Ontogenic changes in multiplication-stimulating activity binding to tissues and serum somatomedin-like receptor activity in the ovine fetus.
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Animals↗
Biomedical subjects
M J Waters
Publications and source records attributed to M J Waters.
Studies on the irreversible nature of prolactin binding to receptors.
Studies on hormone-receptor interactions generally assume that the formation of a hormone-receptor complex is a reversible process. This assumption has been examined directly in three experiments using liver membrane receptor preparations from pregnant rats and ovine PRL (oPRL). In Exp 1, Receptors were preincubated with a range of concentrations of oPRL at 23 C for periods up to 60 min, washed thereafter to remove free oPRL, and subsequently incubated with [125I]iodo-oPRL (23 C) to determine specific binding. Preincubation of receptors (0.25 mg membrane protein) with oPRL (5 ng) for periods as brief as 10 min reduced subsequent binding of [125I]iodo-oPRL to receptor, suggesting incomplete dissociation of oPRL even after 30 h. In Exp 2 after preincubation for 30 min with oPRL and subsequent incubation with [125I]iodo-oPRL for 19 h, membranes were washed, and the dissociation (23 or 37 C) of [125I]iodo-oPRL from the hormone-receptor complex in the presence or absence of 1000 ng oPRL was studied. After 48 h, only 35-50% of the [125I]iodo-oPRL dissociated from the hormone-receptor complex even in the presence or excess oPRL, indicating a heterogeneity of binding sites (i.e. 50-65% irreversible; 35-50% reversible). When pregnant rat serum was used in place of oPRL or when rabbit mammary glands were used instead of rat livers to prepare receptor preparations, results were similar to those described above, except for the nearly complete dissociation (90%) obtained at 37 C using rabbit mammary gland receptors. In Exp 3 after incubation (10 min, 2 h, or 15 h) of rat liver receptors with [125I]iodo-oPRL plus various amounts of oPRL, the hormone-receptor complex could be completely dissociated with 5 M MgCl2, restoring binding affinity and capacity of receptor to their original values. Labeled oPRL dissociated by MgCl2 treatment from such a complex is capable of binding to fresh receptor. These data strongly suggest that the PRL-receptor interaction, particularly the rat liver receptor interaction with PRL under usual in vitro conditions, is not reversible to a significant degree. This is not due to hormone or receptor damage but to a significant number of binding sites (50-65%) in the receptor preparation which are not reversible except under extreme conditions.
Animals↗
Prolactin receptor content of rabbit milk.
Rabbit milk fat globule membranes have been shown to contain PRL receptors by a variety of criteria, including hormonal specificity and inhibition by specific anti-PRL receptor antisera. Collection of milk samples throughout the period of lactation facilitated a temporal study of the receptor content of the membrane fraction of these milk samples by Scatchard analysis after treatment with 5 M MgCl2 to dissociate endogenously bound PRL. Total receptor content was low after parturition (3.3 +/- 1.3 fmol/mg membrane protein) but increased subsequently, reaching maximal levels (43.7 +/- 3.4 fmol/mg) by day 21 of lactation. No significant difference in the Ka (4.9 X 10(9) +/- 0.35 M-1) of the milk receptor was detected over a 4-week suckling period. No apparent relation seemed to exist between rabbit serum PRL values measured by RIA in serum samples taken just before milking and milk PRL receptor content. Milk receptor content, however, was significantly (P < 0.02) correlated with the PRL receptor content of the gland when animals were sacrificed immediately after milking.
Animals↗
Increase in placental lactogen and somatomedins after nephrectomy.
To investigate the relationship between renal agenesis and fetal growth retardation, 6 fetal sheep underwent bilateral nephrectomy and carotid artery catheterization between 105 and 125 days of gestation. Five fetal sheep were catheterized at a similar age as controls and all but one were delivered between 134 days and 147 days gestation. The weights of the fetuses did not differ significantly but the nephrectomized fetuses were shorter than their controls (P less than 0.01). Hydranencephaly was observed in two nephrectomized animals and the mean brain weight was reduced in this group (P less than 0.01). Fetal lung weights were likewise reduced (P less than 0.01). The mean plasma concentrations of maternal and fetal immunoreactive insulin and prolactin did not differ significantly in the two groups, nor did fetal ovine growth hormone and maternal ovine placental lactogen, also measured by radioimmunoassay. However, there were greater concentrations of ovine placental lactogen in the plasma of the nephrectomized lambs than in the controls (P less than 0.005). Somatomedin-like receptor activity was measured by receptor assay using [125I] multiplication-stimulating activity. The mean activity in unextracted serum from nephrectomized lambs was greater than that of the controls (p less than 0.05). When the fetal serum was chromatographed on Sephadex G-150 and the eluted fractions assayed for somatomedin-like activity three peaks of activity were observed. Two, with indicated molecular sizes of approximately 100,000 and 10,000 were greater in serum from nephrectomized animals than from the controls (P less than 0.01). The increased concentrations of placental lactogen and somatomedins after nephrectomy suggest that the kidney has a role in clearance of these hormones in the fetus. The growth retardation after fetal nephrectomy may be due to increased binding of somatomedin peptides by serum proteins or to an inhibition of somatomedin action by an unidentified serum component.
Animals↗
Purification and partial characterization of a nonprimate growth hormone receptor.
Pregnant rabbit liver membranes have been shown to possess two types of receptors by displacement analysis, a growth hormone (GH) receptor which binds bovine growth hormone with an affinity constant (KA) of 3 x 10(9) M-1 and ovine prolactin with a KA of 3 x 10(8) M-1, and a prolactin (Prl)-specific receptor which binds ovine prolactin with a KA of 5 x 10(9) M-1. The prolactin-specific receptor when solubilized with Triton exhibits a 4-fold increase in the its KA while the KA of the growth hormone receptor decreases slightly to 2 x 10(9) M-1 after solubilization. The 10-fold difference in affinity which results has been exploited to facilitate the separation of these two receptors by differential affinity chromatography on human growth hormone (hGH) affinity gels. The growth hormone receptor is eluted from the gel with 4 M urea while 5 M MgCl2 is required to elute the prolactin receptor. Conditions of affinity chromatography have been optimized, and further purification of the GH receptor by preparative isoelectric focusing and Sepharose 6B gel filtration resulted in a more than 8000-fold purification of the receptor. This material had a Stokes radius of 62 A, consistent with a molecular weight of 300,000 and gave one main band (75,000 to 80,000) and two minor bands on sodium dodecyl sulfate (SDS) polyacrylamide gels, which could be interpreted as indicating a tetrameric receptor. The GH receptor was shown to be a sialoglycoprotein (or closely associated with sialoglycoprotein) by analytical isoelectric focusing with an isoelectric point of 4.6. Specificity studies with the highly purified receptor confirmed the initial hypothesis that this receptor is capable of binding bovine growth hormone (bGH) with high affinity and ovine prolactin (oPrl) with low affinity, in contrast to the prolactin-specific receptor.
Animals↗
Studies with anti-growth hormone receptor antibodies.
Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.
Animals↗
Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone.
Binding of growth hormone (GH) to its receptor (GHR) is a well-studied example of molecular recognition between a cytokine and its receptor. Extensive mutagenesis studies and several crystal structures have defined the key interactive amino acid residues that are involved in binding and subsequent receptor dimerization. This review encompasses each of the three molecular recognition events involved in GHR activation, namely binding of GH to its two receptors and the interactions that occur between these receptors. Particular attention is given to species and ligand specificity of hormone binding and to the molecular recognition events involved in receptor activation, including the possibility that a conformational change in the receptor is required.
Animals↗
Growth hormone receptor and serum binding protein: purification, cloning and expression.
A putative growth hormone receptor from rabbit liver and the growth hormone binding protein from rabbit serum have the same amino-terminal amino-acid sequence, indicating that the binding protein corresponds to the extracellular hormone-binding domain of the liver receptor. The complete amino-acid sequences derived from complementary DNA clones encoding the putative human and rabbit growth hormone receptors are not similar to other known proteins, demonstrating a new class of transmembrane receptors.
Amino Acid Sequence↗
Dysgonic fermenter 2 septicemia.
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Animals↗
Epidemiology of hepatitis D virus (delta) infection in Melbourne over a 15-year period.
The prevalence of delta infection was studied in 3986 individuals seropositive for hepatitis B surface antigen (HBsAg), who were seen in Melbourne between 1971 and 1985. The group comprised 2004 patients with acute hepatitis B, 1820 asymptomatic HBsAg carriers, 139 HBsAg carriers with evidence of chronic liver disease, and 23 carriers who had suffered more than two separate attacks of acute hepatitis. Markers of delta infection were found almost exclusively among intravenous drug abusers and their close contacts. Studies of stored sera suggest that delta infection was introduced into this group about 1970. In carriers with no evidence of chronic liver disease, the prevalence of delta infection was highest among intravenous drug abusers (19.2%). In carriers with evidence of chronic liver disease, delta markers were present in 20.1% and all 23 carriers with recurrent acute hepatitis had evidence of infection with the delta virus.
Acute Disease↗
Growth hormone and colorectal carcinoma: localization of receptors.
Growth hormone (GH) plays a crucial role in stimulating and controlling the growth, metabolism and differentiation of many mammalian cell types by modulating the synthesis of multiple mRNA species and a paracrine or autocrine mechanism of action has been proposed. These effects are mediated by the binding of GH to its membrane-bound receptor and involve a phosphorylation cascade that results in the modulation of numerous signaling pathways. To address the side/mode of action through which GH exerts its effects, a panel of well characterized monoclonal antibodies, directed against the hormone binding side of the receptor, was applied to immunohistochemically determine growth hormone receptor (GH-receptor) expression in poorly- moderate- to well differentiated col.orectal adenocarcinomas (n = 40) from the rectum, transverse-, ascending-, descending and sigmoid colons. Of five anti-growth hormone receptor monoclonal antibodies used, human GH- receptor specific Mab 263 consistently resulted in strong receptor expression in colorectal carcinoma tumour cells. Heterogeneity of immunoreactivity was found in primary and secondary tumour lesions with a variable range of positive cells. Staining was mainly intracellular, showing either a monotonous or granular pattern, with some nuclei also reactive. The presence of intracellular GH-receptors has been previously documented and is a result of endoplasmic reticulum and Golgi localization. Immunoreactivity in surface columnar cells, independent from pathological tissue, was weak to moderate. Epithelial cells from normal tissue, adjacent to tumour lesions, were of variable intensity. Goblet and mucous cells located at the crypt base immunostained faintly or were negative for the GH-receptors. Crypt base columnar cells strongly expressed the GH-receptor, but oligomucous cells were less reactive. In conclusion, this study indicates that receptor expression may be associated with malignancy of colorectal carcinoma and supports the hypothesis that GH may act locally in colorectal tissue. The demonstration of the presence of receptors for GH will be useful for site-specific studies of the evolution of gastrointestinal tract tumours, providing valuable information concerning cellular growth kinetics and tumour prognosis. It also raises questions regarding the administration of GH to cancer-induced cachexia patients and the possible oncogenic potential of the GH-receptor.
Adenocarcinoma↗