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Biomedical subjects

G Carpenter

Publications and source records attributed to G Carpenter.

At least 145 records · Page 8Linked to original sources
PubMed1986

A biological assay for epidermal growth factor/urogastrone and related polypeptides.

A bioassay for epidermal growth factor (EFG) which is rapid, specific, and extremely sensitive is described. As the bioassay detects as little as 25 pg of EGF, this assay is more sensitive than commonly used radioreceptor assays and nearly as sensitive as radioimmunoassays. This bioassay involves only the measurement of the proliferation of cultures of an EGF-requiring cell line and can be carried out in a quantitative manner over a 40-fold range of EGF concentrations.

Animals↗
PubMed1986

Epidermal growth factor, its receptor, and related proteins.

An interesting aspect of the developments forthcoming from the study of cell growth control by growth factors is the structural and functional homologies that have been found to exist between growth factors or their receptors and other molecules. The epidermal growth factor (EGF) system has been particularly fruitful in this regard. The information in this article is meant to summarize the relationships that have been recently described between EGF and other EGF-like molecules and between the EGF receptor and related macromolecules.

Amino Acid Sequence↗
PubMed1986

Nasal glioma. A rarity.

Nasal glioma is a rare congenital malformation which most often presents to the otorhinolaryngologist or neurosurgeon. However, occasional cases of the extranasal variant are referred to a dermatologist as an unusual subcutaneous mass over the bridge of the nose, most often in an infant. These lesions may not be recognised clinically, leading to dangerous mismanagement, and may be misinterpreted by pathologists as a neoplasm, either benign or malignant. Three cases are described here with the purpose of focussing the attention of workers in dermatology on this unusual developmental abnormality.

Female↗
PubMed1986

Epidermoid cyst of the spleen.

Epidermoid cysts of the spleen are extremely rare, occurring mainly in people under 20 years of age. They are thought to originate from an abnormality in the development of the spleen during the seventh week of embryological life, when the spleen is close to the mesonephric tissues. Cysts present as an asymptomatic abdominal mass or with pain in the left upper quadrant and/or the left shoulder. In the past the recommended treatment has been splenectomy, but with the changing attitudes towards splenic surgery a more conservative approach is now employed. Drainage under radiographic guidance, deroofing with external drainage or simply deroofing with drainage into the peritoneal cavity are now more popular techniques (along with simple cystectomy). Complications are few, although those associated with deroofing and internal drainage are inadequately investigated.

Adult↗
PubMed1985

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues.

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.

Carcinoma, Squamous Cell↗
PubMed1985

EGF receptor antiserum inhibits bone resorbing activity produced by a rat Leydig cell tumor associated with the humoral hypercalcemia of malignancy.

The humoral hypercalcemia of malignancy (HHM) is a syndrome caused by tumor cells releasing unknown circulating factors which stimulate osteoclastic bone resorption. In the D6 variant of the rat Leydig cell tumor model of HHM, we found that tumor extracts and tumor cell conditioned medium contained a macromolecular bone resorbing factor which coeluted on column chromatography with transforming growth factor activity (TGF). This observation led to the hypothesis that the tumor-derived bone resorbing factor was a TGF which interacts with the epidermal growth factor (EGF) receptor. To test this hypothesis, we examined the effects of two classes of antisera to the EGF receptor on bone resorption stimulated by conditioned medium from Leydig D6 tumor cells using organ cultures of fetal rat long bones. The antiserum which blocks the binding of EGF to its receptor inhibited bone resorption stimulated by tumor conditioned medium and by EGF. The second antiserum to the EGF receptor which does not block EGF binding or biological activity had no effect on bone resorption stimulated by either tumor conditioned medium or EGF. Neither antiserum had any effect on bone resorption stimulated by parathyroid hormone (PTH). These results indicate that the tumor-derived bone resorbing factor is dependent upon the availability of EGF receptors for its activity and are consistent with it being a TGF.

Animals↗
PubMed1985

Biosynthesis of the epidermal growth factor receptor in cultured human cells.

A 160,000 mol wt precursor of the epidermal growth factor (EGF) receptor has been identified in human A-431 carcinoma cells and skin fibroblasts. The presence of one discrete precursor band indicates the presence of a slow processing step. We have determined that this slow processing step involves the conversion of high mannose N-linked oligosaccharides on the receptor precursor to primarily complex oligosaccharides on the mature form of the receptor. This is shown by 1) the presence of fucose, a characteristic terminal sugar of complex oligosaccharides, in only the mature receptor and by 2) the susceptibility of the precursor to digestion with endoglycosidase H, which cleaves high mannose N-linked oligosaccharides, but not complex oligosaccharides from glycoproteins. The precursor to mature receptor transition half-time is 1.7 h in A-431 cells. This long transition half-time causes an accumulation of approximately 7.2 X 10(5) precursor molecules per cell (approximately 12% of the total population of EGF receptors). The net quantity of mature EGF receptors, but not of receptor precursors, is reduced when EGF is added to the culture medium of A-431 cells. The presence of EGF in the growth medium also decreases electrophoretic migration (as a result of increased phosphate incorporation) of the mature receptor, but not that of the precursor. The EGF-insensitive state of the precursor is most likely due to its intracellular location.

Carcinoma↗
PubMed1985

Epidermal growth factor: biology and receptor metabolism.

Epidermal growth factor (EGF) is a small (Mr 6045) protein that stimulates cell proliferation in cell culture systems and in intact animals. This growth factor has been isolated from rodents and human material and probably exists in nearly all animal species. In humans EGF has been detected in many body fluids and receptors for the growth factor are also ubiquitous. While the mitogenic activity of EGF has been most frequently reported, it clearly has other functions, such as the inhibition of gastric acid secretion, that are unrelated to mitogenic responses. Correspondingly, receptors for EGF have been localized on cells that are rapidly proliferating and cells that are essentially non-proliferating. Nevertheless, it has not been possible to define experimentally the biological function(s) of the endogenous EGF present in the intact animal. Studies of the mechanism of action of EGF have concentrated, to date, on the plasma membrane receptor that specifically binds this ligand. The receptor is undoubtedly the first cellular component that mediates the eventual biological response(s) of the cell to this extracellular signal. Studies of the EGF receptor have shown that this molecule, which has no subunit structure, functions not only in ligand recognition, but also may produce an intracellular 'second message'. The receptor contains a protein kinase activity that is activated by the binding of EGF and it is this enzymic function that may yield the critical 'second messenger', by phosphorylation of an intracellular protein. Although intracellular targets of this EGF-sensitive protein kinase have been identified, it has not been possible to demonstrate their relevance as regulatory mediators of EGF activity.

Animals↗
PubMed1984

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function.

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.

Carcinoma, Squamous Cell↗
PubMed1984

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells.

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.

Amino Acid Sequence↗
PubMed1984

Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells.

The metabolism of the receptor for epidermal growth factor (EGF) in A-431 cells has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. The rate of EGF receptor degradation (t1/2 = 20 hr) was faster than the rate of degradation of total cell protein (t1/2 = 52 hr). When EGF was added at the beginning of the chase, the half-life of prelabeled receptor decreased to 8.9 hr. This decrease was specific, as the level of total cellular protein and another plasma membrane protein, the transferrin receptor, were relatively unaffected by EGF. The carbohydrate portion of the receptor is degraded, in the presence or absence of EGF, at approximately the same rate as the protein moiety. The amount of EGF receptor protein in A-431 cells has been quantitated by radiolabeling total cellular protein and quantitating the immunoprecipitable receptor. The EGF receptor constitutes approximately 0.15% of the total cell protein in A-431 cells. These cells, therefore, have approximately 30 times more EGF receptor protein than fibroblasts. The EGF receptor constitutes an even higher proportion of 3H-glucosamine- or 3H-mannose-labeled macromolecules in A-431 cells, 1.5% or 5.2%, respectively. The EGF receptor from A-431 cells can easily be identified by submitting carbohydrate-labeled, solubilized cells to electrophoresis as described by Laemmli (1970).

Animals↗
PubMed1984

Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts.

The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1.2 h in human fibroblasts. These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded. Enhanced receptor degradation was observed 5-10 min after the addition of EGF. The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25 degrees C. We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present. The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor. The EGF receptor constitutes approximately 0.0035% of the cellular protein in human fibroblasts.

Cells, Cultured↗
PubMed1984

Translation in Xenopus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins.

Translation of messenger RNAs (mRNAs) for human epidermal growth factor receptor (EGF-R) proteins was accomplished in a Xenopus oocyte translation system. Translation of these mRNAs in rabbit reticulocyte lysates and wheat germ extracts were unsuccessful, in accordance with the general difficulty in translating mRNAs for membrane-bound glycoproteins in conventional heterologous cell-free systems. Total poly(A)+RNA from a human epidermoid carcinoma cell line A431 was injected into oocytes from Xenopus laevis. After incubation in the presence of [35S]methionine, the oocytes were homogenized and the supernatant incubated with several different polyclonal antibodies (IgGs) against the human EGF-R. Immunocomplexes were bound to protein A-Sepharose, washed extensively, and electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. A protein of Mr = 160,000, corresponding to the EGF-R glycoprotein, was specifically precipitated from RNA-injected oocytes by each of the anti-EGF-R IgGs. This protein was not detectable in the immunoprecipitates from uninjected oocytes nor was it precipitated from RNA-injected oocytes by IgG from normal rabbit serum or IgG to an unrelated protein. A second protein of Mr = 100,000 was also immunoprecipitated from the homogenates of RNA-injected oocytes. This product corresponds to the EGF-R-related protein, present in A431 cells, which is structurally similar to the EGF-R (Weber et al., 1984). Thus, frog oocytes provide an efficient and generally applicable heterologous system for translation of mRNAs for large glycoproteins such as growth factor receptors. In addition, successful immunoprecipitation of translated products in this system provides a method for assay of mRNAs where biological activity of the proteins cannot be monitored.

Animals↗
PubMed1984

Visualization of epidermal growth factor receptors in human epidermis.

The localization of epidermal growth factor (EGF) receptors in normal human epidermis was examined with two independent experimental methods. The distribution of EGF receptor sites was studied using light microscopic autoradiography with [125I]EGF and direct immunocytochemical techniques with EGF receptor antibodies and protein A-colloidal gold complexes. Direct visualization by autoradiography indicated that the concentration of EGF receptors was greatest in the lower epidermal layers. Ultrastructural morphometric analysis of protein A-gold complexes showed that EGF receptors were primarily associated with the plasma membranes although intranuclear and cytoplasmic localization was also evident. This postembedment immunolocalization method also confirmed the relative differences in the number of EGF receptors found in individual epidermal layers (basalis greater than spinosum greater than granulosum greater than corneum layers). This inverse relationship between numbers of EGF receptors and the degree of epidermal differentiation and/or keratinization may suggest a physiologic role for EGF in these processes in human epidermis.

Autoradiography↗