流式细胞术检测风湿性心脏病合并心房颤动患者HCN2蛋白的表达

目的：对风湿性心脏病合并心房颤动患者右心耳组织超级化激活环核苷酸门控阳离子通道-2（HCN2）蛋白的表达进行分析和研究。方法风湿性心脏病患者84例分为研究组（心房颤动）和对照组（窦性心律），每组42例，对两组临床资料进行回顾性分析。结果研究组患者的左心房内径（LAD）、右心房内径（RAD）、左心室舒张末期内径（LVEDD）明显高于对照组，右心室内径（RVD）明显小于对照组，两组比较差异具有统计学意义（P〈0.05）；且研究组HCN2蛋白表达水平高于对照组。结论风湿性心脏病合并心房颤动患者HCN2蛋白的表达与患者的心房颤动具有相关性。     

Disposition kinetics of a dipeptide ester prodrug of acyclovir and its metabolites following intravenous and oral administrations in rat

The objective of this work was to study the disposition kinetics of valine-valine–acyclovir (VVACV), a dipeptide ester prodrug of acyclovir following intravenous and oral administrations in rat. A validated LC-MS/MS analytical method was developed for the analysis VVACV, Valine-Acyclovir (VACV), and Acyclovir (ACV) using a linear Ion Trap Quadrupole. ACV was administered orally for comparison purpose. In the VVACV group, both blood and urine samples and in the ACV group only blood samples were collected. All the samples were analyzed using LC-MS/MS. The LLOQ for ACV, VACV, and VVACV were 10, 10, and 50?ng/ml, respectively. Relevant pharmacokinetic parameters were obtained by non-compartmental analyses of data with WinNonlin. Following i.v. administration of VVACV, AUC_0-inf (min*μM) values for VVACV, VACV, and ACV were 55.06, 106, and 466.96, respectively. The AUC obtained after oral administration of ACV was 178.8. However, following oral administration of VVACV, AUC_0-inf values for VACV and ACV were 89.28 and 810.77, respectively. Thus the exposure of ACV obtained following oral administration of VVACV was almost 6-fold higher than ACV. This preclinical pharmacokinetic data revealed that VVACV has certainly improved the oral bioavailability of ACV and is an effective prodrug for oral delivery of ACV.     

Synergistic allelochemicals from a freshwater cyanobacterium

The ability of cyanobacteria to produce complex secondary metabolites with potent biological activities has gathered considerable attention due to their potential therapeutic and agrochemical applications. However, the precise physiological or ecological roles played by a majority of these metabolites have remained elusive. Several studies have shown that cyanobacteria are able to interfere with other organisms in their communities through the release of compounds into the surrounding medium, a phenomenon usually referred to as allelopathy. Exudates from the freshwater cyanobacterium Oscillatoria sp. had previously been shown to inhibit the green microalga Chlorella vulgaris. In this study, we observed that maximal allelopathic activity is highest in early growth stages of the cyanobacterium, and this provided sufficient material for isolation and chemical characterization of active compounds that inhibited the growth of C. vulgaris. Using a bioassay-guided approach, we isolated and structurally characterized these metabolites as cyclic peptides containing several unusually modified amino acids that are found both in the cells and in the spent media of Oscillatoria sp. cultures. Strikingly, only the mixture of the two most abundant metabolites in the cells was active toward C. vulgaris. Synergism was also observed in a lung cancer cell cytotoxicity assay. The binary mixture inhibited other phytoplanktonic organisms, supporting a natural function of this synergistic mixture of metabolites as allelochemicals.     

Differences between dispersed Leydig cells and intact testes in their sensitivity to gonadotrophin-stimulation in vitro after alteration of LH-receptor numbers

The sensitivity to hCG-stimulation in vitro of intact hemi-testes and collagenase-dispersed Leydig cells has been compared directly following either an hCG-induced loss of LH-receptors or after a hyperprolactinaemia-induced increase in LH-receptors. Injection of hCG 65 h previously, reduced hCG-binding to dispersed Leydig cells by over 84%. The sensitivity of the steroidogenic response of these cells to hCG-stimulation in vitro was reduced 22-fold whereas intact testes from the same animals showed only a 3-fold reduction in sensitivity to hCG. Dispersed Leydig cells from control rats were 8 times more sensitive to hCG-stimulation than intact testes from the same rats, a difference not evident with hCG-injected rats. In contrast, there was no difference between intact testes and dispersed Leydig cells from control and hCG-injected rats in their sensitivity to stimulation with dibutyryl cyclic AMP in vitro. Induction of hyperprolactinaemia increased hCG-binding to dispersed Leydig cells by 55%. The sensitivity of these cells to hCG-stimulation in vitro was increased by a factor of 4.5, a difference not found with intact testes from the same animals. These results show that experimental manipulation of LH-receptor numbers alters the sensitivity of dispersed Leydig cells, but not of the intact testis, to hCG-stimulation in vitro, a difference which appears to reside at the receptor level. Possible explanations for these findings are discussed together with their implications with respect to the distribution of LH-receptors over the Leydig cell surface.     

Nitric oxide generation mediated by β-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetic patients have been shown to have reduced basal platelet nitric oxide synthase activity, which is a possible contributor to the vascular complications seen in the disease. We investigated platelet nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase in Type 2 diabetic patients and control subjects.Methods Platelets isolated from blood taken from nine Type 2 diabetic patients and nine healthy control subjects of similar age were treated with isoproterenol 1 µmol/l, forskolin 1 µmol/l or vehicle. Platelet nitric oxide synthase activity was measured by L-[³H]-arginine to L-[³H]-citrulline conversion, cyclic GMP content by radioimmunoassay, and nitric oxide synthase type 3 expression by western blotting.Results Basal platelet nitric oxide synthase activity was lower in diabetic patients than in control subjects (0.01±0.02 pmol L-citrulline/10⁸ platelets, compared with 0.12±0.05; p<0.05), although no corresponding difference was seen in basal platelet cyclic GMP (0.61±0.39 and 0.13±0.22 pmol cyclic GMP/10⁸ platelets respectively; p=0.37). In control subjects isoproterenol 1 µmol/l and forskolin 1 µmol/l increased platelet nitric oxide synthase activity (to 0.27±0.08 and 0.27±0.07 pmol L-citrulline/10⁸ platelets respectively; p<0.05 for each in comparison with basal) and cyclic GMP (to 1.84±0.41 and 1.86±0.48; p<0.05 for each in comparison with basal). This effect was not achieved in diabetic patients. Isoproterenol- and forskolin-stimulated cyclic GMP correlated inversely with plasma glucose and HbA_1c. Platelet nitric oxide synthase type 3 expression was not different in control and diabetic subjects and was not changed by acute exposure of platelets to isoproterenol.Conclusions/interpretation Nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase is impaired in platelets of people with Type 2 diabetes mellitus, with no corresponding change in nitric oxide synthase type 3 expression. It is possible that this impairment contributes to the thrombotic and atherosclerotic complications of Type 2 diabetes.Abbreviations AR -adrenoceptors - GFP gel-filtered platelets - L-NAME N^G-nitro-L-arginine methyl ester - L-NMMA N^G-monomethyl-L-arginine - NO nitric oxide - NOS2 nitric oxide synthase type 2 - NOS3 nitric oxide synthase type 3     

In Vitro Effects of Estradiol, Testosterone, and Progesterone on 5-Methoxyindole Content, Cyclic Adenosine 3'',5''-Monophosphate Synthesis, and Norepinephrine Release in Different Parts of the Female Guinea Pig Pineal Complex

To examine the effects of estradiol, testosterone, or progesterone on cyclic adenosine 3',5'-monophosphate (AMP) accumulation, 5-methoxyindole levels, and norepinephrine (NE) release by the female guinea pig pineal complex, samples of the deep, intermediate, or superficial portions of the complex were incubated in vitro with varied concentrations of either hormone. Exposure for 10 minutes to physiological amounts of estradiol (10 nM) or to 100 microM NE increased significantly cyclic AMP levels to the same extent in the three pineal regions. A maximal effect on cyclic AMP accumulation was observed at 100-nM concentrations of estradiol, with a tendency to return to basal levels at 1-10 microM of estradiol. Only high concentrations of testosterone or progesterone (i.e., 1-10 microM) increased cyclic AMP accumulation in incubated guinea pig pineal fragments. At a 100-nM concentration estradiol did not affect NE-stimulated cyclic AMP accumulation in guinea pig pineal fragments. In samples of either pineal region incubated for 6 hours in TC 199 medium with 10(-7) M or greater concentrations of estradiol and analyzed for melatonin, 5-methoxyindoleacetic acid, and 5-methoxytryptophol by high-pressure liquid chromatography with electrochemical detection, a significant increase of melatonin levels was found. Neither testosterone nor progesterone modified 5-methoxyindole levels of incubated explants. K+-stimulated transmitter release from guinea pig pineal fragments previously incubated with 3H-NE was not affected by hormone exposure. These results suggest that physiological concentrations of estradiol may exert a postsynaptic stimulation of cyclic AMP and melatonin synthesis to the same extent in all three regions of the female guinea pig pineal complex.     

Recovery from Severe Cyclic Antidepressant Overdose with Hypertonic Saline/Dextran in a Swine Model

Objective:To determine the effect of a hypertonic saline and dextran (HSD) solution on blood pressure and QRS duration during severe cyclic antidepressant (CA) toxicity in swine.
Methods:Ten domestic swine weighing 20–24 kg were anesthetized and placed on mechanical ventilation. Nortriptyline solution was infused intravenously to achieve hypotension (systolic blood pressure equal to 50% of baseline) and a QRS duration of 120 msec. After reaching toxicity, the animals received in a randomized fashion either 10 mL/kg of a 7.5% saline/6% dextran solution or an equal volume of 0.9% saline as a rapid intravenous bolus. The animals were observed for one hour or until they died. Blood pressure and ECG were recorded continuously. Arterial pH was maintained in the physiologic range by controlled ventilation.
Results:Mean systolic blood pressure 10 minutes after treatment was 45 ± 8 torr in the normal-saline group compared with 115 ± 12 torr in the HSD group (p < 0.05). Mean QRS duration 10 minutes after treatment was 180 ± 8 msec in the normal-saline group; it was 88 ± 13 msec in the HSD group (p < 0.05). All normal-saline-group animals died within 20 minutes, and four of the five animals in the HSD group survived to 60 minutes (p < 0.05). The mean peak sodium concentration was 157 mmol/dL (mEq/dL) in the HSD group, and this was transient.
Conclusions:In this swine model of severe CA toxicity, a solution of 7.5% saline/6% dextran significantly reversed hypotension and QRS prolongation. HSD also improved survival to 60 minutes.     

Comparison of ex vivo and in vitro intestinal cystic fibrosis models to measure CFTR-dependent ion channel activity

Background

New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis.

Mink相关肽1对超极化激活的环核苷酸门控的阳离子通道4电生理特性的调节

目的:评价Mink相关肽1(KCNE2或MiRP1)对异源转染的中国仓鼠卵巢(CHO)细胞上的超极化激活的环核苷酸门控的阳离子通道(HCN)4电生理特性的影响。方法:将成功转染HCN4的CHO细胞(n=12)及共转染HCN4+KCNE2的CHO细胞(n=13),即将KCNE2质粒脱氧核糖核酸(DNA)单独转染或和HCN4质粒DNA共转染CHO-K1细胞,用标准微电极全细胞膜片钳记录细胞膜上的HCN4电流。结果:KCNE2对HCN4电流大小的影响:无论是在单独转染HCN4,还是HCN4和KCNE2共转染的CHO细胞中,均能检测到电流的表达。HCN4和KCNE2共转染的细胞中所检测到的电流密度,显著大于单独的HCN4转染细胞中的电流密度,差异有统计学意义(P<0.01)。KCNE2对HCN4激活动力学的影响:KCNE2和HCN4共转染后,其代表通道激活动力学的指标:激活时间常数(Tau)较单独转染HCN4时明显减小,差异有统计学意义(P<0.05)。KCNE2对HCN4激活的电压依赖性的影响:从稳态激活曲线中发现,无论是通道激活50时的脉冲电压(V1/2)还是倾斜因子(S),在HCN4与HCN4+KCNE2两组之...