Coping with stress.
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Adaptation, Psychological↗
Biomedical subjects
C D Scott
Publications and source records attributed to C D Scott.
Purification and characterization of insulin-like growth factor-II receptors.
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Animals↗
Bilateral spontaneous pneumothoraces complicating AIDS-related Pneumocystis carinii pneumonia.
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Acquired Immunodeficiency Syndrome↗
Immunofluorescent localization of type II insulin-like growth factor receptor in rat liver and hepatoma cells.
We have used an immunofluorescent technique to localize type II insulin-like growth factor (IGF) receptors in rat liver, rat hepatocytes and three rat hepatoma cell lines (HTC, H-35 and 5123) using a polyclonal antibody (C-1) raised to purified rat liver type II IGF receptor. Specificity of the antiserum was confirmed by Western blotting of microsomal membranes prepared from hepatocytes and hepatoma cells which showed a single class of receptor in all cells, of Mr approximately 210,000 for hepatocytes, HTC and H-35 cells and approximately 220,000 for 5123 cells, on non-reduced, 4-15% polyacrylamide gradient gels. The specificity of the immunofluorescent technique was also verified by abolition of labelling after preincubation of antiserum with purified type II IGF receptor. Rat liver cryosections contained areas of juxtanuclear labelling in hepatocytes, consistent with the presence of type II IGF receptor in the Golgi region. Brightest immunofluorescence was seen in sections from fetal and neonatal rats with adult rat hepatocytes staining brightly only around central veins. Areas of labelling were also seen in connective tissue surrounding larger veins. Cultured adult rat hepatocytes and rat hepatoma cell lines also showed bright areas of juxtanuclear nuclear immunofluorescence, with HTC and H-35 cells staining more than 5123 and adult hepatocytes. Fetal rat hepatocytes in culture also labelled very brightly both in a juxtanuclear location and in small clusters over the cell, possibly on the cell surface. These observations indicate that type II IGF receptors are located predominantly on intracellular membranes and are most abundant in rapidly growing cells and tissues (such as fetal liver and hepatoma cells).
Aging↗
Managing occupational stress associated with HIV infection. Self-care and self-management skills.
There is a crisis in health care that has to do with the ability of health-care workers to provide the long-term care and help for AIDS patients. Although more severe, it is just an extreme form of the denial among health care professionals of their own needs, and lack of ability to manage stress. The situation also reflects absence of explicit concern in health settings for creating environments that are healthy for staff. This paper has suggested some of the forms that a comprehensive response to this problem can take.
Acquired Immunodeficiency Syndrome↗
Differential regulation of insulin-like growth factor-II receptors in rat hepatocytes and hepatoma cells.
This study compares the regulation of IGF-II receptors in three rat hepatoma lines, HTC, H-35 and 5123tc, and primary rat hepatocytes. In all cell types [125I]IGF-II bound solely to a species of approximately 250 kDa. Cell surface IGF-II receptors in hepatoma cells had slightly lower affinities (1-2 liters/nmol) than in hepatocytes (4 liters/nmol), but slightly higher IGF-I cross-reactivity (2-4% compared to 1% in hepatocytes). In confluent cultures, the three hepatoma lines expressed 5- to 15-fold more cell-surface receptors per cell than hepatocytes. However, while hepatocyte receptors showed marked inverse density-dependence, increasing over 6-fold between dense (3 x 10(5) cells/3.8 cm2) and sparse (0.16 x 10(5) cells/3.8 cm2) cultures, receptors in all hepatoma lines remained at a constant high level regardless of culture density. These distinct regulatory patterns resemble those described for growth-related functions in hepatocytes and hepatoma cells, and are thus consistent with a role for IGF-II receptors in liver cell proliferation.
Animals↗
Exclusive contracts and hospital privileges--a hospital perspective.
Hospitals are increasingly entering into exclusive contracts with physician to operate a hospital department. Physicians who are not parties to the exclusive contract are foreclosed from providing any of the covered services and, in most instances, from access to the hospital equipment and facilities necessary for rendering such services. This article focuses on the substantive and procedural legal issues that arise with regard to physicians whose privileges are reduced or terminated as a result of an exclusive contract arrangement and factors that will be considered by hospitals in negotiating exclusive contracts.
Contract Services↗
Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density.
Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.
Animals↗
Purification and immunological characterization of the rat liver insulin-like growth factor-II receptor.
Rat liver microsomal insulin-like growth factor-II (IGF-II) receptor has been purified to homogeneity using a single step affinity chromatographic procedure on agarose-IGF-II with elution at pH 4. Determined by either IGF-II binding or a direct RIA for receptor, purification of 2000-fold was obtained. The mean recovery was 28% for five such preparations. Sodium dodecyl sulfate-electrophoresis and autoradiography of purified receptor, radioiodinated receptor, and affinity-labeled receptor all indicated a molecular mass of approximately 250K. Scatchard analysis of IGF-II binding to purified receptor, solubilized microsomal membranes, or plasma membranes showed a single class of binding site with an affinity constant of 6 X 10(10) liter/mol in all cases. Potent antibodies to the purified receptor were raised in rabbits, capable of inhibiting 50% of IGF-II binding at dilutions of 1:170,000 and also of fully precipitating IGF-II-prelabeled receptor at 1:50,000. Both types of antibodies reacted with IGF-II receptors in rat adipose tissue, brain, heart, kidney, lung, and spleen. However, little cross-reactivity was seen with other species. Comparison of the ability of receptor antibodies to inhibit IGF-II binding to microsomal and plasma membranes indicated a specific immunological difference between the IGF-II receptors in the two membrane preparations.
Animals↗
Hormonal regulation of the peripubertal surge of insulin-like growth factor-I in the rat.
The marked increase in circulating insulin-like growth factor-I (IGF-I) levels during puberty observed in primates indicates an important functional relationship between hypothalamic-pituitary gonadal function and hormonal regulation of peripubertal circulating IGF-I levels. Recent studies demonstrating local production and secretion of gonadal peptides including IGF-I suggest that increased circulating IGF-I levels during puberty might be due to direct gonadal secretion of IGF-I or alternatively to indirect effects of increased gonadal steroid secretion on nongonadal tissues including the hypothalamus, pituitary, and liver. We therefore studied the effects of prepubertal castration on the pubertal IGF-I surge and demonstrate that castration provokes a further increase rather than ablation of the pubertal IGF-I surge in the rat. Furthermore, neonatal treatment with monosodium glutamate, a hypothalamic neurotoxin, abolishes the pubertal IGF-I surge when commenced on postnatal day 1 but not on day 5, whereas treatment with a GnRH antagonist commencing within 12 h of birth significantly reduces but does not abolish the pubertal IGF-I surge. We therefore propose that the pubertal IGF-I surge in the rat is not due to direct gonadal secretion of IGF-I or other gonadal hormones during puberty but may involve hypothalamic and/or hepatic programming by events during prenatal or very early postnatal life.
Animals↗
Comparison of receptors for insulin-like growth factor II from various rat tissues.
Receptors for insulin-like growth factor II (IGF-II) have been compared in solubilized microsomal membranes from rat lung, brain, kidney, heart, epididymal and subcutaneous fat, ovary, testis and adrenals. Highest binding/microgram protein was seen with testicular membranes. Receptors from all tissues showed high affinity for human IGF-II (mean association constant = 65 litres/nmol) and a high degree of specificity (mean IGF-I cross-reactivity 0.3%; no cross-reactivity with insulin). Affinity labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed binding was only to a type-II IGF receptor, with a major band seen at a molecular weight of about 230,000 in lung, brain, kidney and testis, and 240,000 in heart, fat and adrenal gland. All tissues showed broad or bimodal pH dependence of binding, with optima seen at about pH 6 and pH 9. Mild stimulation of IGF-II binding by low calcium concentrations (1-2 mmol/l) was seen in all tissues, although higher concentrations were inhibitory in the brain. It was concluded that IGF-II receptors from different rat tissues, when studied under uniform conditions, show similar binding affinities but differences in size and regulation which might be missed if receptors are examined in separate studies.
Adipose Tissue↗
Production of insulin-like growth factor I and its binding protein in rat hepatocytes cultured from diabetic and insulin-treated diabetic rats.
This study examines the effect of experimental diabetes on the release of rat insulin-like growth factor I (rIGF-I) and its binding protein (IGF-BP) by adult rat hepatocytes in primary culture. Rats treated with streptozotocin (75 mg/kg) had decreased serum rIGF-I values of 0.37 +/- 0.04 U/ml compared to 1.06 +/- 0.04 in age-matched untreated rats (1 U = 770 ng human IGF-I). Concomitant decreases in hepatocyte production rates for rIGF-I (15% of the rate in cells from normal rats) and IGF-BP (30% of normal) were also observed for hepatocytes isolated from diabetic rats. Insulin replacement therapy (1.2 U/day) for 3-4 days normalized serum rIGF-I levels (0.92 +/- 0.07 U/ml) and increased rIGF-I production by isolated hepatocytes to 67% the rate of normal cells and IGF-BP production to 70% normal. Treatment of streptozotocin-treated rats with rGH (150 micrograms/day) in vivo for 7 days failed to increase serum rIGF-I levels or hepatocyte production of rIGF-I. Insulin in vitro (3 X 10(-7) M) increased rIGF-I release by hepatocytes from nondiabetic rats, but had no effect on cells from diabetic animals, suggesting that factors other than insulin are required to maintain rIGF-I synthesis in diabetes. Serum rIGF-I levels showed a strong correlation with hepatocyte rIGF-I production in the animals used in this study. However, calculation of circulating rIGF-I half-life based on these values showed a 2-fold higher half-life in diabetic rats (7.91 +/- 1.58 h) and rGH-treated diabetic rats (7.52 +/- 1.25 h) than in nondiabetic (2.99 +/- 0.35 h) and insulin-treated diabetic animals (3.85 +/- 0.36 h). This suggests that the rate of clearance of circulating rIGF-I may be slower in diabetic animals.
Animals↗
Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes.
Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.
5-Aminolevulinate Synthetase↗
Interactions between ATP and cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries.
ATP stimulated the rate of [4-14C]cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. The effect of ATP was apparently similar to the stimulatory effect of choriogonadotropin on mitochondrial [4-14C]cholesterol utilization. Enhancement of the rate of steroidogenesis by ATP and choriogonadotropin were not additive. ATP seemed to promote both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for [4-14C]cholesterol utilization from both endogenous substrate and succinate.
2,4-Dinitrophenol↗
Production of insulin-like growth factor I and its binding protein by adult rat hepatocytes in primary culture.
This study examines the production and characteristics of insulin-like growth factor I (IGF I) and its binding protein (BP) from adult rat hepatocytes in primary culture. IGF I and BP in samples of conditioned medium were separated by automated high pressure gel permeation chromatography at pH 2.8, with total recovery of both species. Measured by a specific RIA, with about 2% IGF II cross-reactivity, the rate of production of IGF I [compared with a human IGF I (hIGF I) standard] was 4.39 +/- 0.57 pmol/mg cell protein X 24 h; measured by a radioligand assay using radioiodinated hIGF I as tracer, BP was produced at a rate of 2.97 +/- 0.54 pmol binding sites/mg cell protein X 24 h. High pressure permeation chromatography studies indicated approximate mol wts of 7,500 for hepatocyte IGF I and 50,000 for BP. The pI of hepatocyte IGF I was 8.8, and of the predominant BP species, 6.8. Rat serum treated in the same way as hepatocyte medium showed IGF I and BP activities at identical pI values to those from hepatocytes but, in addition, contained BP activity at more acidic pI values, apparently not produced directly by the liver. Competitive binding studies using acid-chromatographed hepatocyte or serum BP, and hIGF I and IGF II as labeled and unlabeled ligands, also suggested that BP activity in serum was heterogeneous, while BP from hepatocytes appeared to be a single species with similar affinities for IGF I and IGF II. It is concluded that adult rat hepatocytes produce immunoreactive IGF I, pI 8.8, and BP, pI 6.8, which are released into the circulation, but that additional, more acidic, BP species which are also found in serum could result from extrahepatic production or processing.
Animals↗
Rat hepatocyte insulin-like growth factor I and binding protein: effect of growth hormone in vitro and in vivo.
This study examines the regulation of insulin-like growth factor I (IGF I) and binding protein (BP) release by adult rat hepatocytes in primary culture. Increasing the density of plating of cells had a marked positive effect on the IGF I production rate per mg cell protein, with the highest rate, approximately 4 pmol/mg cell protein X 24 h, seen at densities of 1 X 10(5) cells/cm2 or higher. Cycloheximide (15 micrograms/ml) inhibited both IGF I and BP production by more than 90%, while actinomycin D (0.1 microgram/ml) caused less marked, but still significant, inhibition. Over the insulin range 30 pM-300 nM there was a 45% increase in IGF I production, but no effect on BP production. Bovine GH stimulated production of both peptides, significant effects (up to 50% stimulation) being seen at concentrations from 20-500 ng/ml. Cells from hypophysectomized rats, with serum IGF I and GH levels reduced more than 90% from normal, had IGF I and BP production rates only 7% of normal, measured after 48 h in culture. In vivo replacement with rat GH, 150 micrograms/day for 5 days by osmotic minipump, significantly restored the production of both peptides by hepatocytes. Cells isolated from rats bearing the GH-secreting tumor, MtT/W15, had IGF I and BP production rates approximately twice as high as normal. A significant stimulatory effect of bovine GH (200 ng/ml) in vitro was seen on cells from normal and hypophysectomized, GH-replaced rats, but not from unreplaced hypophysectomized, or tumor-bearing rats. The results in hypophysectomized, animals are consistent with known changes in hepatic GH receptors, while the lack of GH responsiveness in tumor-bearing rats indicates a persistence of maximal stimulation in culture. The reflection of in vivo GH status by hepatocytes cultured for 48 h suggests that messenger RNA turnover for IGF I and BP must be slow. The close parallel in the regulation of the two peptides under most conditions might be indicative of coordinated synthesis.
Animals↗