胰岛素及胰高血糖素对HepG2细胞载脂蛋白CⅢ受体功能的影响

为寻求一种可替代人肝细胞研究载脂蛋白（apolipoprotein, apo）CⅢ受体的生理模型,并探讨apoCⅢ受体的生理功能及其在内源性高甘油三酯血症发病机制中的作用,首先以¹²⁵I标记的人apoCⅢ为配体,利用放射性配体结合分析法观察了人肝癌细胞系HepG2细胞是否有apoCⅢ受体存在.结果证实HepG2细胞上存在高亲和力的、可饱和的、特异的apoCⅢ受体结合位点,其受体的亲和力(K_d)和apoCⅢ受体结合容量(B_max)分别为(9.53±1.03)×10^-9 mol/L和(3.28±0.31) μg/g.随后又分别研究了胰岛素及胰高血糖素对HepG2细胞apoCⅢ受体功能的影响,结果表明：胰岛素可使HepG2细胞apoCⅢ受体结合容量（B_max）显著增加,但对受体的亲和力（K_d）无影响;胰高血糖素可使HepG2细胞apoCⅢ受体亲和力显著下降（即K_d值升高）,对受体的结合容量无影响.提示人肝apoCⅢ受体的功能可能受胰岛素及胰高血糖素的不同调节.     

人血管内皮细胞生长因子受体Flt-1胞外区cDNA在毕赤酵母中的表达和鉴定

将编码血管内皮细胞生长因子受体Flt-1胞外区1-3 loop 316个氨基酸残基的cDNA插入到含AOX1启动子和α分泌信号肽序列的Pichia pastoris酵母载体中,构建了重组表达质粒pPIC9K/Flt-1(1-3),转化酵母宿主菌GS115,筛选His⁺Mut^s表型转化子,经摇瓶培养,1%甲醇诱导表达4 d后,SDS-PAGE结果显示,培养上清中Flt1(13)表达量达总蛋白的30%以上。ELISA及Western blot实验表明,表达产物具有良好的抗原性和特异性。生物学活性检测证实其具有结合VEGF的能力和抑制VEGF对HUVEC细胞的促增殖功能。     

人T细胞系Jurkat表达特异性的C1q受体

采用酶联免疫吸附试验、流式细胞术、放射配体结合试验及蛋白质印迹等方法,首次证实了人Ｔ细胞系Jurkat表达特异性C1q受体,并对其特性进行了分析Jurkat可为C1q或抗人C1q受体抗体112识别,其对异硫氰酸荧光黄标记C1q的结合能被未标记C1q所抑制．在生理离子强度和温度条件下,Jurkat与 ¹²⁵I－C1q的结合呈持异、剂量依赖、可饱和及可逆性,每个细胞C1q结合位点数为1.1×10⁶,对C1q的K_a值为1.5×10⁷mol^-1,Hill系数为0.9643．Jurkat C1q受体识别C1q的胶原样区．该受体是分子量约70000的膜蛋白分子     

γ-氨基丁酸受体及其基因研究进展

γ-氮基丁酸(GABA)是脊椎动物脑内一种重要的神经递质.它与其特异性受体(即 GABAR)分子的相互作用,可引起该受体偶联的 Cl^-,K⁺和 Ca²⁺通道传导的改变并产生神经元抑制效应.近年 GABA_AR 基因及其表达的研究,已为不同的种属、不同脑区域和细胞类型中 GABA_AR 的亚基组成、生理功能及其对很多中枢神经系统药物反应的多样性提供了令人信服的依据,可以预见不久这方面深入的探索也必将为有关该受体的神经精神病发病的分子机理研究及其治疗性药物的设计提出新的线索.     

爪蟾卵母细胞表达猫脊髓背根神经节初级感觉神经元的速激肽受体

提取猫的背根神经节 (DRG)中的Poly(A) ⁺RNA ,注射到非洲爪蟾卵母细胞中进行表达 ,2d后通过双电极电压箝技术检测爪蟾卵母细胞对速激肽受体激动剂的反应 .NK- 1受体特异激动剂 [Sar⁹,Met(O₂ ) ¹¹]SP (Sar -SP) ,NK -2受体特异激动剂 [β- Ala⁸] neurokininA ( 4～ 1 0 ) (Ala -NKA)和神经激肽A(NKA)产生相似的内向电流 .由一个快速的锋电流和持续数分钟的振荡电流组成 .Sar- SP的反应只被NK- 1受体特异拮抗剂L- 6 6 8,1 6 9( 1 μmol/L)阻断 ,而NKA及Ala- NKA的反应则仅被NK -2受体特异拮抗剂L -6 5 8,877( 1 μmol/L)阻断 .注射猫脊髓背角Poly(A) ⁺RNA在爪蟾卵母细胞中表达的速激肽受体的反应基本相似 .这些速激肽受体的反应都具有强烈的脱敏性 .这是首次在爪蟾卵母细胞受体表达系统证明DRG神经元有速激肽NK- 1和NK -2受体 ,从而提示在伤害性初级传入末梢上可能存在速激肽突触前自身受体     

枯草杆菌中性蛋白酶基因在大肠杆菌中的表达

蛋白酶是枯草杆菌(Bacillus subtilis)产生的具有重要工业价值的水解酶。对蛋白酶基因的分离与高效率表达一直是基因工程研究领域的重要内容之一^[1-4]。蛋白酶基因的筛选可采用不同的方法,如“免疫法”、“DNA 杂交法”、“遗传互补法”等。大肠杆菌(Escherichia coli)是基因工程中最常用的宿主菌, 若能以E．Coli作为筛选蛋白酶基因的宿主苗,那么使用E．Coli的常规载体,便可直接获得完整的蛋白 酶基因。枯草杆菌的蛋白酶基因能否在大肠杆菌中表达．则是实现这一目标的关键。Koide等人^[5]报道过枯草杆菌的胞内丝氨酸蛋白酶基因在大肠杆菌中的表达。转化细胞在含有脱脂牛奶的平板上可产生十分微弱的水解圈。Ikeraara等人^[6]将Subtilisin E(枯草杆菌蛋白酶E)插人大肠杆菌的表达载体,具有活性的Subtilisin E便可分泌到大肠杆菌的细胞周质中。吴汝平撰文指出^[7]。克隆的枯草杆菌蛋白酶基因不能在大肠杆菌中表达。是因为大肠杆菌不能转录枯草杆菌的促使生长调节基因。Wang等人^[8]则认为,在大肠杆菌中观察不到野生型的中性蛋白酶基因E(nprE)的表达。是因为nprE的表达产物对大肠杆菌有致死作用．除去该基因上的核糖体结合位点,nprE便能在大肠杆菌中低水平表达,并能将表达产 物分泌至胞外。由上可知．枯草杆菌的蛋白酶基因能否在大肠杆菌中表达以及表达的位置仍然是一个众说纷纭的问题,这一问题也正是能否用大肠杆菌作为宿主菌筛选蛋白酶基因的关键。     

显色培养基在几种食源性致病菌快速检测中的应用*

用显色培养基鉴定微生物是一种新的微生物快速检测技术,该技术以生化反应为基础,通过在培养基中加入细菌特异性酶的显色底物直接根据菌落颜色对菌种作出鉴定。常见食源性致病菌检测中,李斯特菌显色培养基(BCM^TMListeriamonocytogenes,Rapid’LMONOagar,CHROMagar^TMListeria)、大肠杆菌显色培养基(CHROMagarTMEcoli)、沙门氏菌显色培养基(Rambachagar)、金黄色葡萄球菌显     

表皮生长因子及其受体和erb-B在鼠视网膜的表达

用免疫组织化学方法,对鼠视网膜的表皮生长因子(EGF)及其受体(EGF-R),和erb-B的表达,作了研究.结果表明:①EGF,EGF-R和erb-B分别在视网膜各层有不同表达;②EGF免疫反应着色不强,但有特异性地均匀分布在节细胞层(GCL)、内核层(INL)、外核层(ONL)和视网膜色素上皮层(RPE);③EGF-R免疫反应着色以GCL和INL的细胞明显,分布于细胞浆和细胞核,以细胞核更明显;④erb-B的分布类似EGF.但在ONL内出现一条较明显的着色带.对以上结果的可能意义进行了讨论.     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

研究: 肺炎衣原体抑制LXRα-ABCA1途径促进巨噬细胞脂质蓄积

为探讨肝X受体α (LXRα)-三磷酸腺苷结合盒转运体A1 (ABCA1)途径在肺炎衣原体 (C. pneumoniae)促巨噬细胞脂质蓄积中的作用和机制,以THP-1巨噬细胞源性泡沫细胞为模型,采用高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量,液体闪烁计数器检测细胞内胆固醇流出,RT-PCR检测ABCA1和LXRα mRNA的表达,蛋白质印迹检测ABCA1和LXRα的蛋白质表达;使用LXRα的特异性激动剂T0901317对细胞进行预处理,再观察上述指标的变化．结果显示,C. pneumoniae可促进THP-1巨噬细胞源性泡沫细胞内总胆固醇、游离胆固醇和胆固醇酯含量增加,抑制胆固醇外流,降低细胞ABCA1和LXRα的表达;使用ABCA1激动剂8-溴-环磷酸腺苷预处理细胞或LXR激动剂T0901317预处理细胞后,可明显减弱C. pneumoniae对THP-1细胞ABCA1的表达抑制,促进细胞胆固醇流出,降低细胞内胆固醇的含量．结果提示,C. pneumoniae促进巨噬细胞脂质蓄积及胆固醇流出障碍,其机制可能与LXRα-ABCA1途径有关．     

Sex differences in nuclear receptor-regulated liver metabolic pathways

Liver metabolism is markedly sex-dimorphic; accordingly, the prevalence of liver diseases is different between sexes. The superfamily of nuclear receptors (NRs) governs the proper expression of key liver metabolism genes by sensing lipid-soluble hormones and dietary lipids. When the expression of those genes is deregulated, disease development is favored. However, we lack a comprehensive picture of the differences between NR actions in males and females. Here, we reviewed explorative studies that assessed NR functions in both sexes, and we propose a first map of sex-dimorphic NR expression in the liver. Our analysis suggested that NRs in the female liver exhibited cross-talk with more liver-protective potential than NRs in male liver. This study provides empirical support to the hypothesis that women are more resilient to some liver diseases than men, based on a more compensative NR network. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.     

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

Astragalar morphology of late Eocene anthropoids from the Fayum Depression (Egypt) and the origin of catarrhine primates

The phylogenetic relationships of the late Eocene anthropoids Catopithecus browni and Proteopithecus sylviae are currently a matter of debate, with opinion divided as to whether these taxa are stem or crown anthropoids. The phylogenetic position of Catopithecus is of particular interest, for, unlike the highly generalized genus Proteopithecus, this taxon shares apomorphic dental and postcranial features with more derived undoubted catarrhines that appear in the same region 1-2 Ma later. If these apomorphies are homologous and Catopithecus is a stem catarrhine, the unique combination of plesiomorphic and apomorphic features preserved in this anthropoid would have important implications for our understanding of the crown anthropoid morphotype and the pattern of morphological character transformations that occurred during the early phases of stem catarrhine evolution.Well-preserved astragali referrable to Proteopithecus, Catopithecus, and the undoubted early Oligocene stem catarrhine Aegyptopithecus have provided additional morphological evidence that allows us to further evaluate competing hypotheses of interrelationships among Eocene-Oligocene Afro-Arabian anthropoids. Qualitative observations and multivariate morphometric analyses reveal that the astragalar morphology of Proteopithecus is very similar to that of early Oligocene parapithecids and living and extinct small-bodied platyrrhines, and strengthens the hypothesis that the morphological pattern shared by these taxa is primitive within crown Anthropoidea. In contrast, Catopithecus departs markedly from the predicted crown anthropoid astragalar morphotype and shares a number of apomorphic features (e.g., deep cotylar fossa, laterally projecting fibular facet, trochlear asymmetry, mediolaterally wide astragalar head) with Aegyptopithecus and Miocene-Recent catarrhines. The evidence from the astragalus complements other independent data from the dentition, humerus and femur of Catopithecus that support this taxon's stem catarrhine status, and we continue to maintain that oligopithecines are stem catarrhines that constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines.