Biomedical subjects
Publications and source records attributed to G N Hendy.
The nucleotide sequence of a novel peptide from a rat Leydig cell hypercalcemic tumor H-500 was determined. This cDNA encodes a peptide of 93 amino acids and contains a heparin binding domain similar to histone 2-B. Northern blot analysis showed tissue specific expression of this peptide mRNA.
Antidiuretic hormone (arginine vasopressin) binds to and activates V2 receptors in renal collecting tubule cells. Subsequent stimulation of the Gs/adenylyl cyclase system promotes insertion of water pores into the luminal membrane and thereby reabsorption of fluid. In congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disorder, the kidney fails to respond to arginine vasopressin. Here we report that an affected male of a family with CNDI has a deletion in the open reading frame of the V2 receptor gene, causing a frame shift and premature termination of translation in the third intracellular loop of the receptor protein. A normal receptor gene was found in the patient's brother. Both the normal and the mutant allele were detected in his mother. A different mutation, causing a codon change in the third transmembrane domain of the V2 receptor, was found in the open reading frame of an affected male but not in the unaffected brother belonging to another family suffering from CNDI.
Previous studies have indicated that 19-day-old fetal long bones of the rat contain an adenylyl cyclase-stimulating activity antigenically related to parathyroid hormone-related peptide. To ascertain its origin, Northern blotting and in situ hybridization histochemistry were performed. Results demonstrate that mRNA of parathyroid hormone-related peptide is present in RNA extracted from fetal long bones of the rat and that cells responsible for its production are localized in the periosteum. These cells are not mature osteoblasts because they do not synthesize mRNA of osteocalcin. Thus the present study shows that parathyroid hormone-related peptide could be produced locally, at least in part, in the skeleton of fetal rats.
We have recently demonstrated that parathyroid hormone-like protein (PLP) production by cultured human squamous carcinoma cells (SCC) can be modulated by co-culture with fibroblasts. The interaction of SCC with fibroblasts, possibly occurring during the invasive phase of SCC, may be the stimulus for enhanced PLP production, thus contributing to the genesis of humoral hypercalcemia of malignancy in this type of cancer (Cancer Res 50:3589-3594, 1990). In the present study we show that the fibroblast-induced increase in PLP level in the medium of SCC-4 cells is paralleled by an increase in PLP messenger ribonucleic acid (mRNA) expression in these cells. We also found that the inhibition of secretion of PLP by monensin for 2 h resulted in a marked increase in immunodetectable PLP intracellularly, suggesting that secretion of PLP was a fast process. The modulation of the production of PLP by calcium and hydrocortisone was further examined in SCC-4 cells and was compared to that in normal keratinocytes and in SCC-9 cells. PLP levels in conditioned media were highest in poorly differentiating SCC-4 cells, intermediate in moderately differentiating SCC-9 cells, and lowest in normal keratinocytes showing high differentiating capacity. Furthermore, in each of the cell types used, PLP production was highest in cultures grown under low calcium conditions; at both calcium concentrations used, the presence of hydrocortisone reduced the PLP release into the medium. This reduction was probably due to a direct effect of hydrocortisone on PLP synthesis because the expression of PLP mRNA was also reduced in the presence of hydrocortisone when tested in SCC-4 cells. In conclusion, our findings indicate that the induction of differentiation in both normal and malignant keratinocytes is associated with the inhibition of PLP production.
The single copy parathyroid hormone-like peptide gene (Pthlh) was mapped to distal mouse chromosome 6 using genetic linkage analysis with a panel of DNA samples from interspecific backcross mice. In all 114 meiotic events examined, the Pthlh locus cosegregated with the locus for the Kirsten ras-2 gene (Kras-2) which was previously localized to distal mouse chromosome 6. In addition, Pthlh was localized to chromosome 6 band F-G and the mouse parathyroid hormone Pth was localized to chromosome 7 band F, by in situ hybridization. These studies confirm the previous localization of Pthlh to mouse chromosome 6 using somatic cell hybrids and show that the Pthlh/PTHLH locus is a part of a conserved linkage group between distal mouse chromosome 6 and the proximal segment of the short arm of human chromosome 12.
Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.
PTH-related peptide (PTHrP) is one of the etiological factors associated with hypercalcemia of malignancy in humans and rodents. In both in vivo and in vitro animal systems its actions mimic those of PTH; however, its bioactivity in humans has not previously been assessed. Therefore, we compared the actions of the synthetic human (h) analogs hPTHrP-(1-34) and hPTH-(1-34) when given by iv infusion to 15 healthy subjects, aged 25 +/- 3 yr. Three 12-h test infusions were given to each subject in the order: hPTH-(1-34) at a dose of 8 pmol/kg.h, an equimolar dose (8 pmol/kg.h) of PTHrP-(1-34) (low dose), and a 10-fold higher dose (80 pmol/kg.h) of hPTHrP-(1-34) (high dose). PTH infusion resulted in significant increases from basal values in serum total ionized calcium, urinary phosphate and cAMP, and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2d3]. No significant increases from basal values in any of these variables were observed during low dose PTHrP infusion. However, a 10-fold higher dose of PTHrP significantly increased serum calcium from 2.36 +/- 0.07 to 2.63 +/- 0.16 mmol/L (P less than 0.003), ionized calcium from 1.22 +/- 0.03 to 1.39 +/- 0.09 mmol/L (P less than 0.003), urinary phosphate from 0.21 +/- 0.19 to 0.31 +/- 0.16 mmol/L glomerular filtrate (P less than 0.05), urinary cAMP from 37 +/- 18 to 53 +/- 28 nmol/L glomerular filtrate (P less than 0.01), and serum 1,25-(OH)2D3 from 29.8 +/- 12.1 to 46.0 +/- 20.3 pmol/L (P less than 0.01). For each variable these changes were statistically equivalent to the increases observed during PTH infusion. The molar concentrations of circulating immunoreactive PTH-(1-34) and PTHrP-(1-34) (at the higher dose) achieved during infusion were at a ratio of 1:3. These results suggest that the in vivo actions of synthetic hPTHrP-(1-34) are comparable to those of hPTH-(1-34), but its biological activity after infusion may be less than that of hPTH-(1-34). Moreover, the increased concentrations of serum 1,25-(OH)2D3 observed with administration of hPTHrP-(1-34) are unlike the changes seen in hypercalcemia of malignancy in which levels of this vitamin D metabolite are frequently depressed.
Our previous studies indicated that regulation of bovine parathyroid chromogranin-A (CgA) by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] may occur at the level of CgA turnover or mRNA translation. In the present study, immunoprecipitation of extracts of bovine parathyroid cells that had been pulse chased with [35S]methionine revealed that 1,25-(OH)2D3 had no effect on the disappearance time of intracellular CgA. Therefore, we examined the effect of 1,25-(OH)2D3 on the polyribosome profile of CgA mRNA analyzed by sucrose density gradients. In the presence of 1,25-(OH)2D3, there was a dose-dependent recruitment of CgA mRNA into the denser polyribosomal fractions by 24 h. To determine whether this increased ribosome loading represents increased or decreased efficiency of mRNA translation, ribosome transit time experiments were conducted. The average ribosome transit time and the specific PTH ribosome transit time were not altered by 1,25-(OH)2D3. However, that for CgA was doubled in the presence of 1,25-(OH)2D3. Thus, parathyroid CgA synthesis is regulated by the vitamin D sterol at the level of peptide chain elongation. These studies, therefore, explain the lack of quantitative correspondence between 1,25-(OH)2D3-induced CgA gene transcription and CgA protein levels by revealing a previously unsuspected level of regulation of mRNA translation in the parathyroid cell.
Molecular biology techniques have been used to examine alternative splicing of the PLP gene, interspecies homology of PLP, the genetic relationship of PLP and PTH. Molecular biology approaches can also be successfully employed to examine both the regulation of PLP production and its function both in emulating PTH and in non PTH-related actions.
Nephrogenic diabetes insipidus (NDI; designated 304800 in Mendelian Inheritance in Man) is an X-linked disorder with abnormal renal and extrarenal V2 vasopressin receptor responses. The mutant gene has been mapped to Xq28 by analysis of RFLPs, and tight linkage between DXS52 and NDI has been reported. In 1969, Bode and Crawford proposed, under the term "the Hopewell hypothesis," that most cases in North America could be traced to descendants of Ulster Scots who arrived in Nova Scotia in 1761 on the ship Hopewell. They also suggested a link between this family and a large Mormon pedigree. DNA samples obtained from 13 independent affected families, including 42 members of the Hopewell and Mormon pedigrees, were analyzed with probes in the Xq28 region. Genealogical reconstructions were performed. Linkage between NDI and DXS304 (probe U6:2.spl), DXS305 (St35-691), DXS52 (St14-1), DXS15 (DX13), and F8C (F814) showed no recombination in 12 families, with a maximum lod score of 13.5 for DXS52. A recombinant between NDI and DXS304, DXS305, was identified in one family. The haplotype segregating with the disease in the Hopewell pedigree was not shared by other North American families. PCR analysis of the St14 VNTR allowed the distinction of two alleles that were not distinguishable by Southern analysis. Carrier status was predicted in 24 of 26 at-risk females. The Hopewell hypothesis cannot explain the origin of NDI in many of the North American families, since they have no apparent relationship with the Hopewell early settlers, either by haplotype or by genealogical analysis. We confirm the locus homogeneity of the disease by linkage analysis in ethnically diverse families. PCR analysis of the DXS52 VNTR in NDI families is very useful for carrier testing and presymptomatic diagnosis, which can prevent the first manifestations of dehydration.
Nine chromosome 10 DNA markers (FNRB, D10S34, D10Z1, MEN203, D10S94, RBP3, D10S15, MBP [48.11], D10S22) were typed in two large Canadian pedigrees with multiple endocrine neoplasia type 2A (MEN 2A). These markers and the gene for MEN 2A (MEN2A) are believed to be in one linkage group spanning approximately 15 cM (male). MEN203 and D10S94 were informative and tightly linked to MEN2A with no recombinants observed in 26 meiotic events. D10S15 (MCK2), widely used in DNA genotyping predictions, demonstrated two recombinants in these two families. The use of multiple flanking markers increases both the likelihood of informativeness and the accuracy of risk assessments for predictive testing. We were able to assign a risk estimate for all 10 at-risk individuals.
Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.
Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.
Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.
1 alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3) has been measured in human serum by radioimmunoassay. The assay uses a high titre antiserum raised in sheep against 1,25-(OH)2D3-25-hemisuccinate, conjugated to bovine serum albumin. The sensitivity of the assay is 10 pg/tube. Other hydroxylated forms of vitamin D3 cross react with the antiserum and are therefore removed from serum extracts by chromatography on Sephadex LH 20 followed by high pressure liquid chromatography. The mean (+/- SEM) serum 1,25-(OH)2D3 concentration for a group of healthy adult subjects was 41 +/- 2.5 pg/ml. None was detected in anephric patients and the concentration was low or undetectable in patients with chronic renal failure.
The immunological properties of a synthetic peptide comprising the carboxyl-terminal 53--84 region of human parathyroid hormone (PTH) have been studied. The immunoreactivity of the synthetic human PTH-(53--84) peptide paralleled that of a 53--84 fragment of the native human hormone prepared by enzymic digestion, in both a standard radioimmunoassay, which was not region-specific, and also a radioimmunoassay specific for the carboxyl-terminal region of PTH. However, in both types of radioimmunoassay the synthetic human PTH-(53--84) peptide was four to five times more reactive than the native human PTH-(53--84) fragment.
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1. 1alpha,25-Dihydroxy-25-hemisuccinate cholecalciferol has been synthesized and conjugated to bovine serum albumin. 2. This conjugate is immunogenic; when injected into rabbits antibodies of high affinity for 1alpha,25-dihydroxycholecalciferol were obtained. 3. Vitamin D metabolites lacking the 1alpha-hydroxy group were of lower cross-reactivity with the antibodies. 4. By using these antibodies and 1alpha,25-[23,24-3H]dihydroxycholecalciferol as tracer a sensitive radioimmunoassay has been developed capable of detecting 20 pg of 1alpha,25-dihydroxycholecalciferol.