Biomedical subjects

J L Stevenson

Publications and source records attributed to J L Stevenson.

29 records · Page 2Linked to original sources

PubMed1987

Partial purification from human serum of a specific binding protein for human growth hormone.

Following the recent identification and characterization of a highly specific binding protein(s) for human growth hormone (hGH) in human sera, we now report our initial studies on its purification. Outdated blood blank serum was first fractionated by ammonium sulphate precipitation. The majority of the binding activity appeared in the 30-45% saturation fraction, which was then applied in sequence to an hGH-affinity column, to strong anion exchange (SAX) chromatography (a Mono Q column 0.01-0.2 M phosphate gradient), to a TSK phenyl hydrophobic interaction column (HIC) (0.75-0 M sulphate gradient in 0.1 M phosphate) and finally to a second SAX step. By Scatchard analysis the final product was purified approximately 16,500-fold compared to serum with an overall recovery of 5%. The binding affinity (Ka) of the purified material was approximately 0.4 X 10(9) M-1, very similar to that of whole serum (approximately 0.5 X 10(9) M-1). On silver-stained, sodium dodecyl sulphate-polyacrylamide gels, the final SAX product yielded a major doublet, under reduced conditions, of molecular weights (MW) 55,000 and 59,000. Although it is not known whether one or both of these bands might have hGH binding activity, the MW 59,000 band is very similar in size to one (MW 60,000) of two specific binding proteins observed by covalent crosslinking techniques. These data indicate that the specific hGH binding activity of human serum can be substantially purified and that it appears to exist in at least two forms. Further studies are required to determine the biochemical and physiological relationship, if any, between the two forms.

Chromatography, Affinity↗

PubMed1986

Affinity purification and structural characterization of a specific binding protein for human growth hormone in human serum.

A highly specific binding protein for human growth hormone (hGH) has been isolated from human serum by hGH-affinity chromatography. A purification of approximately 1500-fold with a 30-40% recovery was obtained with essentially no alteration in binding characteristics. Covalent cross-linking of 125I-hGH to the binding protein, followed by analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, revealed two specifically labeled complexes. Allowing for a 1:1 binding stoichiometry the binding proteins themselves had mean mol wts of 57,000 and 69,300. These increased slightly to mol wt 60,300 and 72,000 respectively in the presence of 100 mM dithiothreitol, suggesting the presence of intramolecular but not inter-subunit disulfide linkages. These data confirm the presence of the hGH binding protein(s) in human serum and define their gross structural nature.

Blood Proteins↗

PubMed1986

Variability of pulmonary responsiveness to aerosolized histamine in normal rabbits.

Considerable between-subject variability in pulmonary responsiveness to histamine has been reported in normal human subjects, dogs, guinea pigs, and rhesus monkeys, but rabbits have not been studied. We determined the between- and within-rabbit variability of pulmonary histamine responsiveness in 34 anesthetized and mechanically ventilated New Zealand White rabbits. In 30 rabbits, 5 breaths of aerosolized histamine were delivered in 9 increasing concentrations ranging from 0.01 to 100 mg/ml. Eleven of 30 rabbits were rechallenged with histamine on 1-4 additional occasions over a 3-week period. In the remaining 4 rabbits, 9 doses of distilled H2O were aerosolized to determine the degree of spontaneous variability in measurements of lung resistance (RL) and dynamic lung compliance (Cdyn). We defined an increase in RL of greater than 50% of baseline (TD50RL) and a decrease in Cdyn of greater than 25% of baseline (TD25Cdyn) as being significant based on observations in these 4 rabbits. These limits exceeded the 99.9 percentile of spontaneous variability in RL and Cdyn. Pulmonary responsiveness to histamine varied widely, with a greater than 10,000-fold range in TD50RL and a 1,000-fold range in TD25Cdyn between the most and least sensitive rabbits. The variability of this responsiveness was log-normally distributed. It was not correlated with age, sex, or baseline RL and Cdyn. In contrast, within-rabbit responses to histamine challenge were quite reproducible. Five of 30 rabbits were killed at the conclusion of their histamine challenges for pathologic examination of their lungs. No evidence of airway inflammation was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗

PubMed1985

Demonstration of in vitro actions of growth hormone on isolated rat adipocytes.

Previous studies have shown that adipocytes isolated from normal rats possess specific receptors for growth hormone (GH). Thus, we have now investigated the direct effects on such cells of GH added in vitro. The biological actions of GH determined were the stimulation of 14C-glucose oxidation to 14CO2 and conversion to 14C-lipid. As in adipose segments, hGH stimulation of these parameters required a 3-4 hr preincubation period in GH-free medium. The effect of hGH or bGH was dose-dependent with maximal effects at 1-2 micrograms/ml (CO2 55 +/- 13% stimulation above basal n = 9; lipid 33 +/- 4% n = 21, mean +/- SEM). The magnitude of the effect in isolated adipocytes was lower than that seen in adipose segments from similar groups of animals (lipid 67 +/- 26%, n = 4). The induction of responsiveness by preincubation was accompanied by a parallel increase in 125I-hGH binding. These studies have demonstrated that adipocytes from normal rats not only possess specific receptors for GH but also are metabolically responsive to GH added in vitro. These data suggest that the isolated rat adipocyte should be a useful model for further investigation of the relationship between GH receptor and post-receptor events.

Adipose Tissue↗

PubMed1985

Acute cigarette smoke exposure increases alveolar permeability in rabbits.

We measured lung clearance of aerosolized technetium-labeled diethylenetriamine pentaacetic acid (99mTcDTPA) as an index of alveolar epithelial permeability in rabbits exposed to cigarette smoke. Eighteen rabbits were randomly assigned to 3 equal-size groups: control, all smoke exposure (ASE), and limited smoke exposure (LSE). Cigarette or sham smoke was delivered by syringe in a series of 5, 10, 20, and 30 tidal volume breaths with a 20-min counting period between each subset of breaths to determine 99mTcDTPA biologic half-life (T1/2). Mean T1/2 minimum (i.e., the smallest T1/2 observed) was significantly lower (p less than 0.05) for ASE and LSE rabbits than by control rabbits. We observed a significant difference at 20 and 30 breath exposures between the control and ASE group mean values (% baseline) for T1/2, arterial blood pressure, and peak airway pressure. A combination of light and electron microscopy showed focal alveolar edema and hemorrhage in the ASE and LSE groups but no alveolar-capillary membrane damage. In summary, acute cigarette smoke exposure increases alveolar permeability as measured by 99mTcDTPA clearance, but there was no detectable ultrastructural alteration of the alveolar-capillary membrane.

Animals↗

PubMed1984

Identification of a rabbit liver cytosolic binding protein for human growth hormone.

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56 X 10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.

Animals↗

PubMed1984