TAT蛋白转导肽介导的秀丽线虫体内外源蛋白的跨膜转导研究

TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana⁺的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。     

新生与成年土拨鼠肝组织中部分差异表达的基因比较分析

目的新生土拨鼠感染土拨鼠肝炎病毒后,大部分发展为慢性肝炎,而成年土拨鼠感染后则多发生急性自限性肝炎。本实验目的就是寻找其肝组织中可能导致这种预后差异的关键基因。方法采用全基因组表达谱芯片技术,对比新生与成年小鼠肝组织基因表达差异,选取目的基因,再通过多个物种序列比对,设计简并引物,在土拨鼠肝组织eDNA中扩增对应基因,测序,再次设计引物,进行实时荧光定量PCR。结果与新生土拨鼠相比,成年土拨鼠肝细胞中与钙离子重吸收相关基因DNMl（Dynamin1）、DNM3（Dynamin3）及Prkcc（proteinkinaseC,gamma）表达率明显升高,分别上升2．65±0．25倍、1．90±0．34倍、2．94±0．54倍。结论在钙离子重吸收通路中,在两组小鼠肝脏中表达差异最明显的上述三个基因,在新生组与成年组土拨鼠之间也有明显差异。此类基因造成肝细胞内钙离子浓度的差别,间接影响其中肝炎病毒的复制。这种表达差异很可能是导致两个年龄段动物感染土拨鼠肝炎病毒后转归不同的原因之一。     

Carbon isotope discrimination in C3-C4 intermediates

Carbon isotope discrimination in C₃–C₄ intermediates is determined by fractionations during diffusion and the biochemical fractionations occurring during CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation occurring in the bundle sheath, the extent of bundle-sheath leakiness and the contribution which C₄-cycle activity makes to the CO₂ pool there. In most instances, carbon isotope discrimination in C₃–C₄ intermediates is C₃-like because only a small fraction of the total carbon fixed is fixed in the bundle sheath. In particular, this must be the case for Flaveria intermediates which initially fix substantial amounts of CO₂ into C₄-acids. In C₃–C₄ intermediates that refix photorespiratory CO₂ alone, it is possible for carbon isotope discrimination to be greater than in C₃-species, particularly at low CO₂ pressures or at high leaf temperatures. Short-term measurements of carbon isotope discrimination and gas exchange of leaves can be used to study the photosynthetic pathways of C₃-C₄ intermediates and their hybrids as has recently been done for C₃ and C₄ species.     

氢火焰气相色谱仪内标法测定反式脂肪酸的条件的建立及验证

目的:研究氢火焰气相色谱仪(GC112A)内标法测定反式脂肪酸的实验室最佳条件。方法:采用气相色谱内标-标准曲线法,通过测定反式脂肪酸标准品工作液的标准曲线、重现性和分离度来确定和验证GC112A的最佳色谱条件。结果:实验室最佳色谱条件:毛细管柱,PC-88(100 m×0.25 mm×0.2μm);进样口温度:260℃;载气:高纯N2,压力220 k Pa,流速41.0 m L/min;检测器(FID)温度:290℃,氢气压力100 k Pa,流速21.0 m L/min;空气压力160 k Pa,流速215.0 m L/min;检测器(FID)灵敏度:1010;手动进样,进样量:1.0μL。结论:该方法避免了外标法(国标方法)的国产仪器局限性和操作者人工进样的不确定性,具有受仪器参数变化的影响较小的特点,适合在基层食品检验机构推广。     

Insects and fungi on a C3 sedge and a C4 grass exposed to elevated atmospheric CO2 concentrations in open-top chambers in the field

The effects of elevated atmospheric CO₂ concentration on plant-fungi and plant-insect interactions were studied in an emergent marsh in the Chesapeake Bay. Stands of the C₃ sedge Scirpus olneyi Grey, and the C₄ grass Spartina patens (Ait.) Muhl. have been exposed to elevated atmospheric CO₂ concentrations during each growing season since 1987. In August 1991 the severities of fungal infections and insect infestations were quantified. Shoot nitrogen concentration ([N]) and water content (WC) were determined. In elevated concentrations of atmospheric CO₂, 32% fewer S. olneyi plants were infested by insects, and there was a 37% reduction in the severity of a pathogenic fungal infection, compared with plants grown in ambient CO₂ concentrations. S. olneyi also had reduced [N], which correlated positively with the severities of fungal infections and insect infestations. Conversely, S. patens had increased WC but unchanged [N] in elevated concentrations of atmospheric CO₂ and the severity of fungal infection increased. Elevated atmospheric CO₂ concentration increased or decreased the severity of fungal infection depending on at least two interacting factors, [N] and WC; but it did not change the number of plants that were infected with fungi. In contrast, the major results for insects were that the number of plants infected with insects decreased, and that the amount of tissue that each insect ate also decreased.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

TPA-induced multinucleation of a mesenchymal stem cell-like clone is mediated primarily by karyokinesis without cytokinesis, although cell-cell fusion also occurs

The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.     

滇姜花抗肿瘤活性二萜及其光敏氧化反应的研究

从滇姜花（ＨｅｄｙｃｈｉｕｍｙｕｎｎａｎｅｎｓｅＧａｇｎｅｐ．）根茎中分离到两个具抗肿瘤活性的二萜成分滇姜花素Ｃ及滇姜花素Ａ,前者为新化合物,其结构经波谱及化学方法鉴定。此新化合物亦可从滇姜花素Ａ的光敏氧化反应产物中分离得到。     

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.     

The level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

 Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms. Received: 10 July 1997 / Accepted: 8 November 1997