Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “COS Cells”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1994

Single-chain mono- and bispecific antibody derivatives with novel biological properties and antitumour activity from a COS cell transient expression system.

Single-chain antibody molecules were expressed from modified eukaryotic expression vectors as individual protein domains encoded on interchangeable cDNA cassettes. Two different single-chain antibody derivatives were constructed by linking individual light- and heavy-chain variable domains. The first was specific for the L6 tumour-associated antigen and the second was specific for human CD3. Each single-chain variable domain was genetically fused with an Fc 'tag' and expressed as a fusion protein in a COS cell transient transfection system. These single-chain antibody derivatives demonstrated specific binding to cells expressing appropriate antigen and bound with affinities similar to native antibody. The CD3 single chain molecule mediated stronger activation of PLC gamma 1 and similar levels of T-cell proliferation compared with native antibody. A bispecific Fv single-chain cassette was created by fusing the expression cassettes encoding the binding domains for L6 and CD3 single-chain molecules using oligonucleotide primers encoding a short 27-residue 'helical' peptide linker. The CD3-L6 variable domains were fused to the Fc tag and expressed in COS cells. The CD3-L6FvIg bispecific fusion protein mediated adhesion between T cells and L6-positive tumour cells, and stimulated potent T-cell proliferation and cytotoxicity against tumour cells expressing the L6 antigen.

Amino Acid Sequence↗
PubMed1995

Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active.

We have expressed a cDNA for virus-induced chick interferon in Escherichia coli. The product, a 19-kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q-Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 10(8) IU/mg and is a powerful inducer of the interferon-responsive promoter of the chicken Mx gene. Culture medium of transfected COS cells expressing full-length chick interferon cDNA contained up to 5 x 10(4) IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23-28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein.

Animals↗
PubMed1995

Morphology and antigenicity of recombinant B19 parvovirus capsids expressed in transfected COS-7 cells.

COS-7 cells transfected with parvovirus B19-simian virus 40 (SV40) hybrid vectors have previously been shown to express B19 structural proteins. In this study the morphology and antigenicity of B19 proteins expressed in these cells were investigated. At 84 h after transfection, approximately 10% of the COS-7 cells expressed B19 antigen, and the yield was equivalent to 2 x 10(3) to 2 x 10(5) B19 particles/transfected cell. The B19 proteins self-assembled into capsids that were morphologically and antigenically similar to native B19 virions, and could substitute for native antigen in a B19 IgM assay. Recombinant capsids lacking the recently described 11 kDa protein also resembled native virions.

Animals↗
PubMed1992

Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes.

We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a lambda gt10 human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IV. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplasmic CAs (CA I, CA II, and CA III), and an additional 27-amino acid C-terminal sequence that ends in a 21-amino acid hydrophobic domain. Of the 17 "active site" residues that are highly conserved in other human CAs, 16 are also present in CA IV. Expression of the cDNA in COS cells produced a 35-kDa enzyme that was membrane associated, resistant to inactivation by SDS, contained no carbohydrate, and reacted on Western blots with antiserum to the 35-kDa CA IV from human lung. Treatment of membranes from transfected COS cells with phosphatidylinositol-specific phospholipase C released 20-30% of the expressed enzyme from membranes, indicating that at least 20-30% of the expressed enzyme was anchored to membranes by a glycosyl-phosphatidylinositol linkage.

Amino Acid Sequence↗
PubMed1995

Novel tricyclic inhibitors of farnesyl protein transferase. Biochemical characterization and inhibition of Ras modification in transfected Cos cells.

Ras protooncogenes encode 21-kDa membrane-associated guanine nucleotide-binding proteins, which play a critical role in control of cellular proliferation and differentiation. Oncogenic, activated forms of Ras proteins are associated with a broad range of human cancers. The elucidation of the post-translational modifications that occur at the carboxyl terminus of Ras and the demonstration that farnesylation of Ras by farnesyl protein transferase is essential for Ras-induced cellular transformation has opened up a new and promising approach to the development of anti-Ras therapeutics. We report here a novel series of potent farnesyl protein transferase (FPT) inhibitors, represented by SCH 44342. This compound inhibits both rat brain and recombinant human FPT with an IC50 of approximately 250 nM, while it is only weakly active against rat brain geranylgeranyl protein transferase-1 (IC50 > 114 microM). FPT inhibition has been observed using both Ha-Ras protein and Ki-Ras-derived peptide substrates in two different assay formats. SCH 44342 and its analogs also inhibit farnesylation of Ras in Cos cells transiently expressing [Val12]Ha-Ras with IC50 values in the low micromolar range. At these concentrations they do not inhibit sterol biosynthesis or geranylgeranylation of protein. In addition, we observed that Cos cells undergo pronounced morphological changes upon overexpression of [Val12]activated forms of Ha-Ras containing COOH-terminal sequences allowing farnesylation (CVLS) or geranylgeranylation (CVLL) but not upon overexpression of activated Ras lacking the isoprenylated Cys (SVLS). Ras-induced morphological changes can be partially reverted with lovastatin. Importantly, SCH 44342 can block morphological changes induced by [Val12]Ha-Ras-CVLS but not [Val12]Ha-Ras-CVLL. Recently, a number of other FPT inhibitors have been reported. Most of the compounds reported to have cell-based activity are peptidomimetic analogs of the CAAX substrate. Our FPT inhibitors are novel in that although they compete with Ras protein in kinetic experiments they are entirely nonpeptidic in nature, they do not have oxidizable sulfhydryl functions, and they are active in cells at low micromolar concentrations.

Alkyl and Aryl Transferases↗
PubMed1995

COS cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies.

We report the deduced protein sequence and preliminary characterization of Pfg377, a novel sexual stage antigen of Plasmodium falciparum. An initial cDNA clone (Pfg377-1) encoding the N-terminal 755 amino acids of Pfg377 was isolated by transfecting a 3D7 gametocyte cDNA library into COS7 cells and selecting using a pool of anti-Pfs230 monoclonal antibodies. The protein encoded by Pfg377-1 included an N-terminal hydrophobic signal sequence, but no apparent transmembrane anchor. Instead, the particular cDNA clone selected was fused in-frame at its 3' end with the coding sequence for the human decay acceleration factor membrane anchor, which had been deliberately placed downstream of the vector polylinker in order to attach potential fusion proteins onto the COS cell surface. Northern blots probed with the Pfg377-1 cDNA demonstrated cross-hybridization to a single approximately 9.5-kb transcript, which was present only in sexual stages, and not in a sexual stages. DNA hybridization was used to obtain a series of overlapping genomic clones which collectively yielded the complete DNA sequence for Pfg377. There are no introns within the gene, which contains a 9360-bp open reading frame and encodes a 377-kDa protein. The Pfg377 protein is highly hydrophilic, and has an essentially non-repetitive structure, with only four very limited regions of tandem repeats. The Pfg377 gene resides on chromosome 12, and immunoelectron microscopy with two different anti-Pfg377 polyclonal antisera raised against two separate recombinant sub-fragments of the protein both indicated that the antigen is located in electron-dense organelles of the gametocytes--the osmiophilic bodies--which are proposed to play a role in parasite emergence from the erythrocyte during gametocyte maturation in the Anopheles mosquito midgut. Although it was selected with anti-Pfs230 antibodies, comparison of the sub-cellular locations and protein sequences of Pfg377 and Pfs2 show them to be completely distinct antigens. We hypothesize that Pfg377-1 was initially isolated because it expresses an epitope which is recognized by (i.e., cross-reacts with) one of the anti-Pfs230 monoclonal antibodies used to select the original transfected COS cells.

Amino Acid Sequence↗
PubMed2003

Ochratoxin A affects COS cell adhesion and signaling.

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.

Animals↗
PubMed1989

Expression and secretion of chicken apolipoprotein AI in transfected COS cells.

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.

Acylation↗
PubMed1995

Cloning of a cDNA encoding a 190-kDa insulin receptor substrate-1-like protein of simian COS cells.

Major insulin signals such as stimulation of glucose uptake and DNA synthesis and modification of hexose metabolism are mediated by the tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1; pp180) in many species of cells. We cloned cDNA encoding a 190-kDa IRS-1-like protein (pp190) in simian COS cells and which is slightly larger than IRS-1 (pp180) of human, rat, and mouse cells. The deduced amino acid sequence of COS pp190 consisted of 1251 amino acids and was 96.4%, 87.9% and 88.7% identical to human, mouse and rat IRS-1. The COS pp190 bound to SH2 (src-homology 2) domains of p85, Grb2/Ash, and SH-PTP2, as did IRS-1. In IRS-1-knockout mice, insulin signals are thought to be mediated by IRS-2 (pp190), which is an alternative signaling molecule and is slightly larger than IRS-1. However, the COS pp190 may be a simian homologue of IRS-1, but not of IRS-2. The results of Southern blotting suggested the possibility that Chinese hamster ovary (CHO) cells have not only the IRS-1 gene but also a gene related to the COS pp190.

Amino Acid Sequence↗
PubMed1998

Studies on intracellular structures of COS cells by X-ray microscopy.

COS-7 cells, fixed with glutaraldehyde, were studied using the transmission X-ray microscope at the electron storage ring BESSY, Berlin. The border of the cell, the nucleus, nucleoli and mitochondria of the cells were clearly visualized with the X-ray microscope. In addition, we found many X-ray dense granules preferentially located around the nucleus. Electron microscopy showed that numerous multivesicular bodies, whose structures belong to the endosome-lysosomal system, were present around the nucleus. The size and localization patterns of the X-ray dense granules were quite similar to those of multivesicular bodies. These results strongly suggest that the X-ray dense granules are multivesicular bodies.

Journal Article↗
PubMed1995

A COS-cell-based system for rapid production and quantification of scFv::IgC kappa antibody fragments.

Single-chain Fv (scFv) antibody (Ab) fragments were transiently produced in COS-1 cells utilizing a mammalian expression vector featuring a murine immunoglobulin (Ig) light-chain leader sequence for efficient secretion and a murine Ig kappa constant domain (IgC kappa) for detection. Several hundred milliliters of supernatants from large-scale COS cell transfections were sufficient to purify the scFv::IgC kappa fusion proteins by one-step affinity chromatography utilizing an immobilized rat anti-mouse IgC kappa monoclonal Ab. Furthermore, the murine IgC kappa domain allowed for accurate quantification of the scFv::IgC kappa fusion protein secreted into the COS cell supernatant by a sandwich enzyme-linked immunosorbent assay (S-ELISA).

Animals↗
PubMed1991

Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells.

cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis.

Adenosine Monophosphate↗
PubMed1992

Parkinsonism-inducing neurotoxin MPP+: uptake and toxicity in nonneuronal COS cells expressing dopamine transporter cDNA.

Expression of a cloned dopamine transporter complementary DNA in COS cells allows these primate kidney cells to accumulate the parkinsonism-inducing neurotoxin metabolite MPP+ (1-methyl-4-phenylpyridinium) avidly, and MPP+ toxicity results. By documenting that the dopamine transporter can confer MPP+ sensitivity to nonneural cells, these results highlight the key role that this transporter could play in mechanisms underlying parkinsonism.

1-Methyl-4-phenylpyridinium↗
PubMed1994

Phosphatidic acid activation of protein kinase C-zeta overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids.

Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.

Animals↗
PubMedDate not supplied

Antisense oligodeoxynucleotide complementary to CXCR4 mRNA block replication of HIV-1 in COS cells.

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatment of acquired immunodeficiency syndrome, we examined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation initiation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2 microM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natural phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This study defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.

Animals↗
PubMed1997

Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells.

The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the anti-depressant drug rolipram (4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone). In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE{HSPDE4A4B[Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell. Biol. 13, 6558-6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 microM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a K(m) for cAMP of 3.3 microM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.

Cell Line↗
PubMed1992

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms.

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulié. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulié. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH-terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:776-785; Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulié. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non-amphiphilic monomers, demonstrating the importance of the T COOH-terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms.

Acetylcholinesterase↗