Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000-26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000-25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1-5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as "CEA distinctive", because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Immunol 1992 Feb
PMID:Epitope mapping of the carcinoembryonic antigen with various related recombinant proteins expressed in Chinese hamster ovary cells and 25 distinct monoclonal antibodies. 137 22

A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The gene is expressed at all developmental stages and in all tissues. Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar. Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development. The RpII15 gene product is thus necessary for viability of D. melanogaster.
Mol Cell Biol 1992 Mar
PMID:The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster. 154 24

To investigate the stage in estrogen receptor (ER) action at which hormone functions, we prepared human ER mutants unable to bind 17 beta-estradiol. In transfected Chinese Hamster Ovary (CHO) cells, two of the ER mutants exhibited less than 5% of the ability to activate transcription shown by wild type ER. Immunoprecipitation followed by Western blotting with monoclonal antibodies was used to examine the ability of the ER mutants to form heterodimers with a truncated form of wild type ER. The non-hormone-binding mutants formed heterodimers with the truncated ER as efficiently as wild type ER. We used a promoter interference assay to measure the interaction of the ER with the estrogen response element (ERE) in vivo. Expression plasmids encoding the ER mutants and wild type ER were transfected into CHO cells across a range of concentrations, resulting in both high and low levels of promoter interference. The ER mutants and wild type ER elicited similar levels of promoter interference, indicating that although they were unable to bind ligand, the ER mutants bound to the ERE in vivo as effectively as wild type ER. Additional evidence that the non-hormone-binding ER mutants are not in a functionally inactive complex comes from their ability to suppress the activity of wild type ER, when they were coexpressed in the same cells. These data support a model for ER action in which the unliganded ER is free to dimerize and bind to the ERE. In this model, the primary role of 17 beta-estradiol in ER action is to induce a conformational change which activates the ligand-dependent transactivation domain.
Mol Endocrinol 1995 Apr
PMID:Estrogen receptor mutants which do not bind 17 beta-estradiol dimerize and bind to the estrogen response element in vivo. 765 89

Somatostatin receptors are abundantly expressed on a variety of human endocrine and epithelial tumors. The ability of these receptors to couple to effector pathways that inhibit the growth of these tumor cells has prompted the use of somatostatin agonists in the treatment of human neoplasms. It has been demonstrated that somatostatin stimulates a phosphotyrosine phosphatase in human tumor cells through a receptor-mediated process. This stimulation may counteract the growth-promoting properties of growth factors and the receptor tyrosine kinases that they activate. The recent cloning and characterization of distinct somatostatin receptor subtypes raise the possibility that different receptor subtypes mediate distinct effector pathways. To determine whether cloned somatostatin receptors could mediate coupling to phosphotyrosine phosphotyrosine phosphatase activity, we examined phosphatase activity after somatotostatin activation of the rat somatostatin receptors SSTR1 and SSTR2 after their stable expression in heterologous Chinese Hamster Ovary (CHO-K1) cells. We found that stimulation of SSTR1 cells was capable of increasing phosphotyrosine phosphatase activity, despite the coupling of both receptors to the inhibition of adenylyl cyclase in these cells. This activation was characterized by an EC50 of 70 nM and was sensitive to pertussis toxin. In addition, we demonstrate that activation of phosphotyrosine phosphatase activity in pituitary cell lines correlates with the endogenous expression of the SSTR1 gene within these cells.
Mol Endocrinol 1994 Oct
PMID:The somatostatin receptor SSTR1 is coupled to phosphotyrosine phosphatase activity in CHO-K1 cells. 785 46

Swiss Mouse 3T3-L1 cells provide a unique model for insulin-sensitive primary fat cells. Under defined conditions this fibroblast cell line can be converted to fully differentiated adipocytes, characterized by increased insulin receptor number and induction of adipogenic-specific proteins. 3T3-L1 cells were therefore transfected with the cDNA for the A/K1018 insulin receptor (alanine substituted for lysine at amino acid 1018 in the ATP binding region of the kinase domain). The cell lines in which the dominant inhibitory effects of the A/K1018 receptor had been previously demonstrated [Rat 1 fibroblasts and Chinese Hamster Ovary cells] have relatively few endogenous insulin receptors; however, the 3T3-L1 cell line has about 200,000 insulin receptors when differentiated. In this study we have used this cell line to explore the molecular mechanisms for the dominant inhibitory effect of a tyrosine kinase-defective insulin receptor on insulin action. The A/K1018 receptor was inhibitory in the 3T3-L1 cells as shown by decreased glucose transport. Further, altered differentiation in the transfected cell implicates the insulin receptor as an important downstream regulator in this process. We were able to demonstrate the presence of numerous hybrid receptors, composed of endogenous mouse heterodimers and human kinase-deficient heterodimers in these cells. Trans phosphorylation did occur within these hybrids as evidenced by autophosphorylation of human beta-subunits; however, these hybrids are unable to phosphorylate substrates. These results establish hybrid formation as an important determinant in the dominant negative nature of the A/K1018 insulin receptor.
Mol Endocrinol 1994 Jun
PMID:Hybrid formation between endogenous mouse and transfected human tyrosine kinase-deficient (A/K1018) insulin receptors leads to decreased insulin sensitivity in 3T3-L1 adipocytes. 793 84

FSH comprises two distinct subunits, both of which contain asparagine-linked carbohydrate residues, located at positions 52 and 78 on the alpha-subunit and positions 7 and 24 on the beta-subunit. These carbohydrate chains have been shown to regulate the biological activity of FSH, including signal transduction and receptor binding. However, the specific roles of the individual carbohydrate chains have been poorly defined. Using site-directed mutagenesis we disrupted the consensus sequences for glycosylation and expressed the mutated cDNAs in Chinese Hamster Ovary cells. Specifically deglycosylated FSH variants were secreted from all clonal cell lines expressing the mutated FSH cDNAs except for the cell line that lacked all four glycosylation sites. Analysis of the singly or doubly deglycosylated FSH mutants revealed that removal of the carbohydrate residue at position 78 on the alpha-subunit significantly increased the receptor binding affinity of human FSH by 72%. Removal of the other carbohydrate residues had no significant effect on receptor binding. The carbohydrate residue at position 52 on the alpha-subunit was found to play an essential role in signal transduction as its removal resulted in a significant decrease in potency to 26% of wild type levels. The other individual carbohydrate residues appear to play a minor role in signal transduction, although removal of each residue results in reduced maximal response. The removal of both alpha-subunit carbohydrates resulted in a significant decrease in biopotency, to 41% of wild type levels; whereas, the removal of both beta-subunit carbohydrate chains resulted in a significant increase in biopotency, to 216% of wild type levels. These studies have allowed the identification of site-specific roles for the carbohydrate residues of human FSH. Our data suggest that the carbohydrate residues play a greater role in determining the biological activity of FSH than has been suggested in similar studies of other glycoprotein hormones.
Mol Endocrinol 1994 Jun
PMID:Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle stimulating hormone in receptor binding and signal transduction. 793 88

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.
Mol Cell Biochem 1996 Jun 21
PMID:Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks. 885 66

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.
Comp Biochem Physiol B Biochem Mol Biol 1996 Apr
PMID:Vitellogenesis-related ovary cathepsin D from Xenopus laevis: purification and properties in comparison with liver cathepsin D. 892 51

In order to obtain homologous follicle-stimulating hormone (FSH) for in vivo and in vitro studies in the rat, rat recombinant (rec) FSH was produced in Chinese Hamster Ovary (CHO) cells. The synthesized rat recFSH was purified and subjected to physico-chemical and biological characterization. including a comparison with two rat pituitary (pit) and reference preparations (NIDDK-rFSH I-8 and NIDDK-rFSH-RP2) as well as with human recFSH (Org 32489). The molecular masses of rat recFSH and human recFSH were determined by SDS-polyacrylamide (SDS-PAGE) and were found to be similar, about 40 kD. The pI distribution of rat recFSH is similar to rat pitFSH, and slightly more acidic than human recFSH (3.6-5.6 vs 3.9-5.5, respectively) as determined by isoelectric focussing in immobilized pH gradients. Rat recFSH displayed dose-response curves parallel and in the same dose range as the rat pitFSH in receptor binding and in vitro bioassays. However, the in vivo activities of rat recFSH and rat pitFSH were 8824 and 3051 IU/mg, respectively, determined by the Steelman Pohley assay. Rat (pit and rec) and human FSH are very different. Human recFSH bound to both calf testicular membranes and CHO cells expressing the human FSH receptor (CHO hFSH-R) with about 10-fold higher affinity (Ka) than pituitary and recombinant rat FSH. In in vitro bioassays with immature rat Sertoli cells and CHO hFSH-R cells human recFSH was also about 10-fold more potent than the rat FSH preparations. In the in vitro bioassays with immature rat granulosa cells the difference was about 5-10-fold. These studies indicate that the receptor binding and in vitro activities of rat pitFSH and rat recFSH are similar. The differences in in vivo activity are probably due to the differences in glycosylation. The biological behaviour of rat FSH (pit and rec) is different from that of human FSH. Therefore, if the rat is used as a model for physiology of gonadotropic action, the results may be greatly influenced by the type (species) of hormone preparation used. The availability of homologous hormone preparations is therefore crucial.
Mol Cell Endocrinol 1997 Mar 14
PMID:Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization. 909 1

Insulin acts on its target tissues by specific interaction with the cell surface insulin receptor (IR). The IR possesses an intrinsic tyrosine kinase (TK) activity which is stimulated by insulin binding. This TK activity is required for many aspects of insulin signalling. We had earlier reported that human plasma alpha 2-HS glycoprotein (alpha 2-HSG) inhibits insulin-stimulated mitogenesis at the level of IR-TK (Mol Endo 7: 1445-1455, 1993). In the present study, using recombinant alpha 2-HSG, which possesses 50-100 times the specific activity of plasma alpha 2-HSG, we have further investigated the molecular basis of this effect. We examined the insulin-stimulated Ras signalling pathway in Chinese Hamster Ovary cells overexpressing the human IR. alpha 2-HSG inhibits insulin-induced tyrosine phosphorylation of IRS-1 and the subsequent association of GRB2, as well as Sos, with IRS-1. This inhibition results in reduced guanine nucleotide exchange in p21ras. alpha 2-HSG also inhibits the stimulation of Raf phosphorylation, in response to insulin, leading to inhibition of MEK activity. In a parallel pathway, alpha 2-HSG also inhibits insulin-induced tyrosine phosphorylation of Shc. However, alpha 2-HSG does not affect any of the metabolic actions of insulin rested in these cells. These results suggest that, while insulin's mitogenic effects can be abolished by inhibition of insulin-induced IR-TK, propagation of signals for metabolic activities might utilize alternate of rescue mechanisms.
 ...
PMID:Recombinant human alpha 2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor. 911 49


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