Search PubMed⌕ Search

Biomedical subjects

C R Savage

Publications and source records attributed to C R Savage.

At least 73 records · Page 4Linked to original sources
PubMed1985

125I-EGF binding to responsive and nonresponsive cells in culture: loss of cell-associated radioactivity relates to growth induction.

A comparison of the binding and overall processing of epidermal growth factor (EGF) was made in ten mammalian fibroblast-like and three epithelial-like cell lines. EGF stimulated the growth of five fibroblastic cell lines (132 to 224%) after 10 days in the constant presence of EGF and were termed "responsive". Eight of the lines did not respond or were growth inhibited by EGF (-64 to + 21%) and were listed as "nonresponsive". Both "responsive" and "nonresponsive" cell lines possessed specific saturable membrane receptors for EGF, but no consistent differences were found between the number of apparent receptors per cell or the concentrations of 125I-EGF required for half maximal binding. However, a consistent difference between the "responsive" and "nonresponsive" cell lines was observed when the amount of cell associated 125I-labeled EGF was measured as a function of incubation time at 37 degrees C in the constant presence of the hormone. In every cell line we classified as responsive, the binding of 125I-EGF reached a maximum after 30 minutes incubation at 37 degrees C and the cell associated radiolabel subsequently decreased by 42-68% within 4 hours. In contrast, the "nonresponsive" cell lines required 1-2 h to reach maximal binding and showed a minimal decrease of 1-10% during the 4-h period. These data indicate that a variety of different cell lines can possess receptors with similar binding properties but process EGF in a dissimilar manner. This difference in processing may reflect the specific events which are necessary for the induction of cell growth.

Animals↗
PubMed1985

Effects of antiinsulin receptor antibodies on amino acid uptake by cultured rat hepatocytes.

The effects of two antiinsulin receptor antisera (AIRA) on alpha-amino-2-[14C]isobutyric acid [( 14C]AIB) uptake by and [125I]insulin binding to cultured rat hepatocytes were examined. Diluted 1:100, both antisera inhibited insulin binding by 75-80%, mainly by decreasing available high affinity insulin-binding sites. Diluted 1:500, they decreased insulin binding and high affinity binding sites by 35-40%. Neither antiserum had an effect on basal [14C]AIB uptake, but decreased insulin-stimulated AIB uptake by 83% (dilution, 1:100; 24-h incubation) and 25% (dilution, 1:500; 24-h incubation), respectively. Insulin stimulation of AIB uptake correlated positively (r = 0.96; P less than 0.01) with insulin binding to high affinity receptors on hepatocytes incubated with normal serum or AIRA. We conclude that: 1) insulin stimulation of amino acid uptake by hepatocytes was mediated through insulin receptors, especially high affinity binding sites, and was inhibited by AIRA in parallel with receptor blockade; and 2) AIRA had no insulin-like effect on basal amino acid uptake by hepatocytes.

Amino Acids↗
PubMed1984

In vivo and in vitro effects of antiinsulin receptor antibodies.

We studied a patient with Acanthosis nigricans and the type B syndrome of severe insulin resistance. The patient's rates of basal glucose disappearance and appearance were both normal (2.2 and 1.7 mg/kg . min, respectively). FFA, betahydroxybutyrate, and acetoacetate concentrations were stable at 0.8, 1.0, and 0.3 mM, respectively, during a 2-h saline infusion after an overnight fast, indicating continued presence of insulin-like activity (ILA) in her serum. Infusion of insulin at rates of 2.7 and 27 U/h, raising peripheral insulin concentrations from 1400 to 4000 and 6000 microU/ml, respectively, had no effect on glucose disappearance and appearance or plasma concentrations of beta-hydroxybutyrate, acetoacetate, and FFA, suggesting that the observed ILA was not caused by the patient's plasma insulin. To determine the source of the ILA we used the patient's serum containing antiinsulin receptor antibodies (AIRA) to study its acute (2 h) and chronic (24 h) effects on insulin binding and glycogen synthesis in rat hepatocytes in primary culture. Preincubation of hepatocytes with AIRA serum (diluted 1:100) inhibited insulin binding by 84% and 88% after 2 and 24 h, respectively. It increased U-[14C]glucose incorporation into glycogen by 40% and 52% after 2 and 24 h, respectively. These effects were not caused by insulin present in the patient's serum. We conclude that AIRA serum, in addition to causing severe insulin resistance through inhibition of insulin binding, also exerted strong and long lasting insulin-like effects. These findings are compatible with the patient's clinical features of absence of ketoacidosis despite severe insulin resistance, decrease in glucose concentrations during fasting, and postprandial hyperglycemia.

Adult↗
PubMed1983

Monoclonal antibody to human epidermal growth factor.

A monoclonal antibody directed to a species-specific determinant of human epidermal growth factor (h-EGF) was obtained by fusing murine myeloma cells with BALB/c mouse splenocytes sensitized to h-EGF. This antibody, referred to as 863.D4, did not react with either rat or mouse epidermal growth factor or with 11 other polypeptide hormones tested as shown by solid-phase radioimmunoassay (SPRIA), and immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scatchard analysis of the antibody binding to purified h-EGF revealed an apparent equilibrium dissociation constant of 1 X 10(-8) M. The antibody blocked both the binding of h-EGF and h-EGF stimulation of 3H-thymidine incorporation into DNA by greater than 90% in confluent cultures of human foreskin fibroblasts.

Animals↗
PubMed1981

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2- to 6-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. 12-O-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF. It did not inhibit the binding of 125I-labeled EGF to these cells. The stimulation of enzyme activity by EGF was not inhibited by cycloheximide or actinomycin D, although these agents did cause a significant decrease in enzyme levels when added without EGF. Also, colchicine, chloroquine, ammonium chloride, and methylamine, compounds that inhibit EGF degradation in various cells types, did not interfere with the ability of EGF to elevate enzyme levels in the human hepatoma cells.

Ammonium Chloride↗
PubMed1980

Regulation of coenzyme A biosynthesis by glucagon and glucocorticoid in adult rat liver parenchymal cells.

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [(14)C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4-2.3) and 2.4-fold relative to cultures containing 1-3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5mum had no effect on the rate of [(14)C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18mum-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [(14)C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.

Animals↗
PubMed1980

Vitamin A potentiates the mitogenic effect of epidermal growth factor in cultures of normal adult human skin fibroblasts.

The polypeptide hormone, epidermal growth factor (EGF) and Vitamin A (retinoic acid) were added to log phase cultures of adult human skin fibroblasts and the effect on cell growth observed. At the end of seven days EGF alone stimulated cell growth 68%, while trans-retinoic acid alone had no significant effect. However, when EGF was added in combination with trans-retinoic acid, cell growth was increased 159 to 214%. Cis-retinoic acid gave similar results. These results indicate that, under certain conditions, a presumed inhibitor of fibroblast growth (retinoic acid) can potentiate the mitogenic action of a hormone, namely, epidermal growth factor.

Adult↗
PubMed1977

Radioimmunoassay for serum transcobalamin II.

A radioimmune assay for transcobalamin II (TC II) was devised from the following: (1) TC II-57Co B12 precipitated from normal serum with (NH4)2SO4 as the labeled ligand; (2) the TC II of whole serum as the standard source of TC II; (3) rabbit anti-pure TC II as the binding agent; (4) separation of the bound and free TC II-B12 by precipitation of the antibody bound with polyethylene glycol. The assay was responsive to either TC II or TC II-B12 and to TC II either pure or in crude preparations. It was not responsive to R-type binders of B12. The median TC II of 10 normal sera was 890 pg. per milliliter and of 10 sera from 10 random hospital patients was 1,010 pg. per milliliter. There was no measurable TC II in the serum of a child with congenital absence of TC II and the assay measured levels greater than 5,000 pg. per milliliter in abnormalities of TC II metabolism.

Antigen-Antibody Complex↗
PubMed1976

Conformation and unfolding thermodynamics of epidermal growth factor and derivatives.

Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.

Amino Acid Sequence↗
PubMed1976

Improved method for the purification of biologically active transcobalamin II.

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.

Blood Proteins↗