酵母双杂交筛选与视黄醇结合蛋白有相互作用的蛋白质

为了研究视黄醇结合蛋白(RBP)在维生素A代谢调控中的作用和与相关疾病发生、发展的关系,以RBP作为诱饵基因构建了融合表达载体pGBKT7-RBP;在排除自身转录激活活性的基础上,与人肾脏cDNA预转库融合筛选(Pretransformed MATCHMAKER Lbraries),经营养缺陷型和X-α-gal显色鉴定,从中筛选出三种与RBP有相互作用的新蛋白质,并从人睾丸cDNA库中克隆了这三个基因的全长序列。     

猪细胞视黄醇结合蛋白基因1（CRBP1）内含子1的克隆测序

类维生素A(维生素A及其代谢产物)在动物的视觉、生长、繁殖、细胞的分化及胚胎发育等方面起重要作用.猪细胞视黄醇结合蛋白基因1(CRBP1)属于猪视黄醇结合蛋白基因家族(CRBPs).本研究将猪细胞视黄醇结合蛋白基因1(CRBP1)的内含子1进行了克隆测序,其大小为453bp.为进一步研究猪CRBP1基因的全序列奠定了一定的基础.     

TRF,Cys-C,RBP和Hcy联合检测对糖尿病肾病的早期诊断价值

目的探讨联合检测尿转铁蛋白(TRF)、血清胱抑素C(Cys-C)、视黄醇结合蛋白(RBP)和同型半胱氨酸(Hcy)对糖尿病肾病(DN)的早期诊断价值.方法依据糖尿病诊断标准和24 h尿白蛋白排泄率(UAER)的检查结果,将早期糖尿病肾病(DN)微量蛋白尿组患者40例(30 mg/24 h≤UAER≤300 mg/24 h)作为观察组A组,临床蛋白尿组患者40例(UAER300 mg/24 h)作为观察组B组,同期健康体检者40例作为对照组.比较3组TRF,Cys-C,RBP和Hcy水平.结果 1)TRF,Cys-C,RBP和Hcy在A,B组中明显升高,与正常对照组比较,差异具有统计学意义(P0.05).2)TRF,Cys-C,RBP和Hcy在B组中明显升高,与A组和对照组比较,差异具有统计学意义(P0.05).结论 TRF,Cys-C,RBP和Hcy联合检测对于早期糖尿病肾病(DN)的诊断具有重要意义.     

血清胱抑素C和视黄醇结合蛋白联合检测对慢性肾小球肾炎诊断价值的研究

目的探讨联合检测血清胱抑素C(Cys C)和视黄醇结合蛋白(RBP)在慢性肾小球肾炎诊断中的应用价值.方法选取50名慢性肾小球肾炎患者为观察组,50名健康体检者为对照组,分别对观察组和对照组血清Cys C以及RBP的含量进行测定,并分析其与慢性肾小球肾炎之间的关系.结果观察组和对照组Cys C的含量分别为(2.97±1.06),(0.95±0.62)mg/L,观察组明显高于对照组;RBP的含量观察组为(105±30.5)mg/L,相比于对照组的(38±10.3)mg/L明显升高;单项指标阳性率分别为Cys C 84%、RBP 76%,联合检测阳性检出率为90%,和单项指标相比阳性检出率明显提高,差异具有统计学意义(P0.05).对慢性肾小球肾炎患者的两项指标研究发现:血清Cys C和尿RBP的含量呈正相关(r=0.921,P0.05).结论慢性肾小球肾炎血清Cys C和RBP明显高于健康对照组,对慢性肾小球肾炎的诊断具有重要的临床价值.联合检测血清Cys C和RBP对慢性肾小球肾炎检出率和敏感性均明显高于单指标检测.     

能诱导糖尿病的蛋白

脂肪组织中的胰岛素作用遭受影响是Ⅱ型糖尿病的一个主要病因,现在一个以前未知的、也许能够促使这一过程发生的机制已被发现。曾有报道说,糖尿病患者血清中维生素A的一种结合蛋白RBP4的含量增加,但没有怀疑到二者之间有因果关系。“全面基因表达分析”方法被用来识别脂肪组织中GLUT4葡萄糖转移因子功能受损（肥胖症和糖尿病的特征）的小鼠体内其表达发生改变的基因,结果显示RBP4含量增加。     

HBV感染者血清RBP4的表达及临床意义

目的探讨乙型肝炎病毒(hepatitis B virus,HBV)感染者血清视黄醇结合蛋白4(RBP4)水平及与不同类型肝脏疾病间的关系,并分析感染者血清谷氨酸氨基转氨酶(ALT)、总胆红素(TBIL)与RBP4水平的相关性。方法对临床已确诊的乙型肝炎肝硬化患者(LC)18例、重型肝炎肝衰竭患者(LF)15例、慢性乙型肝炎(chronic hepatitis B,CHB)患者25例及健康体检者(Con)30例(对照组)用ELISA检测血清RBP4含量,RT-PCR检测血清中HBV-DNA载量,全自动生化仪检测血清中ALT和TBIL含量,并分析RBP4与HBV-DNA、ALT、TBIL的相关性。结果 LC、LF及CHB组患者血清RBP4含量与Con组比较,差异有统计学意义(P0.05);LC组患者血清RBP4含量与血清HBV-DNA载量呈负相关(P0.05);LC、LF组患者血清RBP4含量与ALT、TBIL均呈负相关(P0.05)。结论 HBV感染者血清RBP4水平可反映HBV的感染程度与肝损伤的程度,监测HBV感染者血清RBP4水平对于及时诊断治疗HBV感染具有一定意义。     

拟南芥中一个镍离子转运蛋白的亚细胞定位

离子的跨膜转运是细胞获取养分的重要环节，亦是植物在组织和器官水平上进行养分吸收运移的基础．在植物中镍（Ni）元素主要以Ni^2＋的形式存在，并通过Ni^2＋转运蛋白将其跨膜转运至相应的组织器官，参与氢酶和脲酶的合成．生物信息学分析表明，拟南芥中一个Ni^2＋转运蛋白AT2G16800含有叶绿体定位信息．克隆该基因5’端编码转运肽的272bp片段，与绿色荧光蛋白（GFP）基因融合后，在拟南芥中高效表达，对其进行了亚细胞定位的研究．转基因植株通过共聚焦扫描显微镜的观察，发现GFP荧光信号只存在于叶绿体中，该结果表明A他G16800为叶绿体蛋白．     

2型糖尿病患者性别、肥胖对血中RBP4和TNF-α的影响

目的:检测不同性别2型糖尿病患者正常体重组和肥胖组的血清视黄醇结合蛋白4(RBP4)和肿瘤坏死因子α(TNF-α)水平,探讨两者与体脂,糖类、脂类代谢,胰岛素敏感性等的相关性.方法:采用HOMA-IR评价各组胰岛素敏感性,测定受试者的体重指数(BMI),腰臀比(WHR),检测空腹状态下血清RBP4、TNF-α、血糖、HbA1c、血脂和胰岛素水平.结果:超重或肥胖者,RBP4及TNF-α浓度均比体重正常者高;男性RBP4浓度均比女性高,TNF-α浓度无性别差异.RBP4与TNF-α显著正相关.Pearson相关分析:不论性别,血清RBP4和BMI、WHR、TG及HOMA-IR均有显著正相关(P<0.05).而TNF-均与腹内型肥胖、脂代谢有关,TNF-α     

基于纳米金表面等离子体共振光散射法测定视黄醇结合蛋白

采用柠檬酸钠还原法制备了粒径约为12nm的纳米金颗粒,并用其标记视黄醇结合蛋白抗体(anti-RBP).在pH=5.29的KH_2PO_4-Na_2HPO_4缓冲溶液中,纳米金标记anti-RBP可与视黄醇结合蛋白(RBP)发生特异性结合使纳米金颗粒凝集成网格结构,由于纳米金颗粒的表面等离子体共振效应,使其在620nm处共振光散射信号显著增强.C_(RBP)在0.1~60ng·mL~(-1)范围内与共振光散射增强值ΔI_(620nm)线性相关,检出限为0.02ng·mL~(-1),用于定量分析人血清中的RBP,方法简单、灵敏,结果满意.     

高亲和力钠离子依赖二羧酸转运蛋白基因的克隆、表达及亚细胞定位研究

为了研究高亲和力钠离子依赖二羧酸转运蛋白(high affinity sodium-dependent dicarboxylate transporter,SDCT2,NaDC3)的功能,从小鼠肾组织中克隆出了其cDNA基因,并对其结构特征、基因表达谱及细胞内定位情况进行了分析。结果显示NaDC3蛋白由600个氨基酸组成,同源性分析表明其氨基酸序列与大鼠及人NaDC3分别有97％和87％相同。二级结构分析显示,该蛋白有13个跨膜α-螺旋区。Northern杂交显示该基因可在肾、肝、脑、胎盘等多种组织中表达。激光共聚焦显微镜观察显示该蛋白定位于肾小管上皮细胞膜上。     

Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes

In obesity and type 2 diabetes, expression of the GLUT4 glucose transporter is decreased selectively in adipocytes. Adipose-specific Glut4 (also known as Slc2a4) knockout (adipose-Glut4(-/-)) mice show insulin resistance secondarily in muscle and liver. Here we show, using DNA arrays, that expression of retinol binding protein-4 (RBP4) is elevated in adipose tissue of adipose-Glut4(-/-) mice. We show that serum RBP4 levels are elevated in insulin-resistant mice and humans with obesity and type 2 diabetes. RBP4 levels are normalized by rosiglitazone, an insulin-sensitizing drug. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Conversely, genetic deletion of Rbp4 enhances insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high-fat diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and impairs insulin signalling in muscle. Thus, RBP4 is an adipocyte-derived 'signal' that may contribute to the pathogenesis of type 2 diabetes. Lowering RBP4 could be a new strategy for treating type 2 diabetes.     

血清CYSC,RBP及传统肾功能指标对肾炎的早期诊断价值

目的探讨联合检测血清CYSC,RBP及传统肾功能指标对肾炎早期诊断价值.方法选取肾炎早期患者50例作为观察组Ⅰ组,肾衰患者50例作为观察组Ⅱ组,同时选取健康体检者50例作为对照组,检测3组血清CYSC,RBP及与传统肾功能指标的含量.分析CYSC,RBP对肾炎早期诊断价值.结果观察组Ⅰ组、Ⅱ组血清CYSC,RBP水平明显高于对照组,差异具有统计学意义(P0.05).观察组Ⅰ组BUN,Cr水平与对照组比较差异无统计学意义(P0.05).联合检测血清中CYSC,RBP含量阳性率高于单项检测指标,随着肾功能损害加重血清CYSC,RBP含量逐渐升高.结论血清CYSC,RBP对肾炎早期诊断具有重要价值,同时通过联合检测CYSC,RBP可提高对肾炎早期诊断的灵敏度.     

Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product

The E7 transforming protein of human papilloma virus-16 binds to the retinoblastoma gene product (pRb) through a nine-amino-acid segment of E7 (21-29). This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins. Each of these viral transforming proteins bind to the same region of pRb. To isolate cellular proteins that interact with this viral protein-binding domain on pRb, we used recombinant pRb to screen a human complementary DNA expression library. Two cDNAs were isolated that encode retinoblastoma binding proteins (RBP-1 and RBP-2). We report here that these RBP genes exist in separate loci and produce discrete messenger RNAs. The predicted amino-acid sequence of these genes showed no homology to known proteins, but both RBPs contain the pRb binding motif conserved between E7, large T and E1A14. In vitro expression of the RBP cDNAs yielded proteins that specifically bound to pRb. Recombinant E7 protein, the E7 21-29 peptide and the homologous RBP-1 peptide inhibited RBP-pRb binding. Mutations introduced into the putative pRb-binding segment in RBP-1 impaired its binding activity. These studies indicate that the cellular RBP-1, RBP-2 and viral E7 proteins interact with pRb through similar domains.     

细胞膜上的离子通道

离子通道是细胞膜上控制离子进出的功能蛋白,在细胞生命活动中发挥重要作用．离子通道具有对离子的选择性、通透的饱和性和开关的可控制性等特点;膜电压的变化、机械刺激和某些信号分子都可以调控离子通道开关;离子通道担负着离子吸收、渗透压调控、电冲动的形成和信号转导等重要的生理功能．离子通道的结构或功能失常会导致一些严重的疾病,对离子通道进行研究,寻找和设计调控离子通道的有效药物是治疗相关疾病的重要手段。     

嗜水气单胞菌感染暗纹东方鲀脾脏的蛋白质组学分析

为了解暗纹东方鲀(Takifugu obscurus)受到病原菌感染后参与应答的主要蛋白的功能与调控作用,采用TMT标记、高效液相色谱(HPLC)分级技术以及基于质谱的定量蛋白质组学技术,分析了暗纹东方鲀感染嗜水气单胞菌(Aeromonas hydrophila)12 h后脾脏组织的蛋白表达水平,鉴定差异表达蛋白,并通过亚细胞定位、GO(Gene Oncology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)对差异表达蛋白进行进一步分析.本次实验共鉴定到306个差异表达蛋白,其中120个上调表达蛋白,186个下调表达蛋白;利用平行反应检测实验(PRM)对4个(1个上调,3个下调)与免疫相关的差异蛋白进行验证,其结果与蛋白质测序结果一致;亚细胞定位结果显示:差异表达蛋白主要定位在细胞质、细胞核和胞外;GO分析发现:差异表达蛋白参与的主要生物进程有代谢过程、细胞进程和单一生物进程,主要的分子功能有结合、催化活性和机构分析活性;KEGG通路分析结果显示:差异表达蛋白主要参与了核糖体、视黄醇代谢、卟啉和叶绿素代谢、ECM受体相互作用、吞噬体、叶酸生物合成、药物代谢、酪氨酸代谢等通路.研究结果为进一步探究病原菌感染暗纹东方鲀的致病机理提供了一定的理论基础.     

Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells.

Replication of DNA occurs at discrete sites in eukaryotic cell nuclei, where replication proteins are clustered into large complexes, or 'replicases'. Similarly, viral DNA replication is a highly structured process, notably in herpes simplex virus type-1 (HSV-1; reviewed in ref. 4) in which large globular 'replication compartments' containing the viral replication machinery exist. Replicating cellular DNA redistributes to these compartments upon HSV-1 infection. We have now used antibodies raised against several cellular proteins to detect changes in their subnuclear localization on HSV-1 infection. We found that various proteins involved in cellular DNA replication move to sites of viral DNA synthesis, whereas a selection of non-replication proteins do not. The retinoblastoma protein and p53 (the products of two putative anti-oncogenes) relocate to the same sites as known DNA replication proteins, suggesting that they may be associated with DNA replication complexes in normal, uninfected cells.     

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.