mutationMEK基因的dominantnegative的效应,实验组磷酸化的MEK1减少。EC1-4基因转染的MDA-MB-435细胞表达了EC1-4基因产物。结论:重组小鼠干细胞逆转录病毒载体能高效基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,转染的基因能稳定地表达。     

中国92例HIV/AIDS患者HIV-1 gp41 2F5和4E10中和抗体表位变异研究

目的 探讨92例HIV/AIDS患者HIV-1病毒近膜端(membrane proximal external re-gion,MPER)中和抗体2F5和4E10保守表位ELDKWA、NWFDIT氨基酸变异特点,为中国HIV/AIDS患者免疫治疗以及疫苗设计提供数据.方法 Nest-PCR扩增HIV-1 env区gp41段基因,核酸序列测定,翻译为氨基酸与HIV-1 Sequence Database HXB Ⅱ参考株中和抗体表位数据比对,分析2F5、4E10中和表位氨基酸变异情况.结果 92例HIV/AIDS患者HIV-1外膜蛋白env gp41段中和抗体2F5、4E10保守表位氨基酸均存在突变;2F5中和抗体表位主要有E662A(14.1%)、K665S(17.4%)、A667K(16.3%)突变;4E10中和抗体表位主要有N671S(13.0%)、D674S(3.3%)、T676S(16.3%)突变;CRF_B'C亚型与B'亚型的2F5和4E10表位氨基酸突变差异具有统计学意义(P<0-05);CRF_B'C与CRF01_AE亚型2F5表位突变差异具有统计学意义(P<0.05);B'亚型缓慢进展者、HIV感染者和AIDS患者的4E10表位氨基酸突变差异具有统计学意义(P<0.05).结论 92例HIV/AIDS患者HIV.1包膜蛋白env gp41段中和抗体2F5、4E10中和表位氨基酸存在突变,且变异多样化;不同亚型中和抗体保守表位氨基酸位点变异有差异;B'亚型4E10中和抗体表位变异可能与疾病进展有一定联系.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (41u28u7212/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-escin)-skinned arterial strips, application of TG (0.1–1 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) or CPA (0.5–10 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) or nifedipine (1 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) but was blocked by La³⁺ (50 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) and tyrphostin (2–50 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) and CPA (10 41u28u7212/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Intracerebroventricularly administered corticotropin-releasing factor releases somatostatin through a cholinergic,vagal pathway in freely fed rats

The aim of this study was to investigate whether corticotropin-releasing factor influences the plasma levels of somatostatin, gastrin or cholecystokinin when administered intracerebroventricularly to rats, and if such an effect could be vagally mediated, and dependent on the animals feeding states. Anaesthetized, freely fed rats were given 5 μl intracerebroventricular injections of corticotropin-releasing factor in four doses; 10 pmol-1.28 nmol. Immediately following death, trunk blood was collected for subsequent peptide analysis with radioimmunoassay (RIA). The three higher doses of corticotropin-releasing factor elevated the plasma levels of somatostatin (P < 0.01) after 20 min but left the plasma levels of gastrin and cholecystokinin unchanged. Intraperitoneal injections of 60 and 320 pmol of corticotropin-releasing factor did not influence the somatostatin levels. Further, intracerebroventricular injections of 60 pmol of corticotropin-releasing factor produced a peak increase in somatostatin after 20 min (P < 0.01). After 60 min the somatostatin levels were still increased (P < 0.05). Gastrin and cholecystokinin remained unaltered at these timepoints. Intracerebroventricular administration of 10 nmol of a-helical corticotropin-releasing factor 9–41 attenuated the basal levels of somatostatin and blocked the corticotropin-releasing factor-induced rise in somatostatin. Bilateral truncal vagotomy, as well as pretreatment with atropine (0.05 mg kg^-1, subcutaneously) abolished the effects of corticotropin-releasing factor on somatostatin. In animals which were food-deprived for 24 h, corticotropin-releasing factor did not influence somatostatin, gastrin or cholecystokinin. Pretreatment with cholecystokinin did not potentiate corticotropin-releasing factor-induced somatostatin release in food-deprived rats. These findings suggest that corticotropin-releasing factor acting within the central nervous system may regulate gastrointestinal functions partially through a cholinergic, vagally mediated release of somatostatin in freely fed, but not in food-deprived rats.     

计算流体力学在组织工程中的应用进展

目的综述计算m41;体力学（computational fluid dynamics，CFD）在组织工程中的应用进展。方法广泛查阅 CFD 应用于组织工程的相关文献，主要对 CFD 用于生物反应器设计改良或优化、模拟体外组织再生过程中的m41;体动力学和细胞生长动力学等方面进行综述。结果CFD 的模拟预测能力可为生物反应器的设计优化和体外组织工程组织培养提供重要的指导作用，且结合实验研究能进一步提高模型预测结果的准确性。结论CFD 作为新兴和有效的研究工具，已在组织工程中展现出独特优势并取得显著进展，但更全面、准确地模拟组织再生全过程仍需进一步研究。