黏着斑激酶和细胞外信号调节激酶在SACC-LM和SACC-83细胞中的表达及其意义

目的:检测黏着斑激酶(FAK)及细胞外信号调节激酶(ERK)在涎腺腺样囊性癌(SACC)细胞系SACC-LM和SACC-83中的表达,探讨其与SACC肺转移发生的关系.方法:选择停止在G1/S期的SACC-LM和SACC-83细胞,测定其细胞内FAK和ERK酶活力,采用Western blotting方法检测SACC-LM及SACC-83细胞中FAK及ERK蛋白表达,对其结果进行量化分析.结果:FAK及ERK在SACC-LM细胞中的活性均高于SACC-83细胞(P<0.01),SACC-LM细胞中FAK酶活性与ERK酶活性呈正相关关系(r=0.62,P<0.05).Western blotting方法检测到在SACC-LM细胞中两种蛋白的表达水平均高于在SACC-83细胞中的表达水平(P<0.05).结论:FAK及ERK可能参与了SACC的发生发展过程和转移信号转导通路,FAK及ERK蛋白表达变化与SACC的转移行为有关.     

表达Cre重组酶Cre-pCEP4载体的构建及其在Cre/loxP重组系统中的应用

目的:构建表达Cre重组酶的载体Cre-pCEP4,并验证其能够有效识别loxP位点,为人类疾病动物模型的建立提供依据.方法:构建重组载体Cre-pCEP4和pStop-eGFP,利用Fugene HD转染猪胚胎成纤维细胞(PEF)和MCF-7细胞系,利用荧光显微镜观察绿色荧光蛋白表达情况.结果:成功构建重组载体Cre-pCEP4和pStop-eGFP,将2个载体瞬时共转染PEF;经潮酶素B筛选出Cre重组酶稳定表达的MCF-7细胞系瞬时转染pStop-eGFP,在荧光显微镜下观察2种细胞均有绿色荧光蛋白的表达.而单独转染pStop-eGFP的MCF-7细胞系和PEF均未见绿色荧光蛋白的表达.结论:重组载体Cre-pCEP4在细胞内能够表达Cre重组酶,并且表达的Cre重组酶能够识别loxP位点,删除两同向loxP间的DNA片段.     

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.     

Cloning and squencing of human thrombopoietin (hTPO) cDNA and it''s expression in COS-7 cells]

Viliuisk encephalomyelitis (VE) is an unique neurological disease occurring in the Iakut (Sakha) people of Siberia. Evolution of the disease follows one of three broad clinical forms: subacute, slowly progressive or chronic. Death occurs within 3 to 6 months in subacute cases and within 6 years in the slowly progressive cases. Chronic cases lack a subacute phase but show a slowly progressive dementia associated with bradykinesia, dysarthria and spastic paraparesis that stabilizes late in the disease process. In subacute and slowly progressive cases, focal necrotizing encephalomyelitis is seen at necropsy. Chronic cases show multifocal areas of lysis with a gliotic margin, predominantly within grey matter, lacking associated chronic inflammatory changes seen in the other forms of the disease. Epidemiological studies are consistent with a disease of low-grade communicability, but laboratory studies have so far failed to reveal an infectious organism. The spectrum of neuropathological changes are reviewed in this examination of 11 cases. Although the aetiology of VE remains obscure, further studies are warranted since it may represent a novel disease process.     

cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells

Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.     

Assembly and secretion of recombinant chains of human inter-alpha-trypsin inhibitor in COS-7 cells

The inter-alpha-trypsin inhibitor (ITI) family is a group of structurally related plasma serine protease inhibitors. The ITI family members consist of combinations of mature heavy chains named HC1, HC2, HC3 linked to bikunin (a Kunitz-type protease inhibitor) by a covalent interchain protein-glycosaminoglycan-protein cross-link. The biosynthesis of the ITI family members takes place in the liver. In this report we examine the biosynthesis of these proteins using transient transfected COS-7 cells expressing one or more combinations of human ITI chains. The processing and secretion of alpha1-microglobulin and bikunin does not require the ITI heavy chains. A small proportion of the H3 chain seems to be processed into the HC3 form in the absence of the other ITI chains. In contrast, the processing of H2 into HC2 needs the presence of the L chain. The COS-7 cells are able to link the HC2 and HC3 heavy chains with bikunin by means of a chondroitin sulfate bridge, and thus to generate 260-kDa ITI-like proteins as well as pre-alpha-trypsin inhibitor (PalphaI). However, the maturation of the Hl chain into HC1 and the assembly of HC1 inside multichain proteins may take place according to a mechanism which differs from that of the H2 and H3 chains. These results indicate that the assembly of the constituent chains of the ITI-like proteins and PalphaI is not dependent on the liver machinery.     

CD36 forms covalently associated dimers and multimers in platelets and transfected COS-7 cells

CD36 is a transmembrane glycoprotein expressed on the surface of a number of cell types. The analysis of CD36 from platelets using immunoblotting, gel filtration, and native PAGE indicated the presence of high molecular complexes exceeding the Mr of monomeric CD36. Experiments using transfected COS-7 cells revealed these complexes were homodimers and -multimers of CD36. The multimers could be dissociated by treatment with a reducing agent, indicating they were formed by intermolecular cysteine-bridging. Mutagenesis of the cDNA for CD36 implicated the cysteines in the extracellular domain of the molecule. The potential physiological roles of CD36 multimerisation are discussed.     

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.     

Intracellular retention of the corticotrophin-releasing hormone (CRH) precursor within COS-7 cells

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.     

Lymphocyte-specific protein 1 expression in eukaryotic cells reproduces the morphologic and motile abnormality of NAD 47/89 neutrophils

Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, "hairlike" F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.