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Biomedical subjects

R E Fellows

Publications and source records attributed to R E Fellows.

At least 37 records · Page 2Linked to original sources
PubMed1980

Fibroblastic cultures from the diabetic db/db mouse. Demonstration of decreased insulin receptors and impaired responses to insulin.

Binding of 125I-insulin to cells cultured from the skin of nondiabetic and diabetic (db/db) mice was 80 to 90% specific, time- and temperature-dependent, and maximal at pH 8.0. Porcine insulin and desalanine insulin competed equally for 125I-insulin binding, while proinsulin and desoctapeptide insulin were 35% and 20% as potent, respectively. 125I-Insulin dissociated from both types of fibroblasts with a T 1/2 of 7.5 min. Analysis of the dissociation data resolved two rate constants of 3.0 and 1.0 X 10(-4) s-1 for nondiabetic, and 2.0 and 0.8 X 10(-4) s-1 for diabetic fibroblasts. Binding of 125I-insulin to diabetic fibroblasts was 35 to 50% of that to nondiabetic fibroblasts during at least 17 passages. Scatchard analysis of the binding data resolved high (K1 = 2 X 10(10( M-1) and low affinity K2 = 2 X 10(9) M-1) sites. Nondiabetic fibroblasts possessed 7.7 X 10(4) sites/cell, while diabetic fibroblasts possessed 2.9 X 10(4)/cell. Incubation of nondiabetic fibroblasts with insulin resulted in a time- and concentration-dependent decrease in the binding of 125I-insulin. Binding activity returned to normal when insulin was removed and it was prevented by cycloheximide. In contrast, diabetic fibroblasts did not exhibit down-regulation of receptors. A half-maximal and maximum (85%) stimulation of 2 deoxy-D-glucose uptake was observed with 0.75 nM and 16.7 nM insulin in nondiabetic fibroblasts. In contrast, diabetic cultures required 3.5 nM insulin for half-maximal stimulation of 2 deoxy-D-glucose uptake, and maximum stimulation was 32% with 16.7 nM insulin. Similarly, diabetic fibroblasts required higher concentrations of insulin (20 nM) to stimulate ornithine decarboxylase activity to 42% of nondiabetic cells. These results indicate that in comparison with fibroblastic cultures from nondiabetic animals, those from diabetic animals expressed differences in insulin receptor numbers which are maintained in culture over many generations and are accompanied by diminished insulin responses.

Animals↗
PubMed1980

Purification of somatomedin-C from human plasma: chemical and biological properties, partial sequence analysis, and relationship to other somatomedins.

Somatomedin-C (SM-C) was purified from Cohn fraction IV of human plasma by a series of steps which included cation-exchange chromatography, gel filtration, isoelectric focusing, and reverse-phase high-pressure liquid chromatography. The peptide isolated contained 10,142 units/mg, as judged by a radioimmunoassay, representing a 780,000-fold purification from native plasma. The isolated peptide was basic (pI 8.1-8.5) and was judged to be no less than 90% pure. The best fit for integral amino acids was obtained with 78 residues. Limited sequence data were obtained on the N terminus and on five fragments obtained by tryptic digestion after blocking the lysine residues. Three of these fragments and the N terminus could be aligned with portions of insulin-like growth factor I (IGF-I). Of the 25 residues so aligned, 22 were identical with IGF-I. In addition, two tryptic fragments were obtained which are not present in the sequence of IGF-I. SM-C and IGF-I produced identical curves of displacement in radioimmunoassay and radioreceptor assays for SM-C and in an insulin radioreceptor assay. The potencies of SM-C and IGF-I in these assays differed significantly from IGF-II, somatomedin-A, and several preparations of multiplication stimulating activity. At a concentration of 1 ng/mL, SM-C stimulated the progression of Balb/c 3T3 cells into DNA synthesis and when injected in vivo restored mitosis in lens epithelium of hypophysectomized frogs.

Amino Acid Sequence↗
PubMed1980

Alterations in the responsiveness of diabetic fibroblasts to insulin.

Fibroblastic cultures from the skin of nondiabetic and diabetic (db/db) mice have been used to investigate alterations in the biological responses of diabetic cells to insulin. Confluent cultures from the skin of both nondiabetic and diabetic animals possess specific receptors for insulin. Diabetic fibroblasts exhibit only 36% as mich specific binding of insulin as nondiabetic fibroblasts, because of a decrease in the total number of binding sites, without a change in binding affinity. Insulin caused a time- and dose-dependent increase in the rate of 2-deoxy D-glucose (dGlc) uptake and in ornithine decarboxylase (ODC) activity of both nondiabetic and diabetic fibroblasts. In nondiabetic cells, half-maximal increase in dGlc uptake was obtained with 0.3 nM insulin, and a maximum increase of 120% was obtained with 4.1 nM insulin. In contrast, diabetic cultures required 0.8 nM insulin for a half-maximal increase in dGlc uptake, and maximum stimulation with 4.1 nM insulin was only 50% above control levels. With 4-fold higher insulin concentrations, ODC activity of diabetic cells was only 40% that of non-diabetic cells. In nondiabetic cells, doing regulation of insulin receptors by insulin abolished the ability of insulin to stimulate dGlc uptake. These results demonstrate that cells cultured from diabetic animals, which possess a decreased number of insulin receptors, also exhibit decreased stimulated of deoxy D-glucose uptake and ornithine decarboxylase activity by insulin.

Animals↗
PubMed1980

Increased specific binding of angiotensin II in the organum vasculosum of the laminae terminalis area of the spontaneously hypertensive rat brain.

Three localized sites, the organum vasculosum, lamina terminalis (OVLT) subfornical organ (SFO) and cortex in the brains of spontaneously hypertensive (SH) rats and normotensive Wistar-Kyoto (WKY) rats were micro-dissected out. The membranes were assayed for 125I-labelled angiotensin II specific binding. The only significant difference was a 129% greater specific binding in the OVLT in SH compared to WKY rats. Specific binding of 125I-labelled angiotensin II in the SFO and the cerebral cortex was not significantly different between the two strains. SH rats are known to have a greater sensitivity to centrally administered angiotensin II, which may contribute to the hypertension. This data suggests that a difference in central angiotensin II receptors is at least partially responsible for the increased sensitivity.

Angiotensin II↗
PubMed1980

Presence of angiotensin II in neurons cultured from fetal rat brain.

By means of a specific antiserum to angiotensin II (ANGII), it has been possible to demonstrate the existence of ANGII immunoreactivity in cells cultured from fetal rat brain. Dissociated cells cultured from the whole brains of 20-day-old fetuses were stained according to the peroxidase antiperoxidase method. Immunoreactive ANGII was observed in less than 1% of the total cell population. These cells have been identified as neurons on the basis of morphological criteria. Positively stained neuronal processes with localized concentrations of ANGII immunoreactivity were visualized in association with ANGII-containing soma. The presence of ANGII in cultured brain cells provides an experimental model for investigating the role of angiotensin in the CNS.

Angiotensin II↗
PubMed1980

Stimulation of ornithine decarboxylase activity by insulin in developing rat brain.

Insulin administered ip or intracisternally (ic) increased the activity of ornithine decarboxylase (ODC) in whole brains and brain parts of neonatal rats. Maximal stimulation of activity occurred 4-5 h after ip administration. At the highest doses, insulin stimulated ODC activity by up to 5- and 8-fold after ip and ic injection, respectively. The same amount of insulin given ic caused greater increases in activity than when given ip. Insulin stimulated ODC activity in 2-day-old and in 17- to 60-day-old rats but not in 5- or 9-day old neonates or 80-day-old adults. When insulin-induced hypoglycemia was prevented by giving dextrose, the stimulation of ODC activity was approximately the same as that in animals receiving insulin without dextrose. This indicates that insulin-induced stimulation of brain ODC activity was not caused by insulin-induced hypoglycemia or physiological responses to hypoglycemia. Since ODC is considered an indicator of growth stimulation, these results suggest that insulin or insulin-like peptides have a role in the regulation of brain development.

Adrenocorticotropic Hormone↗
PubMed1980

Cells cultured from the diabetic (DB/DB) mouse have a permanent decrease in insulin receptors.

Investigation of mechanisms responsible for the decreased numbers of insulin receptors observed in obesity and diabetes has been facilitated by the development of cell culture systems permitting study of cellular events independent of fluctuating hormone levels and multiple endocrine interactions present in the whole organism. With such a system, we have found that cells cultured from the skin of diabetic mice have 45-48% fewer receptors for insulin than those from nondiabetic littermates. This difference is maintained in culture over many generations, suggesting that the decreased expression of insulin receptors in these cells is related to the genetic trait for diabetes.

Animals↗
PubMed1979

Alteration of insulin binding and cytoskeletal organization in cultured fibroblasts by tertiary amine local anesthetics.

Tertiary amine local anesthetics cause a time- and dose-dependent, reversible increase in insulin binding sites in cultured chick embryo fibroblasts. Incubation of fibroblasts with 0.2 mM dibucaine for 3 h at 37 degrees C results in a twofold to threefold increase in insulin binding, with an increase in average number of binding sites (Ka = 3.0 X 10(7) M-1) from 9 X 10(3) per cell. Trypsin or ethyleneglycoltetraacetic acid (EGTA) alone increases insulin binding twofold to threefold, but fails to further increase 125I-insulin binding in cells pretreated with dibucaine. Transformation of chick embryo fibroblasts with Rous sarcoma virus causes a threefold to fivefold increase in insulin binding, which is not further increased by incubation with dibucaine. As demonstrated by transmission electron microscopy, dibucaine and trypsin also induce changes in the cytoskeleton of chick embryo fibroblasts, characterized by disorganization and disappearance of microfilament and microtubule bundles. These alterations are accompanied by gross morphologic changes, including rounding of cells and appearance of numerous ruffles and blebs on the cell surface. These observations are consistent with the hypothesis that expression of surface receptors in cultured chick embryo fibroblasts is related to the organization and disorganization of cytoskeletal structures.

Anesthetics, Local↗
PubMed1978

Stimulation of ovine placental lactogen secretion by arginine infusion.

Arginine has been demonstrated to be a potent stimulus to GH and PRL secretion. To determine the effect of arginine on plasma ovine placental lactogen (oPL) concentrations, arginine (50 g in 350 ml distilled water, pH 7.4) or hypertonic saline of identical volume, osmolality, and pH was infused iv over a 30-min period into nine pregnant ewes, and blood samples from chronic indwelling venous catheters were obtained at frequent intervals before and for 8 h after the infusions. After the infusion of hypertonic saline, plasma oPL concentrations (measured by homologous RIA) decreased 20--50% over 1--2 h and then returned to baseline concentrations. After the infusion of arginine, plasma oPL concentrations also decreased by 20--50% for 1--2 h. However, 2--3 h after the infusion, plasma oPL concentrations in creased 79--115% (delta = 204--700 ng/ml) over preinfusion concentrations in seven ewes and 454% (2930 ng/ml) and 1142% (2042 ng/ml) in two ewes and remained elevated for the remainder of the 8-h interval. When the amount of arginine infused was reduced from 50 to 25 g, an increase in plasma oPL concentrations occurred in only one of five ewes. Plasma oPL concentrations increased by 8--58% after infusions of 50 g alanine or glycine but did not increase after 50 g glutamic acid. The delayed oPL response to arginine suggests that the increase in plasma oPL concentrations is not caused directly by arginine but rather by changes in the synthesis, secretion, and/or degradation of oPL induced indirectly by arginine.

Animals↗
PubMed1977

Ovine placental lactogen induces somatomedin: a a possible role in fetal growth.

Ovine placental lactogen (oPL) and bovine growth hormone (bGH) increase the circulating concentration of somatomedin in hypophysectomized rats. This finding indicates that some of the somatotropic properties of oPL may be due to the induction of somatomedin and raises the possibility that oPL has a role in the control of fetal growth.

Animals↗
PubMed1977

Studies on ovine placental lactogen secretion by homologous radioimmunoassay.

The concentrations of ovine placental lactogen (oPL) have been determined in maternal plasma, umbilical cord plasma, and allantoic fluid by an homologous radioimmunoassay for oPL which is sensitive to 0-1 ng hormone. Ovine placental lactogen was first detected in maternal plasma at 41-50 days of gestation and reached a peak concentration of 2547 +/- 226 (S.E.M.) ng/ml at 121-130 days in ewes with singleton gestations. The oPL concentration in cord plasma was 336-4 +/- 60-3 ng/ml and in allantoic fluid was 29-6 +/- 6-4 ng/ml. After surgical removal of the placenta, oPL disappeared from maternal plasma with a half-life of 29-1 +/- 1-3 min.

Allantois↗
PubMed1976

The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.

Amino Acids↗
PubMed1976

Purification and characterization of bovine placental lactogen.

Bovine placental lactogen (bPL), a polypeptide hormone functionally related to bovine growth hormone (bGH) and bovine prolactin (bPL), has been isolated from placentas by pH and ammonium sulfate precipitation, gel filtration, and ion exchange chromatography on DEAE- and CM-cellulose. The hormone has been purified to approximately 99% homogeneity, as determined by end group analysis. On disc gel electrophoresis at pH 9.0 bPL migrates as a pair of closely spaced bands (Rf = 09517 and 0.541) between the positions of bGH and bPR. Its molecular weight, as estimated by gel filtration on Sephadex G-200 in 6 M guanidine hydrochloride and 6.5 mM dithiothreitol, is 22, 150 and its isoelectric point is 5.9. The amino acid composition of bPL closely resembles that of bGH and bPR except for a higher content of serine and glycine and a lower leucine content. Like bPR, it has 2 tryptophans and 6 cysteines, but its COOH-terminal sequence is identical with that of bGH: -Cys-Ala-Phe-OH. By Ouchterlony immunodiffusion, bPL forms lines of partial identity with bGH against bGH antisera and with ovine placental lactogen (oPL) against oPL antisera. In the bPL-antibPL system, oPL forms a line of partial identity while bGH and bPR do not cross-react. However, bPL does not form a precipitin line with bPR antisera. These data would indicate that in terms of structure, and hence molecular evolution, bPL and other subprimate placental lactogens occupy a position more intermediate between growth hormone and prolactin than do the primate placental lactogens.

Amino Acids↗