Molecular characterization of estrogen receptor genes in loach Paramisgurnus dabryanus and their expression upon 17α-ethinylestradiol exposure in juveniles

The full-length cDNAs for estrogen receptor 1 (esr1), esr2a and esr2b were isolated and characterized from the loach (Paramisgurnus dabryanus, Cobitidae, cypriniformes). P. dabryanus Esr1, Esr2a and Esr2b share high amino acids identities with their counterparts of cyprinid species. Quantitative real-time PCR (qRT-PCR) was used to analyze the tissue distribution of esr mRNAs in one-year-old P. dabryanus. The mRNA expression of esr1 in female liver was extremely higher than that in other tissues. esr2a mRNA expression in female intestine and in male muscle was higher than that in other tissues. esr2b mRNA expression was the highest in both male and female intestine. Two-month-old P. dabryanus were exposed to 17α-ethinylestradiol (EE2) for 3weeks and the changes of esr mRNA expression in brain, gonad and liver were analyzed by qRT-PCR. Results showed that EE2 at 1, 5 and 25ng/L significantly suppressed testicular esr1 mRNA expression in male. The ovarian esr2a mRNA expression was significantly up-regulated at 1ng/L EE2. In female brain, esr1 mRNA expression was significantly down-regulated at 5ng/L EE2. Both in males and females, EE2 exposure increased the hepatic esr1 mRNA expression in a concentration-dependent manner. The present study suggests that different esrs in different tissues have differential responsiveness to EE2 and the hepatic esr1 is a sensitive biomarker to EE2 at environmental concentrations in P. dabryanus juveniles. So, the loach P. dabryanus, a typical demersal fish, is a promising ecological model organism to detect estrogenic chemicals in the sediment of aquatic environment by using molecular biomarkers.     

Cardiac specific ATP-sensitive K(+) channel (K(ATP)) overexpression results in embryonic lethality

Transgenic mice overexpressing SUR1 and gain of function Kir6.2[?N30, K185Q] K_ATP channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K_ATP overexpression, wild type (WT) and K_ATP overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&;E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K_ATP overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K_ATP currents were detectable in both WT and K_ATP-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K_ATP-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K_ATP current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K_ATP channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K_ATP channel may have an important physiological role during early cardiac development.     

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.     

烤瓷表面抛光和上釉对其表面粗糙度及细菌黏附的影响

目的比较不同抛光方法对烤瓷表面粗糙度的影响,以及不同粗糙度烤瓷表面对口腔变异链球菌黏附的影响。方法采用原子力显微镜测量不同抛光方法对瓷表面粗糙度的影响,并通过细菌实验观察不同粗糙度的瓷表面对细菌黏附的影响。结果用抛光膏抛光或者上釉后,瓷面平整且有光泽。无论是表面粗糙度还是表面黏附的细菌数,橡皮轮组都大于抛光膏组和上釉组（P<0.05）。结论建议调改过的瓷表面进行抛光膏抛光或上釉以恢复瓷表面的光滑度和减少口腔致龋菌的黏附。     

白色假丝酵母菌pACT1-GFP质粒的构建及表达

目的构建能够稳定表达绿色荧光蛋白（GFP）的白色假丝酵母菌菌株,以便对目的基因进行示踪。方法构建pACT1-GFP质粒,以白色假丝酵母菌CAI4菌株作为感受态进行转化培养后,分别观察绿色荧光蛋白在两相型下的表达情况。结果含有pACT1-GFP的白色假丝酵母菌菌株,99%在两相型下均有较高水平的荧光蛋白表达,且荧光强度没有明显差别。结论pACT1-GFP能够在白色假丝酵母菌体内稳定表达。     

纳米羟磷灰石-脂肪族聚酯酰胺复合材料对成骨细胞的生物学作用

目的研究纳米羟磷灰石-脂肪族聚酯酰胺（nHA—PEA）对成骨细胞的生物学作用。方法以含有nHA—PEA的达尔贝科极限必需培养（DMEM）浸提液作用于试验组细胞,DMEM作用于对照组细胞,以甲噻唑四唑氮检测nHA—PEA对成骨细胞生长的影响,流式细胞计数细胞周期的变化,酶联免疫吸附测定细胞碱性磷酸酶（AKP）活性的变化。将细胞和材料联合培养,观察细胞在复合材料上的黏附和生长情况。结果试验组细胞的相对增殖率为92％．107％且无量效关系,试验组与对照组间差异无统计学意义（P〉0．05）;试验组和对照组细胞的细胞周期及AKP活性表达相似,组间差异无统计学意义（P〉0．05）。成骨细胞直接培养于复合材料上,显现出良好的黏附、铺展和生长行为。结论nHA—PEA对成骨细胞的生长和功能无不良影响,具有骨细胞相容性。     

温敏型壳聚糖/甘油磷酸钠缓释胰岛素凝胶系统的制备

目的:初步探讨制备温敏性壳聚糖/甘油磷酸钠载胰岛素凝胶系统.方法:应用壳聚糖与甘油磷酸钠制备具有温敏性的载有胰岛素的凝胶体系,从凝胶体系的pH值、粘度测定、胶凝时间研究不同配比、不同pH值对壳聚糖/甘油磷酸钠(CS/β-GP) 体系凝胶化性能的影响.结果:壳聚糖凝胶在室温(25℃)下呈液态,56% 甘油磷酸钠(β-GP)与3%壳聚糖(CS)在pH值6.8-7.2,37℃下凝胶化时间(GT) 从1h缩短到8-12min,凝胶形态良好,并且在负载胰岛素溶液后C/GP凝胶形态未受影响.结论:一定配比CS/GPS 体系在37℃具有快速凝胶化性能,在加入胰岛素后并未改变其温敏特性,为后续的温敏性壳聚糖/甘油磷酸钠载胰岛素凝胶系统的体外释药实验打下了基础.