杧果蔗糖合成酶MiSS基因的克隆与表达分析

为克隆杧果(Mangifera indica L.)蔗糖合成酶基因序列,预测其编码蛋白特性,阐明其在果实发育过程中的表达规律和作用.本研究采用同源克隆法和RACE技术克隆了1个编码蔗糖合成酶基因的全长cDNA,命名为MiSS,其cDNA全长2110 bp,开放阅读框为1455 bp,编码484个氨基酸,相对分子量为55.3 kD,理论等电点为6.08.系统进化分析显示,MiSS基因编码的氨基酸序列与温州蜜柑(Citrus unshiu)、荔枝(Litchi chinensis)、龙眼(Dimocarpus longan)氨基酸序列一致性为90％～93％.RT-qPCR分析显示,MiSS基因表达量呈现先上升后下降的趋势,且果实发育各时期果皮内MiSS基因表达量均显著高于果肉,综合分析MiSS基因可能与淀粉的合成密切相关.本研究为进一步了解MiSS基因在杧果蔗糖代谢过程中的作用以及从分子角度阐明植物生长调节剂对杧果蔗糖代谢的影响机理奠定了理论和技术基础.     

光周期对光敏核不育水稻光敏色素A含量及其mRNA丰度的影响

光敏核不育水稻(农垦58S)是我国特有的水稻(Oryza sativa L.)种质材料,光敏色素是光周期诱导其育性转变的受体。报道了育性转换敏感期间的光周期处理对农垦58S及对照“农垦58”叶片中光敏色素A(Phy A)含量及其mRNA丰度的影响。在10个光周期处理的最后一个暗期结束前,收获每株水稻的上部两片叶,用酶联免疫吸附测定法测定Phy A。和长日照(LD)相比,短日照(SD)处理导致农垦58SPhy A相对含量增加38.5％;而“农垦58”只增加18.5％。显然,在较长的暗期中,农垦58S中Phy A的积累比对照快。在水稻幼苗中也得出相似的结果。以光敏色素A基因(phy A)的特异性片段RPA3作探针,用RNA斑点杂交的方法对叶片中Phy A mRNA丰度进行分析的结果表明,光周期处理5d和10d时,两品种水稻的Phy A mRNA丰度都是SD处理的比LD的高,而且SD下农垦58S Phy A mRNA的丰度均比“农垦58”的高。这些结果表明,甲基化水平较低的农垦58S phy A可能比“农垦58”的phy A更活跃地表达。另外,在育性转换敏感期每日主光期结束时(EOD)进行10次短暂的远红光(FR)照射。结果表明,农垦58S植株抽穗和开花期比SD处理推迟2d,而花粉败育率、种子结实率却无变化。暗示农垦58S开花和育性转变过程的光周期反应可能不同。     

基于微流控芯片的温度梯度毛细管电泳检测大肠癌石蜡标本中K-ras基因突变

K-ras基因突变检测可用于大肠癌的早期筛查与诊断,并有利于筛选出抗表皮生长因子受体靶向药物治疗有效的大肠癌患者,以实现肿瘤的个体化治疗．采用以倾斜式热辐射原理建立的微流控温度梯度毛细管电泳(temperature gradient capillary electrophoresis,TGCE)基因突变检测系统,实现了对98例石蜡包埋大肠癌组织中K-ras基因突变的高灵敏度筛查,突变阳性检出率为47.96%,显著高于PCR产物直接测序的23.47%．克隆测序显示该方法至少能检测到2.08%的K-ras基因突变体．K-ras基因突变与临床病理学参数的关系分析显示,直肠癌中K-ras基因突变率明显高于结肠癌(P < 0.05),而与年龄、性别、组织学类型和肿瘤分期等无显著相关性．该检测方法为肿瘤早期诊断和指导临床用药提供了一种灵敏度高、检测速度快、便于大规模筛查的有效手段.     

Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.     

Macrochelid mite fauna in the eastern part of the Lesser Sunda Islands, with description of two new species

Thirteen species in four genera of mites of the family Macrochelidae phoretic on dung beetles were collected in the eastern part of the Lesser Sunda Islands, Indonesia, providing the first record of the family for that area. Of these, two species, Macrocheles entetiensis Hartini and Takaku, sp. nov. and Macrocheles sumbaensis Hartini and Takaku, sp. nov., are new to science. The remaining 11 species are Glyptholaspis fimicola, Holostaspella bifoliata, Macrocheles baliensis, Macrocheles sp. aff. glaber, M. hallidayi, M. kraepelini, M. krantzi, M. limue, M. merdarius, M. oigru and Neopodocinum sinicum.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.     

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.     

北京血站献血员戊型肝炎流行病学调查

为了解献血员戊型肝炎感染情况,对2002年7～8月向北京市血液中心义务献血的所有人员进行整群抽样并抽血,应用ELISA检测戊型肝炎病毒(HEV)IgG的感染率.结果发现:北京献血员HEV IgG总感染率为26.59%,性别、年龄、省份分布存在着差别,男性比女性感染率高,年龄越大感染率越高,来自高感染省份者感染率也高,但ALT与HEV IgG感染无关.因此,男性、年龄大、来自高感染省份具有较高的HEV感染风险,进一步研究献血员的亚临床感染对HEV的输血安全性具有重要意义.     

SHIV1157ipd3N4细胞适应株的制备及其生物特性

目的体外制备SHIV1157ipd3N4病毒中国恒河猴细胞适应株,在细胞水平和中国恒河猴体内评价其生物学特性。方法用SHIV1157ipd3N4病毒阴道感染中国恒河猴,在血浆病毒载量高峰期采血分离外周血单核淋巴细胞（PBMCs）,与正常中国恒河猴PBMCs共培养。定期测定培养液中的P24抗原水平。当病毒复制达高峰期时收集培养上清,分装并冻存。测定病毒RNA载量、P24抗原浓度和TCID50。静脉感染中国恒河猴,研究该批次SHIV1157ipd3N4在体内的病毒学、免疫学指标变化及变异情况,分析其基本的生物学特性。结果本研究共制备了243 mL SHIV1157ipd3N4病毒原液,gp120序列分析表明病毒未发生变异,CCR5的嗜性也未发生改变。病毒载量为1.586×108 copies/mL,P24抗原水平为1.16×103 pg/mL,TZM-bl细胞测定病毒的TCID50为3.16×103/mL。1 mL SHIV1157ipd3N4静脉成功感染中国恒河猴G1004V,高峰期病毒载量达到1.0×106 copies/mL以上。结论此次制备的SHIV1157ipd3N4细胞适应株生物学特性稳定,适合作为毒种库构建SHIV1157ipd3N4/中国恒河猴模型。     

SHIV-XJ02170感染恒河猴后期的传代特点和env基因变异

目的研究SHIV-XJ02170在中国恒河猴感染后期传代过程中病毒和宿主的变化特点,并分析env基因的序列变异。方法将感染中国恒河猴G0401V后期（5年）的SHIV-XJ02170病毒垂直传代2只猴（G0401V→G0402V→0403V）,同时,剔除G0401V猴CD8＋T细胞使潜伏的病毒大量复制后传代1只猴（G0401V→G0404V）,应用流式细胞术、病毒载量测定、序列分析等方法研究该病毒在猴体内长期适应后的病毒和免疫学指标及序列变异特点。结果 G0401V在感染后期仍能稳定传代,且表现出毒力增强的特点。其传代猴G0402V在传代后41 d死亡,外周血CD4＋T淋巴细胞衰竭,仅为43个/mL,符合艾滋病感染猴快速进展型的特征。剔除体内CD8＋T细胞之后的传代猴G0404V的表现类似G0401V,即长期低水平的病毒血症水平。env基因序列分析发现SHIV-XJ02170在G0401V体内长期适应后发生了可遗传的序列变异,并引起糖基化位点的改变。结论 SHIV-XJ02170在猴体长期适应后的传代过程中表现出向强毒株过渡的特征,为进行SHIV-XJ02170感染性克隆的构建奠定了良好的实验基础。