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Introduction
Rickets has long been of interest in Britain, since the original monographs of Whistler and of Glisson, Bate and Regemorter. Both of those works were in Latin but Whistler rapidly had an English translation made which shows that the title of his thesis was 'Inaugural Medical Disputation of the Children's Disease of the English which the inhabitants idiomatically called 'the rickets', which, God aiding him, Daniel Whistler, Eastern Anglo-Saxon, with the authority of the most noble and magnificent rector Dr Johannes Polyander a Kerkchoven, doctor of Holy Theology and Principle Professor of the same faculty in the illustrious Academy of Leyden in Holland propounds there for dispute for the Degree of Doctor and its entailed high privileges in Medicine on the 18th day of October at the accustomed time and place'. His thesis was printed in 1645, a few years before Glisson's book (1650), but even so
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SUMMARY
Porcine parathyroid hormone has been isolated and purified. Its size and charge properties are similar to those of bovine and human parathyroid hormones and in a bioassay system the response to porcine hormone is parallel to that of bovine parathyroid hormone. Porcine parathyroid hormone has been labelled with 125I. The immunological properties of the porcine hormone were compared with human and bovine parathyroid hormone in a radioimmunoassay system using antisera against bovine parathyroid hormone. When labelled bovine parathyroid hormone was used as a tracer in the radioimmunoassay, porcine parathyroid hormone could be shown to differ immunologically from the hormones of the other two species. When labelled porcine hormone was used as tracer, it was displaced equally well by hormone from all three species, so that in addition to regions of difference in the molecule there are probably regions of similarity.
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The metabolism of 25-hydroxycholecalciferol (25-(OH)D3), plasma concentration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the amount of calcium-binding protein (CaBP) in duodenal mucosa were determined in ovariectomized rats and were compared with data observed in normal age-matched cyclic rats. Sephadex LH-20 and high-pressure liquid chromatography were used for the study of the metabolism of 25-(OH)D3. The concentration of 1,25-(OH)2D3 in plasma and prolactin in serum were measured by radioimmunoassay. Calcium-binding protein in duodenal mucosa was determined immunologically using electroimmunodiffusion. The results showed that the lack of ovarian hormones and low prolactin levels observed in ovariectomized rats did not promote a significant change in the metabolism of 25-(OH)D3, in the levels of 1,25-(OH)2D3 in the circulation or in the amount of CaBP in duodenal mucosa. It is possible that the regulation of 25-(OH)D3 by sex hormones is restricted to the state of calcium stress such as during egg-laying in birds or pregnancy and lactation in mammals.
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SUMMARY
An immunoradiometric assay for parathyroid hormone has been developed. Antisera to bovine parathyroid hormone (BPTH) were screened for their ability to bind BPTH and human parathyroid hormone (HPTH). A BPTH-immunoadsorbent was used to extract antibodies from an antiserum which did not discriminate between BPTH and HPTH in a standard radioimmunoassay. These antibodies were labelled with 125I for use in the immunoradiometric assay. With this system as little as 5 pg BPTH and 8 pg HPTH could be detected. The serum concentration of BPTH was shown to rise in a cow rendered hypocalcaemic by an infusion of EDTA. Sera from patients with hyperparathyroidism contained high concentrations of hormone. These sera were found to dilute-out parallel to calibration curves obtained using HPTH extracted from parathyroid adenomata. The advantages of this method over the standard radioimmunoassay are discussed.
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Abstract
To further define the binding site for receptors for 1,25(OH)2D3 (VDR) in the bovine PTH gene and to study the interactions of transcription factors with VDR, Southwestern and gel shift assays were used. Data from the former indicated binding of VDR to DNA fragments spanning the regions −451 to −348 bp and −668 to −452 bp. Studies using gel shift assays confirmed binding to the −451 to −348 bp fragment and specificity was shown by using excess concentrations of unlabelled −451 to −348 bp fragment to compete for binding, whereas excess unlabelled −347 to +50 bp did not compete. Binding was also observed with the −668 to −452 bp fragment but excess concentrations of unlabelled −668 to −452 or −451 to −348 bp fragments did not compete for binding to radiolabelled fragments. These data indicate the presence of two binding domains within this region; the upstream element having a lower affinity for VDR than the downstream element. In addition, there was no interaction between VDR and consensus sequences for AP1, AP2, AP3 and SP1. The putative vitamin D3 response element (VDRE) contains two similar hexameric steroid response element-like half-sites placed as AGGTCA-related direct repeats. The upstream repeat is at −461 to −456 bp and the downstream element is at −449 to −444 bp. The presence of these half-sites is consistent with our experimental data in which cleavage with SspI at −452 bp resulted in two DNA fragments which bound VDR.
Journal of Endocrinology (1994) 142, 53–60
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ABSTRACT
Incubation of bovine parathyroid cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) decreased both preproparathyroid mRNA levels and parathyroid hormone (PTH) secretion. There was a fall to 56·6 ± 13·7% (mean ± s.e.m.) and 65·1 ± 9·3% in mRNA levels and PTH secretion respectively at 1 nmol 1,25-(OH)2D3/l, and 41·1 ± 13·6% and 42·0 ± 12·1% at 10 nmol 1,25-(OH)2D3/l after 24 h. After 48 h in 0·1 nmol 1,25-(OH)2D3/l, mRNA levels had fallen to 35·3 ± 12·6% and PTH secretion to 32·1 ± 5·0%. In human adenomatous cells, however, incubation with 1,25-(OH)2D3 (10 nmol/l) had no effect on either mRNA levels or PTH secretion even after 48 h. This lack of sensitivity of adenomatous cells to 1,25-(OH)2D3 was not due to an absence of receptors (3847 ± 39 receptors/ng cytosolic protein in adenomatous cells compared with 4068 ± 371 in bovine cells) or receptors being of low affinity.
Cortisol (1 μmol/l) caused a reduction in the number of receptors for 1,25-(OH)2D3 in bovine parathyroid cells of approximately 20% within 24 h of incubation, but no change in affinity. This decrease was accompanied by abolition of the response to 1,25-(OH)2D3 and was reversible, in that withdrawal of cortisol for the final 24 h of incubation was sufficient for the response to return, the number of receptors having returned to control values.
These results suggest that only a small percentage of receptors for 1,25-(OH)2D3 in bovine parathyroid cells may be functional at any one time. Furthermore, the insensitivity of human adenomatous cells to 1,25-(OH)2D3 does not seem to be due to a lack of receptors but may be due to a defect in the interaction between the receptor protein and the PTH gene.
Journal of Endocrinology (1989) 123, 137–142
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SUMMARY
Immunoassays specific for limited regions of bovine parathyroid hormone were developed in four ways.
With the heterogeneous antisera produced by immunizing with intact bovine parathyroid hormone (BPTH 1–84), the specificity of radioimmunoassays could be enhanced by presaturating either with an amino-terminal (BPTH 1–34) or carboxy-terminal (BPTH 53–84) fragment. Then, the antibodies which had not been neutralized reacted exclusively with the opposite end of the molecule, even using [125I]BPTH 1–84 as tracer. With some antisera, the appropriate fragment and intact hormone reacted identically. However, with other antisera, the fragment reacted less well than the intact hormone, possibly because these antisera contain antibodies reacting with the middle of the molecule.
Using the labelled fragment ([125I]BPTH 1–34) as tracer, with heterogeneous antisera, radioimmunoassays specific for the amino-terminal region were obtained. With one antiserum, BPTH 1–34 reacted identically with the intact hormone, but with another antiserum, the fragment was more reactive than the intact molecule.
A region-specific radioimmunoassay was also developed using antibodies produced by immunization with a fragment of the hormone. An antiserum raised against BPTH 1–34 had high affinity for the amino-terminal fragment, but reacted less well with the intact hormone.
Immunoradiometric assays, specific for the amino- or carboxy-terminal regions, were developed by using immunoadsorbents consisting of a fragment (either BPTH 1–34 or BPTH 53–84) coupled to cellulose. These were used to fractionate 125I-labelled antibodies. With some of these selected antibodies, the appropriate fragment was of lower reactivity than the intact hormone. This may have been due to the presence of an incomplete antigenic site on the fragment, or to conformational differences between the fragment and the corresponding region of the intact hormone. With other selected antibodies the fragment and the intact molecule reacted identically.
Careful selection of antisera and of technique is necessary to obtain an assay in which a fragment and the intact hormone behave identically.
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SUMMARY
The plasma clearance of calcitonin (TC) was studied by following the disappearance of radioactivity after intravenous injection of labelled TC, and the disappearance of bio-assayable activity after intravenous injection of unlabelled hormone. The turnover was shown to be rapid: two exponential components were involved with half times of 4–5 and 35–40 min. respectively. Similar values were obtained in experiments in which TC was infused to obtain raised, steady concentrations after which the disappearance of biologically active material was followed for up to 100 min. The normal concentration of TC in porcine peripheral plasma was calculated to be about 9 μu./ml.
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SUMMARY
Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1–34 peptide (PTH-(1–34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule.
Highly purified human PTH-(1–84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1–34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.
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SUMMARY
Hypocalcaemia was produced by i.v. infusion of EDTA into pigs from which the thyroid venous effluent could be quantitatively collected. Calcitonin in the effluent was measured by radioimmunoassay. Reduction of plasma calcium concentration rapidly suppressed the secretion of calcitonin. In contrast, as plasma calcium was raised slowly from hypocalcaemic levels by infusion of CaCl2, calcitonin secretion quickly returned and increased rapidly. The secretion rate observed when the plasma calcium concentration had been raised from hypocalcaemia to the initial normocalcaemic level was 3½–10 times greater than that observed during the normocalcaemia which pertained at the start of each experiment. A previous period of hypocalcaemia also produced an exaggerated response to a subsequent hypercalcaemic stimulus. It is suggested that the preconditioned response of calcitonin secretion increases the efficiency of the role of calcitonin in calcium homeostasis.
