ELK-1/JNK/c-Fos对左归降糖解郁方(ZGJTJYF)体外培养的模拟糖尿病并发抑郁症(DD)大鼠海马神经元凋亡中的作用

目的:研究信号通路ELK-1/JNK/c-Fos在左归降糖解郁方(ZGJTJYF)对模拟糖尿病并发抑郁症(DD)环境下抗海马神经元凋亡中的作用。方法:原代培养海马神经元,加入高糖(150 mmol/L)+皮质酮(200μmol/L),构建DD体外细胞模型;将培养的海马神经元细胞随机分为5组:空白血清组、正常组、左归降糖解郁方含药血清组、阳性药(二甲双胍+氟西汀)含药血清组和模型组(每组3个复孔)。模型组和正常组给予等量培养液,其余组加入相应体积分数10%的相应血清,均干预18 h。分别采用Hoechst染色、高内涵细胞成像分析技术和RT-PCR技术分别检测海马神经元凋亡情况及检测凋亡相关ELK-1、JNK和c-Fos蛋白和基因的表达。结果:与空白组比较,模型组细胞凋亡明显,其海马神经元凋亡数量明显增多,ELK-1、JNK和c-Fos的mRNA及蛋白表达水平均显著升高(P<0.05);与模型组比较,左归降糖解郁方含药血清组和阳性药含药血清组大鼠海马神经元可见局部亮点明显减少,凋亡细胞数显著减少,ELK-1、JNK和c-Fos蛋白及mRNA表达均明显下调(P<0.05),且神经网络及树突连接情况得到明显改善。结论:左归降糖解郁方可以减低DD环境下海马神经元中Elk-1、JNK、c-fos表达而起到抗凋亡的效果。     

Temporal correlation of the memory deficit with Alzheimer-like lesions induced by activation of glycogen synthase kinase-3

We have reported that activation of glycogen synthase kinase-3 (GSK-3) by ventricle injection of wortmannin (WT) and GF-109203X (GFX) induces Alzheimer-like memory deficit in rats [Liu et al., J. Neurochem. 87 (2003), 1333]. To further explore the factors responsible for the memory loss, we studied here the temporal alterations of GSK-3, tau phosphorylation, beta-amyloid (Abeta), and acetylcholine (ACh) after injection of WT/GFX, and analyzed their correlation with the memory loss. We observed that the severe memory deficit occurred at 24 and 48 h, and simultaneously, GSK-3 activation, tau hyperphosphorylation at Thr231, Ser396, and Ser404 and decline of ACh in hippocampus were detected, and these changes were mostly recovered at 72 and 96 h after the injection of WT/GFX. Remarkable increase of Abeta and intracellular accumulation of argentophilic substances were detected at 72 h. Pearson analysis showed that the memory deficit was correlated with GSK-3 activation, tau hyperphosphorylation, and decline of ACh but not with Abeta overproduction. Our data provide direct evidence demonstrating that activation of GSK-3 by WT/GFX may cause memory deficit through tau hyperphosphorylation and suppression of ACh in hippocampus.     

Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

A simple liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid–liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C₁₈ column (150 mm × 2.00 mm, 5 μm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl]^? at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.     

利诺吡啶对豚鼠耳蜗外毛细胞和Deiters细胞钾电流的抑制

为探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的功能性表达,我们观察并记录了KCNQ家族钾通道阻滞剂利诺吡啶对豚鼠耳蜗单离外毛细胞(outer hair cells,OHCs)和Deiters细胞总钾电流的影响。采用酶孵育加机械分离法分离豚鼠耳蜗单个OHCs和Deiters细胞：运用膜片钳技术,在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流,并观察100μmol／L和200μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果观察到,在正常细胞外液中的单离外毛细胞,可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential,IKa)两种钾电流,而在单离Deiters细胞中只记录到外向整流性钾电流。在细胞外液中,加入100μmol／L利诺吡啶后,OHCs中的四乙基二乙胺敏感的钾电流峰电流成分被抑制,稳态电流幅值减小,且电流的失活时问常数明显延长;在细胞外液中加入100μmol／L和200μmol／L利诺吡啶后,OHCs的内向性钾电流IKa被完全抑制;而细胞外液中利诺吡啶终浓度为200μmol／L时,Deiters细胞的外向整流性钾电流幅值无明显变化。由此我们推测,KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分,并构成全部的IKn,其功能是介导细胞内K^ 外流和防止细胞过度去极化;KCNQ家族钾通道不存在于豚鼠耳蜗Dciters细胞。     

乙型肝炎血源疫苗不同免疫剂量阻断新生儿母婴传播的效果观察

用乙型肝炎血源疫苗,按0、1、6程序,分5种不同剂量免疫HBsAg和HBeAg均阳性(双阳性)母亲和仅HBsAg阳性母亲的新生儿,井于首针后8～12个月采血,用放射免疫(RIA)法检测他们的HBsAg和抗-HBs、抗-HBc,以比较不同剂量乙肝疫苗阻断母婴传播的效果。结果,10μg×3组对双阳性和仅HBsAg阳性母亲的新生儿的保护率,分别是42.9％和53.5％;20μ×3组为67.4％和69.7％;30μg、10μg、10μg组为75.6％和79.8％,30.20、20μg(含30、30、10μg)组为80.2％和81.5％;30μg×3组为82.3％和83.7％。随疫苗剂量增加保护率逐渐增加,抗-HBs阳转率也是如此。     

MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli

Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.     

甾体1，4-脱氢和11α-羟基化反应的两种不同微生物转化

Two kinds of micro-organism, Arthrobacter sp. AX86(1,4-dehdrogenator)and Absidia sp. A28(11α-hydroxylator) were used in this experiment. Two different fermentation techniques were performed to accomplish the multiple conversional reactions for producing 16β-methyl-11α, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione (Ⅲ) from 16β-methyl-3β, 17α,21-trihydroxy-5α-pregnane-20-one-21-acetate(1) 1)To produce product(Ⅲ)by means of a two-step fermentation method which were independently performed first by Arthrobacter and next by Abslaia, and 2)the product was obtained by a sequential fermentation system of aforesaid two micro-organisms in a single fermentor without isolation of the intermediates from the mixture. Our results showed that in both fermentation systems high yield of product was obtained. However, according to the technical simplicity, shorter duration of fermentation cycle and efficient yield of product, the second method is better than the first one.     

β-葡萄糖苷酶的分离纯化和性质研究

β－葡萄糖苷酶是纤维素酶的重要组分之一,它不仅可水解纤维二糖和寡糖,更可解除纤维二糖对β－１,４－内切葡聚糖酶和外切葡聚糖酶的抑制,提高水解速率和程度．利用ＳｅｐｈａｄｅｘＧ－１５０和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析法从黑曲霉变异株Ｌ－２２中分离提纯了β－葡萄糖苷酶,该酶是由两个分子量相同的亚基组成的二聚体,每个亚基分子量为２０３ｋＤ．该酶最适ｐＨ为４．８,ｐＨ稳定范围在３．６～６．４;最适温度是６０℃,温度稳定范围为４～６０℃;酶分子含糖量为８．３５％．它是一个酸性β－葡萄糖苷水解酶,专一性地水解β－糖苷键．而不水解α－糖苷键,对短链底物表现了相对高的活力．用动力学分析和共价化学修饰方法探讨了与该酶活力有关的必需基团．由ｐＨ对ｌｇＶｍ和ｌｇＶｍ／Ｋｍ的影响,推测出酶活性部位至少有两个可解离基团为酶活性所必需,它们在酶－底物复合物中的ｐＫｅｓ１和ｐＫｅｓ２的值分别为４．０和５．６,在游离酶中的ｐＫ值分别为４．２和５．９．由此可初步判断这两个可解离基团可能为组氨酸和含羧基的氨基酸,它们与酶的催化和底物结合可能有关．     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.