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At least 199 records · Page 11Linked to original sources
PubMed1992

Expression of the high responder/non-responder human Fc gamma RII. Analysis by PCR and transfection into FcR-COS cells.

Distinct differences in the capacity of monocyte Fc gamma RII of different individuals to bind or not bind mouse IgG1 defines a polymorphism of Fc gamma RIIa and has previously been defined as the high responder (HR) or low responder (LR) polymorphism of Fc gamma RII. The precise definition of the molecular basis of the human HR/LR polymorphism of Fc gamma RIIa from the peripheral blood mononuclear cells of normal individuals has been determined by anti-CD3 induction of T cell proliferation, the polymerase chain reaction (PCR), nucleotide sequencing, transfection and IgG binding. Amplification of first strand cDNA from mRNA isolated from mononuclear cells was performed by PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of Fc gamma RIIa, which is normally expressed in monocytes. Sequencing of the PCR products and transfection of these to Fc gamma R- cells indicated that in Fc gamma RIIa of HR or LR individuals: (i) three nucleotide substitutions (CA to TG and G to A) resulted in the change of glutamine to tryptophan at position 27 (first extracellular domain) and arginine to histidine at position 131 (second extracellular domain); (ii) expression of cDNA encoding the various combinations of these indicated that arginine at position 131 was essential for IgG1 binding whereas the amino acid changes at position 27 had no effect; and (iii) IgG1 at high concentration bound to all allomorphic forms of Fc gamma RIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites, Antibody↗
PubMed1988

Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP.

Adenoviruses, Human↗
PubMed1986

Secretion of phytohemagglutinin by monkey COS cells.

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.

Animals↗
PubMed1998

The purification and identification of heme oxygenase isoforms from spleen tissue of rat and the expression of heme oxygenase-1 cDNA in COS-1 cells.

OBJECTIVE: To demonstrate that heme oxygenase (HO) isoforms exist in rat spleen treated with hematin and phenylhydrazine and to confirm that the isolated cDNA actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells) in order to prepare HO-1 mutant for inhibiting the natural enzyme. METHODS: The rat spleen microsomal fractions were first purified by diethylaminoethyl (DEAE)-Sephacel and hydroxylapatite. The activity of two isoforms (HO-1 and HO-2) of enzyme and their apparent molecular weight on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) were measured. Secondly, using the isolated HO-1 cDNA clone, the expression plasmid pcDNA3HO1 was constructed and transfected into cultured COS-1 cells. The transfected cells were collected and disrupted by sonication, and the microsomes were prepared by ultracentrifugation. The activity of HO-1 was measured. RESULTS: Two isoforms were purified and identified in treated rat spleen and HO-1 was the predominant form. The ratio of HO-1 to HO-2 was 3.2:1. The apparent molecular weights of HO-1 and HO-2 were about 30 kD and 36 kD under reducing conditions, respectively. The HO-1 was highly expressed in endoplasmic reticulum of transfected cells. The specific band was located in molecular weight of 30 kD. The specific activity was five times higher than that of the control. CONCLUSION: The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It is suggested that this clone having an insert of 1030 base-pairs encodes HO-1 and that we can prepare HO-1 mutant by site-directed mutagenesis of HO-1 cDNA to prevent and treat hyperbilirubinemia.

Animals↗
PubMed1999

Over-expression of GAPDH induces apoptosis in COS-7 cells transfected with cloned GAPDH cDNAs.

The cDNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) were cloned from cerebellar neurons undergoing age-induced apoptosis and/or healthy cells. COS-7 cells were transfected with the isolated GAPDH cDNAs using to the Lipofectamine method. Assessment of cell death in this paradigm was performed by monitoring the co-transfected luciferase activities and the characterization of cell death was examined by the DNA fragmentation assay and Hoechst dye nuclear staining. These observations show that over-expression of GAPDH occurring from both cDNAs robustly induces apoptotic death in the transfected COS-7 cell cultures. Confocal-immunocytochemical studies using this GAPDH-specific monoclonal antibody revealed that nuclear translocation of overexpressed GAPDH is a primary apoptotic event. Our results directly demonstrate that over-expressed GAPDH functions as a 'killing protein' in apoptosis.

Animals↗
PubMed1996

Negative regulatory activity of a prostaglandin F2 alpha receptor associated protein (FPRP).

The cDNA has been cloned for a protein which copurifies with and colocalizes with [3H]PGF2 alpha binding activity. This cloning was based on prior purification of the [3H]PGF2 alpha binding complex from pregnant corpus luteum, antibody production against the protein of interest, and antibody screening of a rat ovary cDNA expression library. Here I report on the activity of this prostaglandin F2 alpha receptor (FP) associated protein (FPRP). Expression of the FPRP cDNA in COS cells results in production of a full length (approximately 130 kD) immunoreactive molecule with an endoplasmic reticulum and Golgi network distribution similar to that seen in granulosa lutein cells. COS cell expressed FPRP inhibits binding of [3H]PGF2 alpha to FP of COS cell origin or FP expressed from cotransfected rat or mouse FP cDNA in a dose-dependent manner. This inhibition of [3H]PGF2 alpha binding by FPRP occurs only when the FPRP cDNA is expressed in the same cell as the FP resides, reaches a maximum of approximately 80%, and is unaffected by second messenger perturbing agents such as phorbol ester, 8-Br-cAMP, calcium ionophore A23187, and okadaic acid. Scatchard analysis indicates that FPRP induces a decrease in receptor number rather than affinity constant, suggesting a non-competitive means of inhibition. Molecular dissection of the FPRP protein indicates that two portions of the molecule play a role in the inhibition of FP. Whether FPRP is an FP-associated regulatory molecule, an FP subunit, or a receptor for a PGF2 alpha-antagonistic ligand is presently unknown. Physiological relevance and significance of FPRP are discussed. During the course of these experiments it was necessary to clone the rat FP cDNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Comparative levels of CALLA/neutral endopeptidase on normal granulocytes, leukemic cells, and transfected COS-1 cells.

We discovered that the common acute lymphoblastic leukemia antigen, CALLA (CD10), was identical to human neutral endopeptidase 3.4.24.11 (NEP), a Zn-binding glycoprotein with an extracellular active site capable of hydrolyzing several biologically active peptides. In this study we compare the expression of CALLA/NEP in terms of antigenic density and enzymatic activity at the cell surface and of messenger RNA (mRNA) levels on granulocytes, leukemic cells, and CALLA-transfected COS-1 cells. Mature granulocytes, the only readily available source of normal human CALLA, express relatively low but constant levels of antigen, NEP activity (3.5 pmol/min/10(6) cells), and mRNA. The two major CALLA-mRNA species of 6.5 kb and 3.8 kb, observed to date in a variety of cells and tissues, were also found in four independent granulocyte preparations. With leukemia cell lines, a correlation was established between the density of CALLA antigen and the level of enzymatic activity (3.4 to 21.0 pmol/min/10(6) cells). This paper constitutes the first report of NEP activity on blast cells derived from patients with non-T acute lymphoblastic leukemia (ALL); the levels of activity were variable (1.5 to 35.9 pmol/min/10(6) cells for six cases) but correlated with the level of CALLA assessed by flow cytometry. Heterogeneous levels of expression of the CALLA-mRNA species were also observed in non-T ALL cases that correlated with the level of CALLA expression at the surface of these cells. Very high levels of NEP activity were achieved by transfecting COS-1 cells with pSV-CALLA; 20% of the transfected cells were CALLA+ and expressed 550 pmol/min/10(6) cells. Extracts prepared from COS-1 cells transfected with pSV-CALLA (carrying human NEP cDNA) and pSVENK19 (carrying rabbit NEP-cDNA), respectively, gave Michaelis constant (Km) values of 50 mumol/L and similar inhibition curves with thiorphan. Thus the recombinant proteins encoded by these two genes have similar enzymatic properties, confirming the high degree of their structural relatedness. The expression of high levels of CALLA/NEP on COS-1 cells should allow the use of this system to test the effects of specific mutations on activity and might lead to the understanding of the role of CALLA in the onset and/or progression of leukemia.

Animals↗