Search PubMed⌕ Search

Biomedical subjects

R C Baxter

Publications and source records attributed to R C Baxter.

At least 181 records · Page 10Linked to original sources
PubMed1990

Increased expression of insulin-like growth factor-II/mannose-6-phosphate receptor in regenerating rat liver.

This study examined the effect of two thirds hepatectomy on rat liver insulin-like growth factor-II (IGF-II) receptors and IGF-II receptor messenger RNA (mRNA) levels. IGF-II receptor levels were determined in liver plasma membrane and Golgi/endosome fractions from sham-operated and hepatectomized rats by ligand binding and immunoblotting after electrophoresis. After hepatectomy, IGF-II receptors increased initially in the plasma membrane (within 24 h of surgery) to 4-fold control levels, remained elevated for 72 h, and declined to control levels at 120 h when liver regeneration is near complete. However, Golgi/endosome levels of IGF-II receptors did not increase until 48 h, showing a maximum increase of 3.5-fold at 72 h after surgery and returning to control values at 120 h. The 9-kilobase mRNA for IGF-II receptor, determined by slot blotting with a complementary DNA probe, increased 2.5-fold within 24 h of surgery, attaining a maximum stimulation of 4-fold at 48 h, and then decreasing to normal levels by 120 h. These changes show that, after partial hepatectomy, IGF-II receptors increase rapidly at the cell surface, possibly due to receptor translocation from intracellular pools. This is followed by an increase of IGF-II receptor mRNA and increased receptor synthesis, resulting in an increase in total cellular receptors. These results suggest a role for IGF-II receptors in regenerating liver, perhaps in cell proliferation and/or in tissue remodeling.

Animals↗
PubMed1990

Circulating levels and molecular distribution of the acid-labile (alpha) subunit of the high molecular weight insulin-like growth factor-binding protein complex.

A RIA is described for the acid-labile (alpha) subunit of the high mol wt insulin-like growth factor (IGF)-binding protein complex, a glycoprotein of approximately 85,000 daltons (approximately 85K) which combines with the GH-dependent binding protein (BP-53 or IGFBP-3) and IGF-I or IGF-II to form the complex. The assay shows relative specificity for higher primate species. Whereas amniotic fluid, cerebrospinal fluid, and seminal plasma contain virtually no immunoreactive alpha-subunit, the protein is easily detectable in 0.5-microL serum samples. Serum alpha-subunit levels are markedly age dependent, rising over 5-fold from birth to puberty, then remaining relatively constant throughout adulthood. In 170 children, there was a strong association between alpha-subunit and IGFBP-3 levels. Mean alpha-subunit levels (+/- SD) in adults were 24.2 +/- 4.7 mg/L in 93 normal subjects, 54.1 +/- 15.5 mg/L in 12 acromegalics, 6.5 +/- 4.8 mg/L in 10 GH-deficient subjects, and 31.5 +/- 5.7 mg/L in 18 third term pregnant women. In serum fractionated by gel chromatography, alpha-subunit appeared as a broad 100-150K peak. After depleting serum samples of IGFBP-3 by immunoaffinity chromatography, approximately one third of alpha-subunit remained, in a peak of about 100K, suggesting that two thirds of the total alpha-subunit in serum is present in the approximately 150K complex, and one third is uncomplexed. Development of this RIA for alpha-subunit will allow further study of regulation of the GH-dependent complex.

Adolescent↗
PubMed1989

High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum.

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.

Carrier Proteins↗
PubMed1989

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa).

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.

Amino Acid Sequence↗
PubMed1989

Binding proteins for the insulin-like growth factors: structure, regulation and function.

Binding proteins for the insulin-like growth factors (IGF-I and IGF-II) are increasingly being recognized as modulators of IGF actions in both inhibitory and stimulatory ways. At least three distinct classes of binding protein are thought to exist, differing in their primary structures and binding characteristics, although all are able to bind both IGF-I and IGF-II. This review outlines the purification and characterization of the binding proteins that have been identified to date, and describes the regulation of their production and of their levels in the circulation. Current views on their potential biological roles are also discussed.

Amino Acid Sequence↗
PubMed1989

Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling.

To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit.

Acromegaly↗
PubMed1989

IGF binding proteins in growth-retarded children with chronic renal failure.

Changes in the normal hormonal regulation of linear growth which lead to growth failure in children with chronic renal failure (CRF) are not well understood. Previous studies indicate that serum levels of growth hormone and IGF-I and II are normal or elevated in this population; and that serum levels of poorly defined inhibitors of IGF action are increased. Using a recently developed RIA for the 25-kD IGF-binding protein (IGF-BP25), we report significant elevations of this protein in children with CRF when compared to age- and sex-matched controls. IGF-BP25 levels correlate positively with IGF-binding activity in both populations, indicating that the RIA reflects levels of bioactive protein. Although the variation of serum IGF-BP25 with chronologic age and IGF levels are preserved in CRF, the quantitative interrelationships are disrupted. Levels of the 53-kD IGF-binding protein, an IGF-binding protein derived from the high mol wt IGF complex, were also found to be elevated in the CRF population and, unlike in the control population, did not vary with age or IGF levels. IGF-BP25 has been shown to inhibit IGF mitogenic action in vitro. Our finding of elevated levels of IGF-BP25 in children with CRF suggests that this protein may play a role in the growth retardation of pediatric chronic renal failure. The significance of the elevated IGF-BP53 levels in CRF remains uncertain.

Adolescent↗
PubMed1989

Structural and immunological comparison of insulin-like growth factor binding proteins of cerebrospinal and amniotic fluids.

The insulin-like growth factors (IGFs) found in plasma and a variety of other body fluids are complexed to specific binding proteins (BPs). The cDNA for a 25K IGF-BP was recently cloned and sequenced, and the primary structure of the BP deduced. This BP is found in amniotic fluid, decidual tissues, conditioned medium from HepG2 human hepatoma cells, and fetal plasma. An additional small IGF-BP has been identified in human cerebrospinal fluid (CSF). We now demonstrate that the IGF-BP found in CSF is structurally and immunologically distinct from that found in HepG2 conditioned medium. While the latter BP has approximately equal affinities for IGF-I and -II, the CSF BP has a 10- to 20-fold greater affinity for IGF-II. In affinity cross-linking studies under reducing conditions, the CSF BP had an apparent mol wt (Mr) of 32,000, while the HepG2 BP migrated as a doublet, with apparent Mr values of 30,000 and 28,000. On Western ligand blots, CSF BPs migrate as five discrete bands, with the most prominent band at an apparent Mr of 34,000, while HepG2 medium yielded a single band at an apparent Mr of 30,000. A polyclonal antibody developed against the human amniotic fluid BP immunoprecipitated the HepG2 BP and reacted with both the HepG2 and amniotic fluid BPs on Western blots, but failed to react with the CSF BP. These data indicate that the CSF and amniotic fluid/HepG2 BP are structurally and immunologically distinct small IGF-BPs.

Amniotic Fluid↗
PubMed1989

Regulation of growth hormone-independent insulin-like growth factor-binding protein (BP-28) in cultured human fetal liver explants.

Cultured human fetal liver explants were used to study regulation of the GH-independent insulin-like growth factor-binding protein (BP-28). BP-28 was produced by fetal liver explants at approximately 200 ng/mg total tissue protein.day between days 3 and 6 in culture and was inhibited 60% after 3-day exposure to cycloheximide (10 micrograms/mL). BP-28 production was stimulated over 3-fold by 20 mmol/L theophylline, over 2-fold by 30 mumol/L forskolin, and 90% by 10 micrograms/mL glucagon. The glucose concentration in the medium inversely regulated BP-28 production, which was 68% higher in 2 mmol/L glucose than in 30 mmol/L glucose (P less than 0.001); in contrast, albumin production was 40% lower in 2 mmol/L glucose than in 30 mmol/L (P less than 0.05). BP-28 levels were inhibited 50% by 300 nmol/L insulin or 3 mumol/L dexamethasone. In acid extracts of cultured tissue, BP-28 levels were less than 10% of the amount secreted in 48 h. These results are consistent with the conclusion that intracellular glucose availability is an important regulator of hepatic BP-28 synthesis.

Amniotic Fluid↗
PubMed1989

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↗
PubMed1989

Production of insulin-like growth factor-II by human fetal pancreas in culture.

Insulin-like growth factor-II (IGF-II) is a polypeptide hormone thought to be involved in fetal development because of its wide distribution in fetal tissues and its presence in the fetal circulation. We have developed a highly sensitive radioreceptor assay for IGF-II using rat liver microsomal membranes and have used this assay to test for the presence of IGF-II in the human fetal pancreas and the release of IGF-II by the human fetal pancreas in organ culture. IGF-II was present in extracts of pancreatic tissue (0.056 +/- 0.012 pmol/mg tissue, n = 5) and was released in culture at the rate of 0.027-0.134 pmol/mg tissue per day with release being maintained for at least 3 weeks in culture. The rate of release was not affected by the gestational age of the fetus over 14 to 20 weeks but was significantly related to the rate of insulin release (r = 0.712, P less than 0.001, n = 34). Chronic exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), which inhibits insulin release in the human fetal pancreas, caused an 85% drop in IGF-II production, which was reversed when TPA was removed. These studies demonstrate that IGF-II is produced by the human fetal pancreas in a pattern similar to that of insulin. We suggest that control of IGF-II release, like that of insulin, may involve protein-kinase C and that IGF-II may have a paracrine or autocrine role in the development of fetal pancreatic function.

Fetus↗
PubMed1988

Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities.

Competitive binding experiments with insulin-like growth factor (IGF)-1, IGF-2 and des-(1-3)-IGF-1 have confirmed the interpretation based on limited amino-terminal sequence analysis that at least three types of IGF binding protein occur. In addition to the acid stable subunit of the large serum binding protein which exhibits des-(1-3)-IGF-1 binding only slightly less than IGF-1, the small IGF binding proteins can be separated into two classes based on differences in des-(1-3)-IGF-1 and IGF-2 binding potencies.

Animals↗
PubMed1988

Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts.

This study investigates the effects of BP-53, the acid-stable IGF-binding subunit of the circulating 150 kDa IGF-binding protein complex, on IGF-I-stimulated thymidine incorporation by neonatal human skin fibroblasts. When cells were incubated for 24 h with IGF-I in serum-free medium, and thymidine incorporation studied over the final 4-h period, maximal stimulation (4- to 7-fold) was seen with 30 ng/ml IGF-I, with a half-maximal effect at 6.8 +/- 1.2 ng/ml (SD, n = 4). Co-incubation of IGF-I with increasing concentrations of pure BP-53 caused dose-dependent inhibition of IGF-I-stimulated thymidine incorporation, which was complete when IGF-I and BP-53 were equimolar. In contrast, preincubation of cells with BP-53 for 8-48 h before adding IGF-I resulted in a potentiation of the subsequent IGF-I effect. The potentiation was maximal (2-fold) at a BP-53 concentration of 150 ng/ml, and appeared to act by increasing the maximal rate of thymidine incorporation rather than the sensitivity of this process to IGF-I. Since neonatal fibroblasts produce a protein which is identical to BP-53 in size and immunoreactivity, these results suggest an autocrine role for BP-53 in regulating fibroblast responsiveness to IGF-I.

Cells, Cultured↗
PubMed1988

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↗
PubMed1988

The insulin-like growth factors and their binding proteins.

1. This review provides a brief overview of the structure of the insulin-like growth factors (IGFs or somatomedins), their mRNA and genes; the regulation and sites of production of these peptides; their binding and actions in target tissues; and the structure and biological role of their binding proteins. 2. Molecular cloning techniques have allowed the prediction of precursor forms of IGF-I and IGF-II, have provided tools to study the regulation of the synthesis and translation of IGF mRNAs, and have recently yielded the primary sequence of the IGF-I receptor, supplementing other rapidly-accumulating structural data. 3. Several of the IGF binding proteins have also been purified, and initial structural studies performed. 4. The increased knowledge of the structures of the IGFs, their receptors and binding proteins should now permit rapid progress in understanding the physiology and functions of these proteins.

Animals↗
PubMed1988

A comparison of the insulin and insulin-like growth factor I receptors from rat brain and liver.

Recent studies of the human brain insulin receptor raise the possibility that some of the differences described between rat brain insulin receptors and rat liver insulin receptors may have been due to the presence of insulin-like growth factor I (IGF-I) receptors in the brain. This study details side by side comparison of the alpha-subunits from insulin and IGF-I receptors in rat brain and liver. Insulin and IGF-I receptors are present in rat liver and brain, although IGF-I receptors are at a very low concentration in rat liver. These insulin and IGF-I receptors are clearly separate entities based on relative ligand affinities, antigenicity, and apparent molecular size. The alpha-subunits of the brain receptor for both insulin and IGF-I are about 10K lower in molecular size than the corresponding alpha-subunits from liver. These brain alpha-subunits are less sensitive to neuraminidase digestion than the corresponding liver alpha-subunit, although they do bind to wheat germ agglutinin. After treatment with endoglycosidase-F the deglycosylated apoproteins for both receptors from liver and brain have similar mol wt, suggesting that the observed differences were due to differences in N-linked glycosylation. The significance of differences in N-linked glycosylation between the brain receptors for insulin and IGF-I and the corresponding receptors in liver is not known.

Animals↗