Biomedical subjects

G N Hendy

Publications and source records attributed to G N Hendy.

At least 19 recordsLinked to original sources

PubMed2001

Association between total serum calcium and the A986S polymorphism of the calcium-sensing receptor gene.

Serum calcium is under tight physiological control, but it is also a quantitative trait with substantial genetic regulation. Mutations of the CASR gene cause familial hypocalciuric hypercalcemia or autosomal dominant hypoparathyroidism, depending on whether they decrease or increase, respectively, ligand binding to the receptor protein. We described an association between ionized calcium and a common polymorphism (A986S) found in the cytoplasmic tail of this G protein-coupled receptor. We report here on an independent study of 387 healthy young women. Genotyping was performed by allele-specific amplification and serum chemistries were measured by automated clinical assay. Frequencies of SS, AS, and AA genotypes were 6, 107, and 274, respectively, yielding a 986S allele frequency of 15.4%. Mean total serum calcium (Ca(T)) was significantly higher in the SS (9.88 +/- 0.29 mg/dL, P = 0.015) and AS groups (9.45 +/- 0.05 mg/dL, P = 0.002), than in the AA group (9.23 +/- 0.04 mg/dL). In multiple regression modeling, the A986S genotype remained an independently significant predictor of Ca(T) (P < 0.0001) when serum albumin, globulin, inorganic phosphate, and creatinine covariates were included. These data are the first to show significant association between a common polymorphism and concentrations of a serum electrolyte. The A986S polymorphism is also a potential predisposing factor in disorders of bone and mineral metabolism.

Adolescent↗

PubMed2001

25-hydroxyvitamin D 1alpha-hydroxylase: structure of the mouse gene, chromosomal assignment, and developmental expression.

The murine homologue of the 25-hydroxyvitamin D [25(OH)D] 1alpha-hydroxylase gene [1alpha(OH)ase; Cyp27bl], which is mutated in humans with vitamin D-dependent rickets type I (VDDR-I; also known as pseudovitamin D-deficiency rickets [PDDR]) was cloned and characterized. Like the human, the mouse gene has nine exons, and the exon-intron organization is well conserved. By interspecific backcross analysis, the Cyp27bl gene was mapped to 70.5 cM on mouse Chr 10. This is in a region syntenic with human Chr 12q13.1-q13.3 to which the human 1alpha(OH)ase gene was previously mapped. Kidney expression of the 1alpha(OH)ase was localized to cortical tubules and was higher in the adult mouse than in the fetus, consistent with the increased role of its product as a circulating hormone postnatally. Prenatally, the 1alpha(OH)ase gene, together with the vitamin D receptor (VDR) gene, was expressed in embryonic stem cells, and expression of 1alpha(OH)ase in bone and intestine was higher in the fetus than in the adult. These observations suggest that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays a role in fetal development. In view of the fact that humans lacking 1alpha(OH)ase have apparently normal prenatal development, this may point to functional redundancy in the fetal vitamin D system, which now can be explored further in mouse models in which the 1alpha(OH)ase gene has been deleted.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2000

Extracellular calcium-sensing receptor is expressed in rat hepatocytes. coupling to intracellular calcium mobilization and stimulation of bile flow.

Liver cells respond to changes in Ca(2+)(o). The hepatic functions affected include bile secretion, metabolic activity, liver regeneration, and the response to xenobiotics. In the present study, we demonstrate the presence, in the liver, of the extracellular calcium-sensing receptor (CASR), described previously in the parathyroid and thyroid glands and kidney. CASR mRNA was specifically expressed in hepatocytes and was absent in nonparenchymal liver cells (stellate, endothelial, and Kupffer cells). Western blot analysis using a specific CASR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CASR protein and mRNA by a subset of hepatocytes. The known agonists of the CASR, gadolinium (Gd(3+); 0.5-3.0 mm) and spermine (1.25-20 mm), in the absence of Ca(2+)(o), elicited dose-related increases in Ca(2+)(i) in isolated rat hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a greatly attenuated response to a second challenge with either agonist. The response was also abrogated when inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine, an alpha(1)-adrenergic receptor agonist known to mobilize Ca(2+)(i) from IP(3)-sensitive pools. Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not the S enantiomer, NPS S-467, increased the sensitivity of the Ca(2+)(i) mobilization response to 1.25 mm spermine. Bile flow ceased after Ca(2+)(o) withdrawal, and its recovery was enhanced by spermine in isolated perfused liver preparations. The CASR agonists Ca(2+) and Gd(3+) increased bile flow, and the response to a submaximal Ca(2+) concentration was enhanced by NPS R-467 but not the S compound. Thus, the data indicate that rat hepatocytes harbor a CASR capable of mobilizing Ca(2+)(i) from IP(3)-sensitive stores and that activation of the CASR stimulates bile flow.

Aniline Compounds↗

PubMed2000

Methylation patterns of human parathyroid hormone (PTH)/PTH-related peptide receptor gene promoters are established several weeks prior to onset of their function.

Expression of the human parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR) gene is controlled by three promoters, P1-P3. P1 functions specifically in kidney, whereas P2 is ubiquitously active. P3 is also widely active, although more so in kidney than other tissues. However, only P2 functions at midgestation. We examined the role of methylation in controlling PTHR promoter activity. Function of all promoters was inhibited by CpG methylation in vitro. Significantly, P1 is selectively hypomethylated in adult kidney in vivo, strongly suggesting that demethylation is required for renal P1 function. Moreover, this pattern is established by 11. 75 weeks of fetal age, several weeks prior to the onset P1 activity. P3 is unmethylated at midgestation, although it is inactive at this stage of development, and thus exhibits characteristics of both tissue-specific and ubiquitously active promoters. These results show that adult methylation patterns of P1 and P3 are established several weeks prior to their induction, indicating that their function requires factors expressed late in development.

Adult↗

PubMed2000

Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia.

The calcium-sensing receptor (CASR) is a plasma membrane G protein coupled receptor that is expressed in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland and the cells lining the kidney tubule. By virtue of its ability to sense small changes in circulating calcium concentration ([Ca(2+)](o)) and to couple this information to intracellular signaling pathways that modify PTH secretion or renal cation handling, the CASR plays an essential role in maintaining mineral ion homeostasis. Inherited abnormalities of the CASR gene located on chromosome 3p13.3-21 can cause either hypercalcemia or hypocalcemia depending upon whether they are inactivating or activating, respectively. Heterozygous loss-of-function mutations give rise to familial (benign) hypocalciuric hypercalcemia (FHH) in which the lifelong hypercalcemia is asymptomatic. The homozygous condition manifests itself as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism which occur in infancy. The disorder autosomal dominant hypocalcemia (ADH) is due to gain-of-function mutations in the CASR gene. ADH may be asymptomatic or present with neonatal or childhood seizures. A common polymorphism in the intracellular tail of the CASR, Ala to Ser at position 986, has a modest effect on the serum calcium concentration in healthy individuals.

Calcium↗

PubMed2000

Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation.

We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75-95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12-16 h. Nuclear localization of the receptor is achieved approximately 16-20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1-34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.

3T3 Cells↗

PubMed2000

Transferrin-mediated uptake of aluminium by human parathyroid cells results in reduced parathyroid hormone secretion.

BACKGROUND: The present study investigates whether aluminium-transferrin (Al-Tf) uptake by Tf receptor-mediated endocytosis induces hypoparathyroidism and thus might contribute to the increasing prevalence of adynamic bone disease (ABD) in the current dialysis population. METHODS AND RESULTS: Human parathyroid glands as well as in vitro cultured human parathyroid cells were shown to express Tf receptors. Five-day-old cultures of parathyroid cells were incubated for 48 h in serum-free DMEM/F12 supplemented with 12 microM apo-Tf: 12 microM Tf to which 150 microg/l Al or 150 microg/l Al-citrate (Al-ci) was bound. The amount of Al taken up by the parathyroid cells either as Al-Tf or Al-ci did not differ. However, incubation of cell cultures with Al-Tf showed a significant proportional decrease (mean+/-SEM, -23.1+/-4.5%) in iPTH secretion as compared to the reference apo-Tf cultures. Al-ci did not suppress PTH secretion (+3.4+/-6.5%). The Al uptake after incubation with Al-Tf was found to be dose-dependent. With regard to iPTH secretion, a tendency toward a dose response relationship was observed. Northern blot analysis of parathyroid cells incubated in 12 microM apo-Tf or 12 microM Al-Tf demonstrated that the PTH mRNA synthesis was unaffected by the Tf-mediated uptake of Al. These observations suggest an effect of Al on PTH release rather than on PTH synthesis. Since the cytoskeleton can play an important role in the release of secretory vesicles, the influence of Al on the structure of actin, beta-tubulin and vimentin was investigated by confocal microscopy. Comparison of cultures incubated with apo-Tf and Al-Tf revealed no difference in the organization of these cytoskeletal proteins in relation to the inhibitory effect of Al-Tf on PTH secretion. CONCLUSION: In summary, data in the present paper demonstrate that the (i) human parathyroid gland/parathyroid cells exhibit Tf receptors; (ii) Al-Tf complex is taken up by the parathyroid gland in a dose-dependent manner; and (iii) uptake of Al by Tf receptor-mediated endocytosis reduces the secretion of PTH but not its synthesis. These in vitro findings allow us to suggest that Tf receptor-mediated uptake of Al might, besides other factors such as vitamin D, high calcium dialysate or CaCO(3) intake, play a role in the development of hypoparathyroidism associated with ABD. The exact mechanism by which Al-Tf suppresses iPTH secretion remains to be elucidated.

Aluminum↗

PubMed2000

Dissection of differentially regulated (G+C)-rich promoters of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene.

The PTH/PTH-related peptide (PTHrP) receptor (PTHR) is required for normal skeletal development, and a wide array of physiological responses mediated by PTH and PTHrP. We have previously identified three promoters, P1-P3, which control human PTHR gene transcription. P2 and P3 are (G+C)-rich, function in a number of tissues, lie within the same CpG island, and display many hallmarks of housekeeping promoters. However, they are differentially regulated during development as P2, but not P3, functions in fetal tissues. Here, we have used both stably and transiently transfected human osteoblast-like cells to delineate regions of P2 and P3 required for promoter activity. Deletion analyses performed in stably transfected cells indicated that sequences extending from -91 to -12 relative to the transcription start site were required for function of the P2 promoter. No negative regulatory elements were detected in P2. In contrast, deletion of an A-rich region of P3 extending from -147 to -115 was required for optimal basal activity, suggesting that this sequence acts as a repressor of P3. Strikingly, however, whereas the A-rich region also functioned as a negative element when inserted upstream of the (G+C)-rich P2 promoter, it enhanced expression from the thymidine kinase promoter, suggesting that its function depends on other transcription factors bound to promoter sequences. Fine deletion of P3 sequences proximal to -115 implicated Spl motifs and downstream initiation sites in P3 function. These studies indicate that function of P2 and P3 is controlled by ubiquitously expressed transcription factors and raise the possibility that P3 activity is repressed during fetal development.

Base Sequence↗

PubMed2000

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

Amino Acid Sequence↗

PubMed1999

Peptide hormone precursor processing: getting sorted?

Processing of proproteins to biologically active peptides and, in the case of peptide hormones and neuropeptides, their sorting to granules of the regulated secretory pathway, requires the concerted action of a cascade of enzymes and chaperones. The purpose of this review is to summarize the recent emerging knowledge of how these molecules affect specific endocrine systems. This has come about through the study of gene knockout mice as well as endocrinopathies resulting from mutated genes in humans.

Animals↗

PubMed1999

Cell cycle regulation of menin expression.

The multiple endocrine neoplasia type 1 gene product, menin, interacts with Jun D. The physiological role of menin in cell cycle control and the manner in which its inactivation contributes to tumorigenesis remain unknown. In the present study, the expression of menin was examined at various cell cycle stages in GH4C1 cells, a rat pituitary cell line. Cells synchronized at the G1-S-phase boundary expressed menin at a lower level than G0-G1-synchronized cells. The expression of menin increased as the cells entered S phase, at which time Jun D expression also increased. In contrast, cells synchronized at the G2-M phase expressed lower levels of menin. At G0-G1, G1-S, and G2-M phases of the cell cycle, menin was found predominantly in the nucleus. In summary, we show that in pituitary cells, menin is a nuclear protein whose expression is cell-cycle regulated. The data suggest that menin has an important role in cell growth regulation.

Amino Acid Sequence↗

PubMed1999

A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations.

BACKGROUND: The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration. METHODS: We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o. FINDINGS: The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p<0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013). INTERPRETATION: The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

Adolescent↗

PubMed1999

1,25-dihydroxyvitamin D3 inhibits parathyroid hormone-related peptide mRNA expression in fetal rat long bones in culture.

When fetal rat long bones are incubated in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], steady-state parathyroid hormone-related peptide (PTHrP) mRNA levels are decreased. This decrease is temporary: it is observed as soon as after 3 h of exposure and reaches a nadir after 6 h. At that time, PTHrP mRNA levels are significantly lower in the experimental than in the control bones. However the inhibitory effect vanishes after 24 h, despite continuous exposure to 1,25(OH)2D3 for even 48 h. This is the first report showing that PTHrP mRNA expression can be regulated in rat fetal long bones in vitro by 1,25(OH)2D3.

Animals↗

PubMed1999

Rabbit calcium-sensing receptor (CASR) gene: chromosome location and evidence for related genes.

Diverse cellular functions are regulated by the calcium-sensing receptor, encoded by the CASR gene, which plays an important role in calcium homeostasis. Here we provide the sequence for exon VII of the rabbit CASR gene and show that it is 91% identical to the human gene at the nucleotide level, and 95% identical at the amino acid level. The gene was mapped by fluorescence in situ hybridization, using a cosmid isolated from a genomic library, to chromosome 14q11 and this was confirmed independently by PCR amplification of flow sorted chromosomes. In addition, the cosmid detected sites with lower frequencies on four other chromosomes: 3q, 5p, 8p, and 13p. Two of these sites (5p and 13p) were also detected by a related but unidentical cosmid, and map to regions that are homologous to the mouse calcium-sensing receptor related sequences (Casr-rs); suggesting that they may represent CASR-related sequences in the rabbit. The data support the presence of a family of genes related to the calcium-sensing receptor in the G-protein coupled receptor (GPCR) superfamily, as well as extend the existing knowledge of homology for several human and rabbit chromosomes.

Amino Acid Sequence↗

PubMed1999

Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3.

We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic beta-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.

Animals↗

PubMed1999

Comparison of the biochemical responses to human parathyroid hormone-(1-31)NH2 and hPTH-(1-34) in healthy humans.

The 1-31 fragment of human PTH [hPTH-(1-31)NH2] has been shown, like hPTH-(1-34), to have anabolic effects on the skeletons of ovariectomized rats when given intermittently, but, unlike hPTH-(1-34), it does so without affecting serum calcium concentrations and does not activate the protein kinase C second messenger pathway in some target cells. To investigate the biochemical responses to hPTH-(1-31) in humans, we have directly compared it to hPTH-(1-34) during the course of slow infusions of each. Ten healthy adults, five men and five women, aged 26+/-5 yr (range, 22-37), each received 8-h continuous infusions of 8 pmol/kg.h hPTH-(1-34) and hPTH-(1-31) given in random order at least 2 weeks apart. During the infusions there were significant increases in both plasma and urinary cAMP (P < 0.05), but there were no differences in the responses between the two peptides (P = 0.362 for plasma; P = 0.987 for urine). There were also significant phosphaturic and natriuretic responses to the two peptides, which again were not different between peptides. During the infusion of hPTH-(1-34) serum ionized calcium (Ca2+) increased from 1.21+/-0.033 to 1.29+/-0.046 mmol/L (P < 0.01), and endogenous hPTH-(1-84) decreased from 29.6+/-9 to 15.0+/-5.7 pg/mL (P < 0.01), such that there was a negative correlation between them (r2 = 0.45). However, when hPTH-(1-31) was infused, neither serum Ca2+ (1.24+/-0.03 vs. 1.25+/-0.03) nor hPTH-(1-84) (26.8+/-5 vs. 30.7+/-12 pg/mL) was affected. Circulating concentrations of 1,25-dihydroxyvitamin D3 increased from 92+/-42 to 131+/-63 pmol/L (P < 0.05) during infusion of hPTH-(1-34) and from 92+/-27 to 110+/-42 pmol/L (P = NS) during hPTH-(1-31) infusion. There was also a significant increase in the urinary measure of type I collagen degradation of aminoterminal telopeptides from 78+/-45 to 101+/-51 nmol/mmol creatinine (P < 0.05) when hPTH-(1-34) was infused, but it was not affected (68+/-30 vs. 66+/-24 nmol/mmol creatinine) by hPTH-(1-31). Therefore, hPTH-(1-31) appears to be equivalent and equipotent to hPTH-(1-34) in the release of cAMP from target tissues and the renal handling of phosphate and sodium. However, at the doses employed, it does not increase serum calcium, is a weaker stimulator of the 25-hydroxyvitamin D-1alpha-hydroxylase, and does not induce rapid bone resorption.

Adult↗

PubMed1999

The baboon: a model for the study of primate growth hormone receptor gene expression during development.

In subprimates, significant onset of growth hormone receptor (GHR) expression occurs only after birth whereas, in the human, GHR mRNA and protein are widely manifest from the first trimester of fetal life. Thus, it is likely that subprimates are not the best models for studying regulation of human GHR gene transcription, especially during early stages in development. Here we have explored the potential of the baboon as a more appropriate model. Baboon GHR cDNAs were cloned from postnatal liver by reverse transcription (RT)-PCR, using human GHR-specific primers. The encoded baboon GHR precursor protein has an identical signal peptide sequence to that of human and rhesus monkey GHRs, and the mature baboon GHR is also 620 amino acids long, with 95% and 98.5% amino acid identity to the human and rhesus monkey receptors respectively. Previous studies in the human have identified eight 5' untranslated region (5' UTR) variants of the GHR mRNA (V1 to V8, numbered according to their relative abundance). We cloned the baboon V1, V3 and V4 homologues by RT-PCR: these variants have a high degree (>92%) of sequence identity with their human counterparts and also diverge at an identical point, 12 nucleotides upstream of the start of translation. The expression pattern of these three GHR mRNA isoforms in baboon liver during development was characterized. Strong expression of baboon V1 and V4 was evident by 49 days of postnatal life (n=5, 49 days and adult (18.6-19.6 kg)); very low levels of V1, but not V4, were observed in younger animals (n=2, 6 and 30 days). In contrast, V3 5' UTR variant mRNA was present in all fetal (n=4, 141-155 days gestation) and postnatal (n=7, 6-19.6 days and adult (18.6 kg)) hepatic specimens examined. Analysis of postnatal kidney and lung (n=2, 19 and 19.6 kg) revealed that V3 transcripts are present in these tissues, but not V1 and V4. Together, these data demonstrate that, as in the human, baboon V1 and V4 expression is developmentally regulated and tissue specific, while the V3 isoform is more widely expressed. Therefore, it is likely that the regulatory regions of the baboon and human GHR genes are well conserved. Our findings suggest that the baboon is an appropriate animal model in which to define the mechanisms regulating GHR gene transcription during primate development.

Adult↗

PubMed1998

The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3.

Hypocalcemia and hyperphosphatemia caused by parathyroid hormone (PTH)-resistance are the only discernible abnormalities in pseudohypoparathyroidism type Ib (PHP-Ib). Because mutations in the PTH/PTH-related peptide receptor, a plausible candidate gene, had been excluded previously, we conducted a genome-wide search with four PHP-Ib kindreds and established linkage to a small telomeric region on chromosome 20q, which contains the stimulatory G protein gene. We, furthermore, showed that the genetic defect is imprinted paternally and thus is inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or pseudo-pseudohypoparathyroidism, two related disorders caused by different stimulatory G protein mutations.

Chromosome Mapping↗