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At least 649 records · Page 36Linked to original sources
PubMed1991

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.

Amino Acid Sequence↗
PubMed1989

Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum.

The primary event in the pathogenesis of severe malaria in Plasmodium falciparum infection is thought to be adherence of trophozoite- and schizont-infected erythrocytes to capillary endothelium, a process called sequestration. Identifying the endothelial molecules used as receptors is an essential step in understanding this disease process. Recent work implicates the membrane glycoprotein CD36 (platelet glycoprotein IV; refs 2-5) and the multi-functional glycoprotein thrombospondin as receptors. Although CD36 has a widespread distribution on microvascular endothelium, it may not be expressed on all capillary beds where sequestration occurs, especially in the brain. The role of thrombospondin in cell adhesion, in vitro or in vivo, is less certain. We have noticed that some parasites bind to human umbilical-vein endothelial cells independently of CD36 or thrombospondin. To screen for alternative receptors, we have developed a novel cell-adhesion assay using transfected COS cells, which confirms that CD36 is a cell-adhesion receptor. In addition, we find that an endothelial-binding line of P. falciparum binds to COS cells transfected with a complementary DNA encoding intercellular adhesion molecule-1. As this molecule is widely distributed on capillaries and is inducible, this finding may be relevant to the pathogenesis of severe malaria.

Antibodies, Monoclonal↗
PubMed2006

The dynamin-related mouse mitochondrial GTPase OPA1 alters the structure of the mitochondrial inner membrane when exogenously introduced into COS-7 cells.

Mutations in the dynamin family GTPase OPA1 are reportedly the cause of autosomal dominant optic atrophy, the most frequently occurring form of hereditary optic neuropathy. But although the involvement of structural abnormalities of the enzyme in this neurodegenerative disease is clear, little is known about the cell biological and biochemical functions of OPA1. Therefore, to better understand the pathogenesis of autosomal dominant optic atrophy, we precisely analyzed the effects of exogenously introducing mouse OPA1 (mOPA1) on mitochondrial morphology in COS-7 cells. We found that exogenously introducing wild type mOPA1 caused the mitochondria to become fragmented, and moreover caused the intermembrane space to accumulate on one side of the ring-shaped mitochondrial fragments. Immunoelectron microscopic observation of the mOPA1 transfectants confirmed that the structure of the mitochondrial inner membrane had changed dramatically, accumulating on one side of the mitochondrial structures. When cells were transfected with mOPA1 containing a loss of function mutation (K301A) within the G1 GTP-binding domain, mitochondrial fragmentation still occurred. The markers for intermembrane space and matrix showed the similar morphology, which was distinctly different from the finding obtained with wild type mOPA1 transfectants. Notably, we also observed that the effect of two OPA1 missense mutations (E270K and D273A) associated with autosomal dominant optic atrophy elicit effects similar to those seen with the dominant negative K301A mutant.

Animals↗
PubMed2000

Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

Animals↗
PubMed2002

Upregulation of stress response mRNAs in COS-7 cells exposed to cadmium.

Exposure of cells to cadmium (Cd) is known to stimulate the expression of various types of genes. These changes in gene expression are presumed to be related to the cellular response to Cd toxicity. To better understand the mechanisms related to Cd toxicity, suppression subtractive hybridization was carried out on COS-7 cells (African green monkey kidney cells) and the gene expression induced by Cd exposure was investigated. Heat shock protein (hsp) 10, 40, 60, 70, 89alpha and metallothionein II (MTII) mRNAs were found to be induced by Cd. This is the first report to describe the Cd-inducibility of hsp10, 40 and 89alpha mRNAs. Semi-quantitative reverse-transcription polymerase chain reaction showed the diverse expression patterns of these genes, depending on Cd concentration and exposure time. A marked elevation of hsp70 mRNA and induction of mRNA for the co-chaperone, hsp40, were detected. A relatively low level of hsp10 and hsp60 mRNAs was induced, with only a 2-fold increase within 24 h. Hsp89alpha mRNA was induced shortly after Cd exposure. These various induction patterns suggest that hsps play different roles in the cell against Cd toxicity.

Animals↗
PubMed1996

Molecular cloning of two novel transmembrane ligands for Eph-related kinases (LERKS) that are related to LERK-2.

A search of the nucleic acid database of expressed sequence tags (ESTs) revealed several partial cDNA sequences that could encode proteins homologous to the ligands for Eph-related kinases (LERKs). Oligonucleotides designed from the ESTs were used to probe a human brain cDNA library and obtain overlapping clones that encoded two different novel LERKS (NLERK-1 and NLERK-2). NLERK-1 and NLERK-2 are most closely related to human LERK-2/Elk-ligand and they form a subclass of LERKs that contain a transmembrane domain and a conserved cytoplasmic domain. Full-length NLERK-1 was expressed as a glycosylated membrane protein in COS cells and was not secreted into the medium. Full-length NLERK-2 was similarly expressed in COS cells but both membrane-bound and a truncated, proteolytically-released form were detected. Engineered forms of both NLERK-1 and NLERK-2 lacking transmembrane and cytoplasmic domains were also expressed in COS cells and each was detected in the extracellular medium.

Amino Acid Sequence↗
PubMed1996

Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins.

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.

Amidohydrolases↗
PubMed1991

Biosynthesis of reovirus-specified polypeptides: expression of reovirus S1-encoded sigma 1NS protein in transfected and infected cells as measured with serotype specific polyclonal antibody.

Polyclonal monospecific antibody was prepared against the reovirus serotype 1 Lang strain nonstructural sigma 1NS protein encoded by the S1 gene. The antibody was serotype-specific. The sigma 1NS protein of reovirus serotype 1, but not reovirus serotype 3, was recognized by the polyclonal antibody in both immunoprecipitation and Western immunoblot assays. The sigma 1NS protein expressed in vector-transfected COS cells was indistinguishable by immunoprecipitation and immunoblot analyses from the authentic sigma 1NS protein synthesized in virus-infected mouse L or monkey COS cells. The temporal appearance of sigma 1NS protein in virus-infected cells was similar to that of the other reovirus proteins. Both sigma 1NS and sigma 1, the two S1 gene products, were observed in the cytoplasm of COS cells by immunofluorescent microscopy, although their staining patterns were distinct from each other. However, sigma 1NS, but not sigma 1 or the other reovirion structural proteins, was also detected in the nucleoli of COS cells. These results suggest that sigma 1NS, like sigma 1, is a serotype-specific reovirus protein, but unlike sigma 1 is localized in part to the cell nucleus.

Animals↗
PubMed1991

A single amino acid distinguishes the high-responder from the low-responder form of Fc receptor II on human monocytes.

The low-affinity Fc receptor on human peripheral blood monocytes (Fc gamma RIIA) is polymorphic with respect to its ability to bind murine IgG1. The two allelic forms of the receptor, high responder (HR) and low responder (LR), yield characteristic patterns after isoelectric focusing and react differently with the anti-Fc gamma RII monoclonal antibody (mAb), 41H16. We recently cloned cDNA encoding the extracellular domains of Fc gamma RIIA on monocytes from one HR and two LR donors, and found that they differed at only a single base. The cDNA isolated from the HR donor had a G at position 519 and would be expected to encode an aginine at residue 133 in the mature protein, while the cDNA isolated from both LR donors had an A at position 519 and would be expected to encode a histidine at the same residue. To determine whether this single amino acid substitution actually accounts for the functional polymorphism involving Fc gamma RIIA, we transfected COS cells with full-length HR and LR Fc gamma RIIA cDNA, and examined them for their ability to react with anti-Fc gamma RIIA mAb and to bind red blood cells (RBC) coated with either murine IgG2b or murine IgG1. Whereas COS cells transfected with either the HR cDNA or the LR cDNA reacted with the anti-Fc gamma RII mAb, IV.3, and bound murine IgG2b-coated RBC, only COS cells transfected with the HR cDNA formed rosettes with murine IgG1-coated RBC and reacted strongly with mAb 41H16. A total of nine LR donors were identified, and all were homozygous for the A substitution at position 519. We conclude that at an A at position 519 in the cDNA encoding Fc gamma RIIA is the primary molecular basis for the LR form of the receptor, and that the amino acid at residue 133 determines whether Fc gamma RIIA efficiently binds murine IgG1.

Adult↗
PubMed2003

Mice expressing only covalent dimeric heparin binding-deficient lipoprotein lipase: muscles inefficiently secrete dimeric enzyme.

Lipoprotein lipase (LpL) hydrolyzes triglycerides of circulating lipoproteins while bound as homodimers to endothelial cell surface heparan sulfate proteoglycans. This primarily occurs in the capillary beds of muscle and adipose tissue. By creating a mouse line that expresses covalent dimers of heparin-binding deficient LpL (hLpLHBM-Dimer) in muscle, we confirmed in vivo that linking two LpL monomers in a head to tail configuration creates a functional LpL. The hLpLHBM-Dimer transgene produced abundant activity and protein in muscle, and the LpL was the expected size of a dimer (approximately 110 kDa). Unlike the heparin-binding mutant monomer, hLpLHBM-Dimer had the same stability as nonmutated LpL. The hLpLHBM-Dimer transgene prevented the neonatal demise of LpL knockout mice; however, these mice were hypertriglyceridemic. Postheparin plasma LpL activity was lower than expected with the robust expression in muscle and was no longer covalently linked. Studies in transfected cells showed that Chinese hamster lung cells, but not COS cells, also degraded tandem repeated LpL into monomers. Thus, although muscle can synthesize tethered, dimeric LpL, efficient production of this enzyme leading to secretion, and physiological function appears to favor secretion of a noncovalent dimer composed of monomeric subunits.

Amino Acid Sequence↗
PubMed1998

Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells.

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (approximately 5 microM) than those in the cytoplasm (approximately 100 nM); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (approximately 420 nM) were twice those measured by connexin-32 chimera (approximately 200 nM); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (approximately 130 nM). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.

Aequorin↗
PubMed2001

Use of human recombinant DNase I expressed in COS-7 cells as an immunogen to produce a specific anti-DNase I antibody.

To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.

Animals↗
PubMed1997

Ras-dependent signaling by the GTPase-deficient mutant of Galpha12.

Galpha12 and Galpha13 regulate diverse responses through the small GTPases Ras, CDC42, Rac, and Rho. Whereas they activate similar responses in many different cell types, they also activate more specific and critical signaling pathways in other cell types. In COS cells, in which both Galpha12 and Galpha13 stimulate Na+/H+ exchange, they do so by activating different signaling pathways. Here we report that the differential recruitment of specific small GTPases by Galpha12 and Galpha13 defines the molecular basis for their functional differences. We have observed that the stimulation of Na+/H+ exchange by the GTPase-deficient mutant of Galpha12 (Galpha12QL) requires a functional Ras and is independent of Rac/CDC42 and Jun kinase signaling module. By contrast, the stimulation of Na+/H+ exchange by Galpha13QL requires a functional Rac/CDC42 and the Jun kinase signaling module. Our results also indicate that Galpha12QL-Ras stimulation of Na+/H+ exchange involves a D609-sensitive phospholipase and protein kinase C. These studies, for the first time, describe a novel Galpha12-specific signaling pathway involving Ras, phosphatidylcholine hydrolysis, and protein kinase C in the regulation of Na+/H+ exchange.

Animals↗
PubMed1998

Ectopic expression of Nramp1 in COS-1 cells modulates iron accumulation.

Nramp1 (natural resistance-associated macrophage protein) controls innate immunity and encodes a transporter of unknown function. Here we describe an antibody to Nramp1 displaying immunoreactivity towards a mature heavily glycosylated polypeptide of 90-100 kDa and a precursor form of 45 kDa in macrophages. Ectopic expression of the Nramp1 cDNA in COS-1 cells demonstrates that Nramp1 modulates cellular iron levels following loading with low molecular weight iron chelates. Surprisingly, Nramp1 does not enhance iron uptake, but expression is associated with reduced cellular iron loads. We propose Nramp1 may play a role in a salvage pathway of iron recycling.

Animals↗
PubMed1990

Human papillomavirus type 16 DNA expresses a replication modulation factor in cos-1 cells.

Plasmid DNA including the simian virus 40 (SV40) origin of replication undergoes uncontrolled, runaway replication in cos-1 cells owing to the intracellular production of SV40 large T antigen. Covalent linkage of such plasmids with human papillomavirus type 16 (HPV-16) DNA was found to prevent runaway replication. Replication control was found to be dependent on the presence of HPV-16 DNA sequences including the E1 open reading frame and part of the non-coding region.

Animals↗
PubMed1994

DNA sequence affects nucleosome ordering on replicating plasmids in transfected COS-1 cells and in vitro.

Nucleosome ordering on a variety of replicating plasmids, assembled into chromatin in transfected COS-1 cells, was studied by micrococcal nuclease digestion of isolated nuclei. Generally, no more than three well defined multiples of a unit nucleosome repeat, which resembled the first three bands of the (187 +/- 5 base pair (bp)) cellular chromatin ladder, could be detected in constructs that contained a near-minimal SV40 replication origin. In contrast, constructs that additionally contained the SV40 early region exhibited significantly more regular nucleosome arrangements. In some cases, eight to nine multiples of a 203 +/- 5-bp repeat could be resolved. The presence of the SV40 early region was necessary for physiological nucleosome alignment over the SV40 late and ori regions, or onto adjacent pBR327 DNA. In an in vitro chromatin assembly system, using purified chicken erythrocyte histones plus polyglutamic acid, a portion of SV40 DNA became packaged into a highly ordered 200 +/- 5-bp nucleosome array, which encompassed the early region and extended for about 2800 bp. The data suggest that in the cell nucleus, nucleosome ordering on SV40 DNA might spread from sequences in the early region, close to where transcription terminates, probably as a consequence of histone H1-nucleosome interactions.

Animals↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8.

In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.

3',5'-Cyclic-AMP Phosphodiesterases↗