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Biomedical subjects

R C Baxter

Publications and source records attributed to R C Baxter.

At least 199 records · Page 11Linked to original sources
PubMed1988

Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs.

We have characterized the insulin-like growth factor-binding protein (IGF-BP) produced by neonatal human skin fibroblasts in monolayer culture using antibodies specific for the acid-stable subunit of the 150K GH-dependent IGF-BP complex, BP-53, and the amniotic fluid IGF-BP, BP-28. Fibroblasts produced 65.3 +/- 10.4 ng/ml.72 h (SE; n = 6) immunoreactive BP-53 in serum-free medium; this was stimulated by increasing fetal bovine serum in the medium up to 385.3 +/- 49.0 ng/ml.72 h at 10% serum. Epidermal growth factor (EGF) also caused dose-dependent stimulation of BP-53 production, with a maximal effect (3-fold increase) at 30 ng/ml EGF. No immunoreactive BP-28 production was detectable in the presence or absence of serum or EGF. Neutral gel chromatography of serum-free medium revealed a peak of immunoreactive BP-53 at about 50K, with a smaller species at 20-30 K. Serum- and EGF-stimulated cells produced higher levels of about 50K BP-53, and an additional peak of immunoreactivity at 150K was present in serum-stimulated, but not EGF-stimulated, samples. Comparison of IGF-I and IGF-II binding by fibroblast BP-53 revealed slightly higher IGF-II than IGF-I binding, and association constants of 3-4 x 10(10) liter/mol for both IGFs, similar to BP-53 from human plasma. Affinity labeling of acid-stripped medium followed by nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specifically cross-linked IGF-binding species of 60K (identical to labeled plasma BP-53), 42K, and 37K. Only the 60K and 42K complexes were precipitable by antiserum to plasma BP-53, and none was precipitable by anti-BP-28 serum, suggesting that the 37K band might represent a third class of IGF-BP. We conclude that neonatal skin fibroblasts produce no BP-28, but do produce two IGF-BPs immunologically homologous to human plasma BP-53, one of which shows size and IGF-binding characteristics identical to the plasma protein.

Antibodies↗
PubMed1988

Characterization of the acid-labile subunit of the growth hormone-dependent insulin-like growth factor binding protein complex.

The insulin-like growth factors (IGFs) circulate predominantly in a 150,000-dalton (150K) complex, the IGF-binding component of which appears to be an acid-stable 53K glycoprotein (BP-53). This study tested the hypothesis that an acid-labile subunit (ALS) reacts with the binding subunit to form the 150K complex. ALS activity was quantitated by the conversion of covalent BP-53-[125I]IGF-I tracer from about 60K to about 150K. DEAE-Sephadex chromatography of serum at pH 8.2 yielded separate peaks of 30-60K BP-53 immunoreactivity and 100-110K ALS which, when mixed, converted the BP-53 to 150K. Whole serum also contained 100-110K ALS not complexed with BP-53. ALS was markedly acid labile, irreversibly losing activity below pH 4.5. In an assay involving competition between test substances and BP-53-IGF-I tracer in the reaction with partially purified ALS, serum samples, acidified to inactivate endogenous ALS, reacted with potencies proportional to their immunoreactive BP-53 content. Pure BP-53 alone was inactive, but after preincubation with IGF-I or IGF-II, competed with a potency identical to that of BP-53 in acidified serum. Amniotic fluid IGF-binding protein, with or without IGFs, had no activity. These results confirm that serum contains an acid-labile protein which interacts with the acid-stable IGF-binding protein BP-53, when it is occupied by IGFs, to convert it to the 150K form.

Amniotic Fluid↗
PubMed1988

Regulation of the growth hormone-independent growth factor-binding protein in children.

Regulation of the diurnal variation of the GH-independent insulin-like growth factor-binding protein (BP-28) was studied in 53 children who underwent various investigations for possible endocrine abnormalities. The plasma BP-28 levels increased 12-fold from 8 +/- 2 (+/-SE) micrograms/L at 2100 h to a peak level of 109 +/- 15 micrograms/L between 0600 and 0800 h. This rise was inversely related to plasma insulin levels and was unrelated to plasma cortisol levels. The overnight rise of plasma BP-28 was significantly altered in children who ate a light meal at 0130 h; in them BP-28 levels started to fall after 0300 h, reached nadir levels at 0400 h, began to rise again by 0700 h, and returned to control levels by 0800 h. Such changes did not occur in children given water alone. From the peak early morning level, plasma BP-28 fell to basal levels in children given oral glucose at 0800 h; the t1/2 of the fall was 55 +/- 9 (+/-SE) min. In children who continued to fast, plasma BP-28 did not fall but, rather, increased from 144 +/- 12 micrograms/L at 0800 h after 10 h of fasting to 239 +/- 30 micrograms/L by 1600 h. The induction of hypoglycemia by insulin given at 0945 h after an overnight fast caused a similar but more rapid rise in plasma BP-28 to 668 +/- 317 micrograms/L (range, 208-1763 micrograms/L) by 1230 h. These results suggest that the diurnal variation of plasma BP-28 concentrations in children is not due to an intrinsic rhythm, but is regulated by the metabolic status of the child.

Adolescent↗
PubMed1988

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein.

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.

Amino Acid Sequence↗
PubMed1988

Metabolic regulation of the growth hormone independent insulin-like growth factor binding protein in human plasma.

This study examines the regulation of circulating GH-independent insulin-like growth factor binding protein, BP-28. Commencing at 22.00 h, BP-28 in 5 normal adults rose 11-fold to peak values of 120 +/- 12 micrograms/l, remained elevated between 01.00 and 08.00 h, then fell rapidly following a meal. If meals were omitted, BP-28 remained at peak levels throughout the day. The fasting BP-28 level was higher in women (141 +/- 22 micrograms/l, N = 5) than men (59 +/- 14 micrograms/l, N = 7), and pregnancy caused a further 2-fold elevation. Oral glucose rapidly lowered BP-28 in diabetic and nondiabetic pregnant women, nonpregnant women, and men. In a heterogeneous group of 18 subjects, insulin (0.1 U/kg iv), with or without simultaneous administration of GnRH and TRH, elicited a 3- to 4-fold rise in BP-28, commencing 60 min after the nadir of plasma glucose, and independent of the response in GH, PRL, TSH, LH or cortisol. We conclude that BP-28 levels in adults are metabolically regulated, and postulate a role for this protein in the maintenance of glucose homeostasis.

Adolescent↗
PubMed1987

Production of IGF-binding proteins by vascular endothelial cells.

Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.

Animals↗
PubMed1987

Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins.

Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 X 10(10) l/mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs.

Amino Acid Sequence↗
PubMed1987

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↗
PubMed1987

Relationship of somatomedin-C/insulin-like growth factor I levels to conventional nutritional indices in critically ill patients.

Twenty ICU patients, with varying diagnoses and degrees of catabolism, were studied prospectively to determine whether somatomedin-C/insulin-like growth factor I (SMC/IGFI) is related to the conventional nutritional indices, plasma prealbumin, transferrin and albumin, and nitrogen balance (NB) in critical illness. Mean SMC/IGFI concentration in these critically ill patients was below the lower limit of the reference range. SMC/IGFI concentrations correlated with NB for the 24 h before measurement (r = .38, p less than .01) and with cumulative NB for the previous 2 (r = .50, p less than .01), 3 (r = .34, p less than .05), and 5 days (r = .46, p less than .05). Prealbumin correlated with cumulative 5-day NB (r = .39, p less than .05). Plasma albumin and transferrin concentrations did not correlate with NB for any of these time periods. SMC/IGFI concentrations correlated with cumulative protein (r = .59, p less than .01), carbohydrate (r = .63, p less than .01), and energy intake (r = .64, p less than .01). SMC/IGFI was the only index which consistently correlated with NB. We conclude it is a useful index of nutritional status in critically ill patients.

Adult↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb.

The role of the pituitary gland in the regulation of skeletal growth and plasma insulin-like growth factor (IGF)-I and IGF-II concentrations in the late gestation sheep fetus has been studied. Singleton fetuses were either hypophysectomized (n = 14) or sham operated (n = 8) between days 110 and 125. Fetal and maternal blood samples were collected three times weekly through the remainder of gestation. In some hypophysectomized fetuses (HXT4 group, n = 4), T4 was administered (100 micrograms L-T4/day) to overcome the hypothyroidism caused by hypophysectomy. The other hypophysectomized fetuses received no replacement therapy (HXNR group, n = 10). Six HXNR fetuses were allowed to remain in utero post term and were killed at day 163 of gestation. All other animals were killed at day 147. All values are group means +/- SE. Hypophysectomized fetuses had significantly shorter limbs and long bones and delayed osseous maturation at term compared to sham controls. Plasma free T4 concentrations in HXT4 fetuses were not significantly different from those measured in sham fetuses (P greater than 0.05). Bone maturation at term was normal in HXT4 fetuses although there was no improvement in limb or bone elongation. Retention of hypophysectomized fetuses in utero until 16 days past term yielded fetuses which were heavier than controls but whose limb and bone lengths were no greater than hypophysectomized fetuses killed at term. Osseous maturation was appropriate for term in five of the six postterm hypophysectomized fetuses. The plasma IGF-I and IGF-II concentrations were not significantly affected by hypophysectomy, hypophysectomy with T4 replacement, gestational age, or prolonged gestation. The plasma IGF-I concentrations in the sham, HXNR, and HXT4 fetuses were 35.4 +/- 6.6, 28.2 +/- 3.0, and 34.4 +/- 1.7 ngeq human (h)IGF-I/ml, and the IGF-II concentrations were 656.3 +/- 59.2, 635.3 +/- 56.3, and 645.5 +/- 71.9 ngeq hIGF-II/ml, respectively, and remained within these ranges throughout the experiment. Fetal IGF-I concentrations were significantly lower than mean maternal IGF-I concentrations (88.0 +/- 6.8 ngeq hIGF-I/ml, P less than 0.05), and fetal IGF-II concentrations were significantly higher than mean maternal IGF-II concentrations (362.4 +/- 24.0 ngeq hIGF-II/ml, P less than 0.05). We conclude that in the late gestation fetal sheep, elongation of the appendicular skeleton is under some direct pituitary control whereas appendicular maturation exhibits some dependence on circulating T4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

The insulin-like growth factor-II (IGF II) receptor from rat brain is of lower apparent molecular weight than the IGF II receptor from rat liver.

The binding subunits of the insulin and insulin-like growth factor-I (IGF I) receptors from rat brain are of lower molecular weight than the corresponding receptor in rat liver, possibly due to variations in sialic acid content. We have compared the IGF II receptor from rat brain and rat liver. The brain receptor is of smaller apparent mol wt (about 10 K) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is independent of ligand binding as it persists in iodinated and specifically immunoprecipitated receptors. From studies of wheat germ agglutinin binding and the effect of neuraminidase on receptor mobility, we conclude that this difference is not simply due to variations in sialic acid content. Treatment with endoglycosidase F results in reduction in the molecular size of both liver and brain receptors and after this treatment the aglycoreceptors are of similar size. We conclude that in rat brain tissue the IGF II receptor like the binding subunits of the insulin and IGF I receptors is of lower molecular size than the corresponding receptors in rat liver. This difference is due to differences in N-linked glycosylation.

Animals↗
PubMed1987

Two immunoreactive binding proteins for insulin-like growth factors in human amniotic fluid: relationship to fetal maturity.

A binding protein for insulin-like growth factors (IGFs) has been purified from human amniotic fluid by IGF-I affinity chromatography and high performance reverse phase chromatography. This protein, with an apparent molecular mass of 28K nonreduced and 34K reduced, had an identical amino-terminus to a previously purified binding protein from amniotic fluid and to placental protein 12. The purified preparation (BP-28) bound both IGFs with high affinity [Ka, 6.55 +/- 2.24 (+/- SD) L/nmol for IGF-I and 3.23 +/- 1.05 L/nmol for IGF-II], with approximately 0.5 mol binding sites/mol BP-28 for either ligand. A 53K IGF-binding protein purified from human plasma (BP-53) did not cross-react in a RIA for BP-28, and BP-28 had less than 0.1% molar cross-reactivity in a RIA for BP-53. Human amniotic fluid reacted strongly in both assays. Fractionation of amniotic fluid samples by reverse phase chromatography showed that BP-28 and BP-53 immunoreactivities were present on separate proteins. In 40 third trimester amniotic fluid samples selected to cover a wide range of lecithin to sphingomyelin ratios, the mean concentrations of BP-28 and BP-53 were 37.6 +/- 17.6 (+/- SD) and 4.6 +/- 1.6 mg/L, respectively. Significant negative correlations were found between the levels of both BP-28 and BP-53 and the lecithin to sphingomyelin ratio, suggesting an association between the levels of both proteins and the degree of fetal maturity. A significant positive association was also found between the levels of BP-28 and BP-53. We conclude that the 28K IGF-binding protein from amniotic fluid, like the previously purified 53K binding protein, has high affinity for both IGF-I and IGF-II, that it coexists in amniotic fluid with BP-53 or a related protein, and that the levels of both proteins decline with increasing fetal maturity.

Amniotic Fluid↗
PubMed1987

Diurnal rhythm of growth hormone-independent binding protein for insulin-like growth factors in human plasma.

An antibody raised against the major insulin-like growth factor (IGF)-binding protein in amniotic fluid (BP-28) was used to measure the levels of a cross-reacting protein in human plasma by RIA. Plasma BP-28 immunoreactivity had an apparent mol wt of 35,000 by high performance permeation chromatography. Sampled hourly for 12- or 24-h periods in 15 children with a wide range of GH secretory activity, plasma BP-28 levels showed a marked diurnal cycle, rising 10- to 20-fold between 2400 and 0600 h to a peak of 50-500 micrograms/L, then falling to basal levels (0-40 micrograms/L) by 1200 h. Plasma GH levels were measured at 20-min intervals in the same subjects. Neither peak nor basal BP-28 levels were significantly associated with chronological age, bone age, mean or peak nocturnal GH secretion, relative height, or relative growth velocity in tall, normal, short, or GH-deficient children. Plasma proteins measured in a RIA for 53,000 mol wt GH-dependent IGF-binding protein (BP-53) did not vary diurnally. The IGF-binding activity of plasma BP-28, measured by incubating plasma with IGF tracer and precipitating the BP-28-IGF complex with anti-BP-28 antiserum, closely paralleled the morning rise in BP-28 immunoreactivity. Immunoprecipitable BP-28 bound both IGF-I and IGF-II tracers, with a clear preference for IGF-I, and unlabeled IGF-I was more effective than IGF-II in displacing either tracer IGF. We conclude that plasma BP-28 levels in children have a marked diurnal rhythm which is unrelated to GH secretory activity.

Adolescent↗
PubMed1987

Short-term exposure to insulin-like growth factors stimulates testosterone production by testicular interstitial cells.

The effect of short-term exposure to the insulin-like growth factors (IGF-I and IGF-II) on testosterone production by rat testicular interstitial cells in primary culture has been examined. Both peptides, when present during a 1-h pre-incubation period, increased human chorionic gonadotropin (hCG)-stimulated testosterone release over the following 16-h period. The effect of exposure to IGFs was most marked on maximally hCG-stimulated testosterone release. Maximal stimulation following IGF exposure was 80-85% above that seen without IGFs, and the IGF effect was half-maximal at 1.5-2 micrograms/l of IGF-I or IGF-II. Pre-incubation with IGFs did not alter the concentration of hCG (0.1 microgram/l) at which half-maximal stimulation of testosterone release was seen. Increasing cell density had a marked effect on the testosterone production rate per 10(5) cells, and the stimulatory effect of IGFs was only seen at relatively high cell density (2.8 X 10(5) cells/ml). Varying the period of pre-incubation with IGFs between 0.5 and 16 h, it was found that a 1-h period gave maximal stimulation. We conclude that a short exposure to IGFs is capable of increasing hCG-stimulated steroidogenesis in Leydig cells, and postulate that this effect may be part of an intratesticular paracrine control mechanism.

Animals↗
PubMed1987

High-affinity receptor for insulin-like growth factor II in rat liver: properties and regulation in vivo.

Receptors for insulin-like growth factor II (IGF-II) have been identified in many tissue types and have been shown to differ widely in their specificities and affinities. We have characterized the IGF-II receptor in rat liver microsomal membranes, both in the intact membrane and in a solubilized extract. Binding was time- and temperature-dependent and was unaffected by changes in pH in the range 6-9. Half-maximal displacement was obtained with 0.33 ng IGF-II/ml standard, and Scatchard analysis showed a class of receptors with an affinity for IGF-II of 1.33 +/- 0.36 X 10(10) litres/mol which increased threefold in the presence of Ca (1 mmol/l) to 3.74 +/- 0.89 X 10(10) litres/mol. There was also a threefold decrease in the rate of dissociation in the presence of Ca. Cross-reactivity with IGF-I was less than 1% and there was no cross-reactivity with insulin. Infusion of rat GH or prolactin for 1 week, at the rate of 175-200 micrograms/day, into female rats had no effect on IGF-II binding in control animals, but rat GH infusion caused a 60% increase (P less than 0.001) in binding in hypophysectomized rats by increasing the number of receptors. These studies demonstrate that rat liver microsomal membranes contain a highly specific, high-affinity receptor for IGF-II which may be under partial GH control.

Animals↗