Biomedical subjects

J L Stevenson

Publications and source records attributed to J L Stevenson.

At least 19 recordsLinked to original sources

PubMed2006

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Chloride Channels↗

PubMed2005

Expression of GLUT12 in the fetal membranes of the human placenta.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

Adult↗

PubMed2003

GLUT12 expression in human placenta in first trimester and term.

The aim of this study was to characterize the expression of a novel glucose transporter protein GLUT12 in human placenta. GLUT12 mRNA expression was identified by RT-PCR in extracts from five normal term placentae and in extracts from cultured cells of the JAR, JEG-3 and HTR-8Svneo cell lines. In further studies, paraffin sections of first trimester tissue from chorionic villus sampling and term tissue obtained after delivery were analysed by immunohistology with a GLUT12 specific polyclonal antibody. GLUT12 immunoreactivity was expressed predominantly in the syncytiotrophoblast and in extra-villous trophoblast cells in first trimester tissues at 10, 11 and 12 weeks' gestation. In term tissue, however, GLUT12 staining was not detected in syncytiotrophoblast and was found predominantly in villous vascular smooth muscle cells and villous stromal cells. These results suggest that there is a dynamic spatial and temporal expression pattern for the novel glucose transporter GLUT12 in human placenta.

Adult↗

PubMed2000

Evidence for two distinct classes of high affinity growth hormone binding proteins in pregnant rat serum.

These studies have established the presence of two major classes of high affinity growth hormone binding proteins in pregnant rat serum, designated GHBPa and GHBPb, with apparent native Mr of 257 K and 98 K respectively. GHBPa, which has not been identified previously, exhibits a binding affinity (2-5 nM(-1)) that is up to 20-fold higher than GHBPb (0.2-0.8 nM(-1)) and is the least abundant form, being approximately 15-20% of total serum GH-binding capacity. Western immunoblot analysis revealed that each GHBP is composed of several immunoreactive proteins which were reactive with carboxy-terminal (RB1615) and/or N-terminal (MAb263) domain antibodies, suggesting the presence of GHBPs with and without the hydrophilic tail. Of importance is that GHBPa exhibited significantly higher Mr (78-182 K, +DTT) than that predicted by GHBP cloning, suggesting that they may be covalently bound to other non-GH-binding proteins or may be distinct entities. GHBPb, on the other hand, was composed of smaller Mr (43/48 K, +DTT) "hydrophilic" tail-containing proteins, some of which were disulphide linked to a larger complex of approximately 110 K. These novel findings challenge the current view of the mechanism for generation of the rat serum GHBP and raise the intriguing possibility that the two classes of GHBP may play distinct and important roles in GH physiology.

Animals↗

PubMed2000

Magnesium regulates hypoxia-stimulated apoptosis in the human placenta.

Apoptosis (programmed cell death) in the human placenta is likely to play a major role in determining the structure and function of that organ. Fetal growth restriction (FGR) has been shown to be associated with increased levels of placental apoptosis. Altered regulation of apoptosis may play an important pathophysiological role in FGR. As reduced placental perfusion and reduced oxygenation are features of FGR, one aim of this study was to determine the effects of hypoxia on apoptotic activity, as assessed by DNA laddering, of placental tissue in vitro. In addition, levels of placental apoptosis may be affected by pharmacological agents routinely used in obstetric patient management. Thus an additional aim of this study was to determine the effects of several relevant pharmacological agents on the levels of DNA laddering during in vitro incubation of human placentae under hypoxic conditions. Incubation of normal placental explant tissue at 37 degrees C for 1-2 h under hypoxic conditions significantly increased placental DNA laddering compared with that in non-incubated tissue, whereas levels of DNA laddering during incubation for up to 2 h under normoxic conditions were not significantly higher than those in non-incubated tissue. The DNA laddering activity of placental explants after 2 h of incubation under hypoxic conditions was significantly increased with increased concentrations of magnesium, but remained unchanged by the inclusion of pethidine, aspirin, nifedipine, dexamethasone, heparin or indomethacin in the incubation mixture. These results suggest that hypoxia may stimulate apoptotic activity in cultured human placental tissues, and that hypoxia-stimulated placental apoptosis may be further increased by increasing the extracellular magnesium concentration.

Apoptosis↗

PubMed1998

NMR structure of human erythropoietin and a comparison with its receptor bound conformation.

The solution structure of human erythropoietin (EPO) has been determined by nuclear magnetic resonance spectroscopy and the overall topology of the protein is revealed as a novel combination of features taken from both the long-chain and short-chain families of hematopoietic growth factors. Using the structure and data from mutagenesis studies we have elucidated the key physiochemical properties defining each of the two receptor binding sites on the EPO protein. A comparison of the NMR structure of the free EPO ligand to the receptor bound form, determined by X-ray crystallography, reveals conformational changes that may accompany receptor binding.

Binding Sites↗

PubMed1997

Guinea pig serum contains a specific high affinity growth hormone-binding protein with novel ligand specificity.

Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current studies, however, have identified and characterized the presence of GH-binding activity in guinea pig serum using gel chromatography to separate bound and free hormone. The detection of GH-binding activity is critically reliant on the type of radioligand used to measure binding. Clear identification of GH-binding activity was demonstrated with [125I]ovine GH (oGH), but specific binding could not be measured with [125I]human GH. The novel specificity was also shared by guinea pig liver membrane GH receptor (GHR) and cytosol GHBP, suggesting structural similarity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 microl serum produced 16.03 +/- 0.5% specific binding; mean +/- SEM; n = 11) and incubation time (equilibrium was reached by approximately 6 h at 21 C) and was completely reversible (t(1/2), approximately 2 h). Scatchard analysis revealed linear plots with an affinity constant (Ka) of 0.59 +/- 0.09 x 10(9) M(-1) and a capacity of 23,181 +/- 4,474 fmol/ml serum. Similar association constants were obtained for liver membrane GHR (0.79 +/- 0.22 x 10(9) M(-1)) and cytosol GHBPs (0.99 +/- 0.15 x 10(9) M(-1)), but the capacity, when expressed as femtomoles per g tissue, was significantly increased (4-fold) in cytosol (4,303 +/- 505) over that in membranes (1,071 +/- 257). There was no sex difference in Ka or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity was very low to undetectable in pregnant guinea pig serum. Characterization of the native structure of guinea pig GHBPs has indicated the presence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large Mr range (approximately 70-350 kDa) the major form of the circulating GHBP identified by gel chromatography had an apparent native Mr of 150-170 kDa. Partially purified GHBP (approximate Mr, 170 kDa) was covalently cross-linked to [125I]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP complexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated noncovalently with a non-GH-binding protein. "Pore limit" native PAGE (cathodic and anodic) revealed the presence of specific GHBPs of 363, 158, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-specific mAb 4.3. Interestingly, although GH binding was undetectable in pregnant guinea pig serum, Western immunoblot analysis with mAb 263 demonstrated the presence of a major immunoreactive GHBP band of 105 kDa in addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no evidence to support the presence of a hydrophilic tail sequence homologous to that in the rat GHBP. These studies have identified in guinea pig serum GHBPs that exhibit novel ligand specificity, structural heterogeneity, and an immunological relationship to the rat liver membrane GHR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.

Animals↗

PubMed1996

Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Base Sequence↗

PubMed1994

Growth hormone receptor/binding protein: physiology and function.

Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

Animals↗

PubMed1992

Regulation of prolactin receptor gene expression by thyroid hormone status in the rat.

The nature and tissue distribution of prolactin receptor (PRL-R) mRNA in both male and female rats was studied. A single mRNA species of 2.2 kb was identified in the liver, kidney, adrenal, prostate, lactating mammary gland and ovary but not in the male lung, heart, skeletal muscle, thymus, adipose tissue or brain. There were distinct and contrasting sex differences in abundance of PRL-R mRNA in some tissues: liver (female much greater than male), kidney and adrenal (male much greater than female). A mRNA species of 4 kb was occasionally detected in the male adrenal and female liver. Given previous reports on the effects of thyroid status on PRL binding, the effects of thyroxine (T4), propylthiouracil (PTU) or combined treatment on PRL-R mRNA were assessed. In the male rat, PTU treatment markedly increased (three- to fourfold) PRL-R mRNA in the liver but decreased it (approximately 50%) in the kidney. These changes were reflected in similar changes in lactogenic binding activity. T4 or PTU treatment increased PRL-R mRNA in the prostate, with no obvious changes in binding. No major changes were seen in adrenal glands. In the female rat, PTU had little effect on PRL-R mRNA in any tissue, although binding of 125I-labelled lactogen was decreased in both the liver and kidney. There was an unexpected threefold rise in PRL-R mRNA in the female kidney following combined T4 and PTU treatment. Overall, there was a quite close correlation between the effects of thyroid status on PRL-R mRNA levels and specific lactogenic binding to membranes prepared from the same tissue samples. These studies provide data on the tissue distribution and size of PRL-R mRNA in rats and suggest a novel and complex tissue- and sex-dependent regulation by thyroid hormone.

Animals↗

PubMed1991

Elevation of growth hormone (GH) and prolactin receptors in transgenic mice expressing ovine GH.

The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein 1(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors.

Animals↗

PubMed1991

Reliability of stimulated and spontaneous growth hormone (GH) levels for identifying the child with low GH secretion.

The reliability of stimulated and spontaneous GH levels for identifying the child with low GH secretion has been the subject of debate. We compared the ability of GH concentrations after pharmacological stimulation with levodopa and clonidine and of spontaneous peak and 12-h pooled GH concentrations during sleep on a single night to estimate the maximum spontaneous GH secretion from 2 nights in 55 children, aged 5-16 yr, with heights below the 3rd percentile and/or height velocities below the 25th percentile for age, who had two consecutive overnight GH secretory profiles. Maximum stimulated GH concentrations correctly categorized 80% of children who had maximum spontaneous GH concentrations above and below 4 micrograms/L using a double monoclonal immunoradiometric assay for GH (Tandem-R HGH, Hybritech). The remaining 20% of children had stimulated GH concentrations below but spontaneous GH concentrations above 4 micrograms/L. Using this cut-off, the maximum GH concentrations from the first and second nights correctly categorized 98% and 95% of the children, respectively. Night to night variation in GH secretion was low in children who had low spontaneous GH secretion (maximum spontaneous peak and pool GH concentrations, less than 4 and less than or equal to 0.7 micrograms/L, respectively), and pooled GH concentrations from the 2 nights were concordant in 98% of the cases. We conclude that it is not uncommon for stimulated GH concentrations to underestimate spontaneous GH secretion. Even without acclimatization to the hospital setting, measurement of spontaneous GH secretion on a single night was more reliable for identifying the child with low endogenous GH secretion than was GH stimulation testing alone.

Adolescent↗

PubMed1991

Kidney insulin-like growth factor-I mRNA levels are increased in postpubertal diabetic rats.

Diabetes-associated renal enlargement is more marked in postpubertal than prepubertal rats, and in the postpubertal rat, is associated with increased kidney insulin-like growth factor-I (IGF-I) levels for the first 2 days. In order to determine whether local IGF-I production is the cause of this increase in tissue levels, IGF-I mRNA levels were determined in pre- and postpubertal Sprague-Dawley rats made diabetic with streptozotocin (STZ) and in control rats. RNA was extracted from kidneys and livers of rats at 0 h, 6 h, 12 h and days 1, 2, 3 and 7 after STZ injection. After Northern blotting and hybridization with an oligonucleotide probe complementary to an E domain of the IGF-I cDNA, four distinct bands (7.4, 4.8, 1.8 and 1.0 kb) were found. Densitometric analyses of the most prominent bands (7.4 and 1.0 kb), after normalization for 18S ribosomal RNA content, revealed a 50-100% increase in the kidneys of postpubertal diabetic rats compared with postpubertal controls 12 h after STZ injection (P less than 0.05, diabetes vs control). Between days 2 and 7, kidney IGF-I mRNA levels in postpubertal diabetic rats fell to approximately 50% of control levels (P less than 0.05, diabetes vs control). In contrast, kidney IGF-I mRNA levels in the prepubertal diabetic rats remained unchanged over the 7 days. Liver IGF-I mRNA levels did not rise during the first 24 h and fell to approximately 60% of control levels by day 7 in both pre- and postpubertal diabetic rats (P less than 0.05, diabetes vs control). Increased local IGF-I production may underlie the initiation of renal enlargement associated with diabetes mellitus.

Animals↗

PubMed1991

Effects of intravenous and aerosolized arachidonic acid on alveolar epithelial permeability in rabbits.

To determine whether arachidonic acid (AA) alters alveolar epithelial permeability, we studied the effect of both continuous intravenous and aerosolized AA on clearance of [99m]Tc-DTPA from lung to blood in rabbits. Although intravenous AA increased prostacyclin production and aerosolized AA decreased systemic blood pressure, neither continuous intravenous nor aerosolized AA augmented alveolar epithelial permeability.

Aerosols↗

PubMed1990

Correlation of urinary albumin and beta-2-microglobulin and growth hormone excretion in patients with diabetes mellitus and short stature.

We examined the correlation between urinary GH, urinary albumin, and beta-2-microglobulin excretion to determine how the excretion of GH relates to markers of renal glomerular and tubular function. Urinary albumin and GH excretion was determined in timed daytime and nighttime urine collections obtained from both subjects with diabetes mellitus and subjects with short stature. For subjects with diabetes, urinary GH excretion rate correlated highly with urinary albumin concentration and excretion rate in both the range of 0 to 1.6 g/L (r = 0.75), P less than 0.001) and in the microalbuminuria range, 0 to 0.4 g/L (r = 0.53, P less than 0.001). Changes in GH and albumin excretion occurred in parallel in 71% of the subjects with diabetes and elevated albumin excretion. The mean GH excretion rate was higher in the group with elevated albumin excretion rate (AER) during both day and night compared to the group with microalbuminuria during the day and normal AER at night. For subjects with short stature, the mean albumin excretion rate was 0.7 +/- 1.3 micrograms/min (range 0.05-8.3 micrograms/min) using a sensitive enzyme-linked immunosorbent assay to measure albumin concentration. The correlation of GH and albumin excretion rates for the subjects with short stature was not statistically significant (r = 0.14, P less than 0.5). About half of the subjects with diabetes and elevated AER (greater than 10 micrograms/min) had a GH excretion rate within the range observed in subjects with short stature. The GH and albumin excretion rate were not correlated in this group. There was a positive correlation of both albumin and GH excretion rate with age in the subjects with diabetes. Urinary GH and beta-2-microglobulin excretion rates were determined in a larger group of subjects with diabetes and a separate group with short stature. Urinary GH and beta-2-microglobulin excretion were correlated both in subjects with diabetes (r = 0.46, P less than 0.001) and with short stature (r = 0.64, P less than 0.001). The association was present in urine collected either during the day or night. The mean GH excretion rate of the group with diabetes was greater than the group with short stature. In conclusion, there was an association of urinary GH and albumin excretion rate in subjects with abnormal glomerular function as indicated by elevated albumin excretion rate. An association of urinary GH and beta-2-microglobulin excretion was observed in subjects with normal tubular function.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1989

Differences in the developmental patterns of somatotrophic and lactogenic receptors in rabbit liver cytosol.

These studies have examined the ontogeny of specific GH- and PRL-binding proteins in rabbit liver cytosol across the fetal, early neonatal, and adult periods. The precise hormonal specificity of binding of the somatotropic/lactogenic ligand, [125I]human GH, was determined by displacement with monoclonal antibodies against either rabbit mammary gland PRL receptors or liver GH receptors. The developmental changes were evaluated by gel filtration chromatography, which allowed a measure of both the extent of binding and the molecular size (Mr). Scatchard analysis indicated that fetal liver cytosol contained mostly high affinity PRL receptors (Ka, 13.78 +/- 0.98 nM-1; capacity, 127 +/- 24.0 fmol/g tissue; mean +/- SEM; n = 5) and was low in GH-specific binding. This contrasts markedly with adult liver cytosol, which is a rich source of GH receptors (Ka, 2.44 nM-1; capacity, 20,765 fmol/g tissue), but is low in PRL-specific binding. Despite the deficiency of GH receptors in fetal liver, an abundant receptor-like GH-binding protein was present in fetal serum. Of several fetal tissue cytosols examined, only the placenta contained significant GH-specific binding; no tissues other than liver contained PRL-specific binding. After parturition PRL receptors in liver cytosol increased and predominated up until day 3 (Ka, 27.97 +/- 6.27 nM-1; capacity, 495 +/- 95.09 fmol/g tissue; n = 7), a period during which GH-specific binding was increasing only slightly. By day 6 a striking switch-over occurred, and GH receptors, which had a 4-fold lower affinity, became predominant (Ka, 6.89 +/- 0.63 nM-1; capacity, 600 +/- 47 fmol/g tissue; n = 5), while PRL-specific binding fell dramatically to levels observed in adult liver cytosol. After day 6 GH receptors increased steadily, reaching the high levels observed in adulthood by 2 months (Ka, 3.95 nM-1; capacity, 16,300 fmol/g tissue; n = 2), while PRL-specific binding appeared to change little. Structural and immunological analyses of the cytosolic GH and PRL receptors in the fetal/early neonatal period revealed similarities with the adult liver cytosolic GH receptor and mammary gland cytosolic PRL receptor, respectively. The increase in GH receptors on day 6 was clearly illustrated by cross-linking studies which showed the emergence of a GH-specific binding protein structurally distinct from PRL-specific binding proteins. These studies have demonstrated that in the rabbit major changes occur in the GH/PRL receptor profile during the early period of growth and suggest that important developmental changes occur in the requirement for GH and/or PRL action during this period.

Animals↗

PubMed1989

Binding sites for growth hormone in rabbit placental cytosol.

A soluble GH-binding protein, which cross-reacted with a monoclonal antibody (Mab) to the rabbit liver membrane GH receptor, has been identified in cytosol preparations from both fetal and maternal portions of rabbit placenta. Structural studies using gel filtration chromatography and chemical covalent cross-linking techniques have shown that the GH-binding protein in fetal/maternal placental cytosol has a native mol wt of 104,000 and a denatured subunit of about 57,000 mol wt (with or without dithiothreitol). Very low levels of GH-specific [125I]human (h) GH binding were observed in membrane preparations from the corresponding placentae, even after desaturation of any endogenously bound hormone by 5 M MgCl2. No PRL-specific binding was observed in placental membranes or cytosols. Scatchard analysis of [125I]hGH binding to fetal and maternal placental cytosol revealed linear plots with Ka values of 6.1 +/- 1.1 nM-1 (fetal) and 5.31 +/- 0.63 nM-1 (maternal; mean +/- SEM; n = 5). The binding capacity of maternal placental cytosol, when expressed as femtomoles per mg protein (170 +/- 10.10) or femtomoles per g tissue (3245 +/- 123), was about 3-fold higher than that for fetal placental cytosol. Northern blot analysis of fetal and maternal placental mRNA probed with a GH receptor oligonucleotide probe revealed hybridization to a 4.4 to 4.7-kilobase and a 2.2-kilobase species in fetal placenta only. The level of GH-specific binding observed in fetal and maternal placental cytosol did not correlate directly with the level of mRNA expression. A GH-binding protein has also been shown to be present in fetal rabbit serum and is known to be structurally and immunologically related to the rabbit placental and liver cytosolic GH-binding proteins. Scatchard analysis of [125I]hGH binding to fetal serum GH-binding protein revealed a single class of high affinity sites with a Ka of 4.64 +/- 1.29 nM-1 and a capacity of 338 +/- 167 fmol/ml serum (mean +/- SEM; n = 4). Given the relative binding capacities and the demonstration of GH receptor mRNA in fetal placental cytosol, it is highly unlikely that contamination of fetal placental cytosol by fetal serum accounts for all of the placental binding capacity observed. However, no such definitive conclusion regarding contamination by maternal rabbit serum of maternal placental cytosol can be made. The presence of GH-binding proteins in placental cytosol has not been described previously, and these observations suggest that GH may have a role in placental metabolism.

Animals↗

PubMed1988

Binding proteins for growth hormone and prolactin in rabbit kidney cytosol.

Two soluble, receptor-like binding proteins with apparent somatotrophic [growth hormone (GH)] and lactogenic [prolactin (PRL)] specificities, respectively, and that are present in rabbit kidney cytosol have now been examined in more detail using specific GH receptor and PRL receptor monoclonal antibodies (MAb). Gel chromatography of 125I-labeled human GH (125I-hGH) kidney cytosol complexes in the absence of these MAbs revealed two specifically bound regions of radioactivity at molecular weights (MW) of approximately 120,000 and approximately 60,000, which are similar in size to complexes formed by the native GH receptor of rabbit liver cytosol and the PRL receptor of mammary gland. Co-incubation with GH-receptor MAb inhibited 125I-hGH binding only to the higher MW (120,000) species, whereas the PRL-receptor MAb inhibited only the lower MW (60,000) species, thus establishing definitively the hormonal specificities of the two binding proteins. The presence of both GH- and PRL-specific binding subunits in cytosol was confirmed using covalent cross-linking techniques. No GH binding protein was detected in kidney membranes. The presence of naturally soluble, receptor-like binding proteins for GH and PRL in kidney cytosol preparations raises the possibility of their playing a role in the intracellular regulation of kidney function and/or metabolism.

Animals↗