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Biomedical subjects

R E Gates

Publications and source records attributed to R E Gates.

16 recordsLinked to original sources
PubMed1992

Altered distribution of phospholipase C-gamma 1 in benign hyperproliferative epidermal diseases.

Phospholipase C-gamma 1 (PLC-gamma 1) is a well characterized substrate for the epidermal growth factor receptor tyrosine kinase and has been implicated in the intracellular biochemical signaling cascade which occurs following stimulation of cells with epidermal growth factor. The in vivo localization of PLC-gamma 1 was examined by immunohistochemistry in sections of normal human skin and in skin sections from a diverse series of hyperproliferative epidermal conditions (psoriasis, seborrheic keratoses, acrochordons, and margins near second-degree burns). Immunoreactive PLC-gamma 1 was detected only in the basal compartment of normal skin but was readily detectable in both the basal and outer epidermal compartment in hyperproliferative skin conditions. In addition, immunoreactive PLC-gamma 1 colocalizes with immunoreactive epidermal growth factor receptor in both normal and hyperproliferative epidermis.

Amino Acid Sequence↗
PubMed1991

Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay.

Substantial time and effort are necessary to eliminate interference due to thiols and reducing sugars in the bicinchoninic acid protein assay when detergents such as sodium dodecyl sulfate (SDS) are present. A method to eliminate these interfering substances based on the binding of proteins to positively charged nylon membranes at pH 8.5 followed by methanol and aqueous washes is described. Color development using bicinchoninic acid occurs in solution with only a minor contribution from the nylon membrane itself. The retention of most proteins on the membrane is nearly quantitative and is only slightly decreased by high salt or detergent in the assay mixture. The interference due to thiol compounds such as beta-mercaptoethanol and dithiothreitol is completely eliminated while that due to reducing sugars such as glucose is substantially reduced. Therefore, the amount of protein dissolved in the sample buffer used for SDS-polyacrylamide gel electrophoresis (2% SDS and 5% beta-mercaptoethanol) can be determined with this bound protein assay.

Chemistry Techniques, Analytical↗
PubMed1990

The EGF/TGF alpha receptor in skin.

In responsive cells, all known effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and related proteins are mediated through binding to a specific membrane receptor. The EGF/TGF alpha receptor is a single-chain glycoprotein (1186 amino acids) containing three functional domains: 1) an extracellular, glycosylated portion that binds EGF; 2) a small transmembrane portion; and 3) a cytoplasmic portion that has the intrinsic tyrosine kinase activity and multiple sites that can be phosphorylated. When EGF binds to the receptor its intrinsic tyrosine kinase is activated, resulting in increased phosphorylation of intracellular tyrosine residues both on the receptor (autophosphorylation sites) and on exogenous proteins involved in regulating cellular functions. Site-specific mutagenesis has established that the tyrosine-kinase activity of the receptor is essential for nearly all of the effects of EGF including its ability to elevate cellular calcium levels and to induce DNA synthesis. The binding of EGF and the kinase activity of the receptor are both regulated by the phosphorylation of the receptor on specific threonine/serine sites catalyzed by other protein kinases. Specific lipids such as sphingosine also can regulate kinase activity. Tyrosine-specific phosphoprotein phosphatases and perhaps proteases must be important in terminating the cellular response to EGF. In human skin, the response to EGF/TGF alpha is determined by the location and number of receptors and is modulated by processes affecting the binding affinity, internalization, and tyrosine-kinase activity of the receptor. Specific patterns of EGF binding and of immunoreactive receptors characterize normal growth and differentiation and these are altered during the abnormal growth and differentiation associated with diseases such as psoriasis, viral infections, neoplasms, and paraneoplastic syndromes. It is not clear if the altered patterns reflect the consequence of the disease or are the cause of the disease. As a cause, the EGF receptor may have undetected point mutations that result in internalization and degradation defects, aberrant phosphorylation, and dephosphorylation or abnormal glycosylation.

Animals↗
PubMed1987

Human neonatal keratinocytes have very high levels of cellular vitamin A-binding proteins.

Since cellular retinol- and retinoic acid-binding proteins (CRBP and CRABP) mediate the effects of vitamin A on epidermal differentiation, the levels of these binding proteins were measured in the epidermal and dermal layers of newborn, human foreskin as well as in primary cultures of keratinocytes and fibroblasts from these layers. Ligand binding assays with saturating concentrations of all trans-[3H]retinol or of all trans-[11-3H]retinoic acid were used to quantitate amounts of binding proteins in cytosols prepared from these skin layers or cultured cells. The epidermal levels of CRABP and CRBP (60.9 +/- 14.4 and 7.3 +/- 1.7 pmol per mg cytosol protein, respectively) were markedly higher than that reported for adult epidermis but were comparable to levels in keratinocytes cultured from neonatal foreskin epidermis (61.8 +/- 7.8 and 10.7 +/- 2.5, respectively). The levels of CRABP were much lower in the foreskin dermis than in the epidermis and the levels measured in the fibroblasts cultured from this dermis were consistent with the dermal levels. However, CRBP levels in cultured dermal fibroblasts were very low and could not account for the dermal CRBP levels, suggesting that another dermal cell type has high levels of CRBP.

Carrier Proteins↗
PubMed1985

Altered vitamin A-binding proteins in carcinoma of the head and neck.

Since vitamin A inhibits the terminal differentiation of keratinocytes, elevated levels of cytoplasmic vitamin A-binding proteins could explain the reduced tendency of squamous cell carcinomas to undergo terminal differentiation. The levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were determined in 37 squamous cell carcinomas of the head and neck and were compared with the corresponding levels in adjacent normal tissue. The CRBP levels expressed per milligram of DNA were increased threefold (P = 0.0005) in the tumor as compared with normal tissue. In contrast, CRABP levels expressed per milligram were decreased 2.5-fold (P = 0.0007) in the tumor as compared with normal tissue. Despite significant differences in retinoid-binding protein levels between tumor and adjacent normal tissue, there was no significant correlation of these differences with any of the patient clinical parameters or any of the tumor growth characteristics. The increased CRBP levels may explain why terminal differentiation, an important mechanism for limiting growth in normal keratinocytes, is less readily induced in squamous cell carcinomas.

Adult↗
PubMed1985

Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites.

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.

Animals↗
PubMed1985

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors.

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.

Animals↗
PubMed1985

Cytoplasmic vitamin A binding proteins in chick embryo dermis and epidermis.

Excess vitamin A has striking morphologic and developmental effects on chick embryo skin. While cytoplasmic retinoic acid-binding protein (CRABP) was known to be abundant in chick embryo skin, neither quantitative values nor the distribution between dermis and epidermis have been established. We determined CRABP levels in collagenase-separated dermis and epidermis from 8-day-old embryos using specific binding of all-trans-[11-3H]retinoic acid in cytosols prepared from gram quantities of these tissues. The level of CRABP in dermis was twice the level in epidermis whether calculated on the basis of wet weight, cytosol protein, or DNA. When averaged over many preparations, 3 times as much dermis as epidermis was recovered from a single piece of skin. Therefore, the dermis contained 85% of the extremely high CRABP levels found in collagenase-treated skin, while epidermis contributed only 15%. Cytoplasmic retinol binding protein (CRBP) was also detected in chick embryo skin, but the binding was low and the levels in epidermis and dermis were not significantly different. The amount of CRABP in chick embryo skin (1600 pmol/g wet weight or 100 pmol/mg cytosol protein) is the highest level reported in any tissue and suggests an important role for vitamin A in the normal development and maturation of skin.

Animals↗
PubMed1975

The distinguishing characteristics of the plasma membrane are its exposed proteins.

The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.

Blood Platelets↗