Biomedical subjects

E T Cox

Publications and source records attributed to E T Cox.

5 recordsLinked to original sources

PubMed2000

Structure and expression of a membrane component of the inhibin receptor system.

The purification and cloning of a membrane-anchored proteoglycan with affinity for inhibin A are described. Bovine pituitary membranes were isolated, and membrane-anchored proteins were solubilized and used as an enriched source of inhibin binding protein. The extract was passed over an inhibin A affinity column, and a protein, designated p120, was identified as an inhibin-binding moiety. A partial amino acid sequence was determined for the protein, which matched two human complementary DNAs (cDNAs) in the database. The full-length cDNA predicts a 1336-amino acid glycoprotein. Full-length p120-encoding cDNAs were isolated from human testis RNA and cloned into expression vectors. Two p120 messenger RNA transcripts of 4.6 kb and 2 kb are detected in rat pituitary by RNA blot analysis. Similar analysis of rat testis RNA revealed transcripts of identical molecular mass, albeit at lower abundance. To determine the cellular localization of p120 in pituitary and testis, an antibody directed against the predicted extracellular domain of the protein was generated and used in an immunohistochemical analysis of thin tissue sections. p120 immunostaining is coincident with FSHbeta immunopositive gonadotrope cells in rat pituitary. p120 staining is intense in the testicular Leydig cells, which bind iodinated inhibin but not iodinated activin. In summary, an inhibin-binding protein has been isolated that is produced in tissues that are targets of inhibin action.

Activin Receptors↗

PubMed1993

Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

Amino Acid Sequence↗

PubMed1993

Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids.

Inhibins and activins are produced by a variety of tissues and may have important endocrine and paracrine roles in development, reproduction, and hematopoiesis. However, little is known regarding the physical properties or concentrations of inhibin and activin in biological fluids. Binding proteins for inhibin or activin in serum or at production or target sites may have important implications for restricting the bioactivity of these hormones and may alter the immunoreactivity of these molecules in biological fluids. The objective of this study was to identify inhibin- and activin-binding proteins in human serum (HS) and follicular fluid (hFF) and determine the ability of these proteins to alter biological or immunological activity. In HS, [125I]activin and inhibin bound to a protein identified as alpha 2-macroglobulin (alpha 2M) using three criteria: 1) [125I]inhibin and activin bind purified alpha 2M, but not several other serum proteins tested; 2) complexes formed by [125I]inhibin and activin in HS and in the presence of purified alpha 2M elute with similar retention times on HPLC; and 3) preadsorption of HS with alpha 2M antiserum inhibits inhibin and activin binding to this protein while antiserum directed against follistatin or other serum proteins had no effect. A small amount of a lower mol wt [125I]activin-follistatin complex was also found in HS. This complex eluted with a retention time similar to that of activin bound to purified porcine follistatin. Binding of inhibin to follistatin could not be detected in HS. In contrast, follistatin was the major binding protein of both activin and inhibin in hFF. Concentrations up to 100 micrograms/ml purified alpha 2M had no effect on the bioactivity or immunoreactivity of either inhibin or activin. In contrast, follistatin inhibited both activin-stimulated pituitary FSH release and K562 hemoglobin production as well as antiserum binding in a specific activin-A immunoassay. Follistatin did not interfere with inhibin immunodetection. These data indicate that two inhibin- and activin-binding proteins are present in different relative amounts in HS and hFF, alpha 2M, the primary binding protein in HS, did not alter inhibin or activin bio- or immunoactivity under the conditions of these experiments, while follistatin, the major binding protein in hFF, may mask activin's bio- and immunoactivities.

Activins↗

PubMed1992

Stage-specific binding of inhibin and activin to subpopulations of rat germ cells.

Flow cytometry was used to separate and identify Sertoli and germ cell populations in primary rat testicular cultures derived from animals of different ages on the basis of cell size and DNA and lipid content. Multiparameter fluorescent evaluation of each cell preparation resulted in the assignment of specific staining patterns to Sertoli cells (diploid, high lipid content), spermatogonia (diploid, low lipid content), spermatocytes (large, tetraploid, high lipid content), and round spermatids (haploid, low lipid content). Each field was separately analyzed for inhibin and activin binding. Fluorescein isothiocyanate-conjugated activin bound with greatest intensity to spermatogonia, with little binding to leptotene or zygotene spermatocytes. Fluorescein isothiocyanate-conjugated inhibin bound to all stages of germ cells tested. Cross-competition data indicate that at least two and probably three distinct receptors exist for these peptides.

Activin Receptors↗

PubMed1990

Assay requirements for cell culture process development.

Because the structural parameters which influence product "quality" will vary from protein to protein, the screening methods used during development of each production process will also differ. Thus, not all of the methods described above will be needed for each product. One of the challenges of cell culture process development is choosing the correct structural features to monitor. The next level of process development may involve on-line control of many of the parameters discussed above. This would allow product quality-driven manipulations of culture conditions. The major factor limiting this advance may not be the techniques for real-time analysis, but rather a more thorough understanding of the structural features which describe a high-quality pharmaceutical protein.

Animals↗