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A. R. McGIVEN
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D. D. ADAMS
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H. D. PURVES
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SUMMARY

Studies of the heat stability of a human antibody against thyroglobulin showed that after heating at 70° for 10 min. its potency was reduced to 2% of the initial value. Similarly, the potency of a preparation of long-acting thyroid stimulator (LATS) was reduced to significantly less than 10% by this treatment, whereas the potency of a human thyroid-stimulating hormone (TSH) preparation was reduced only to 45%, with a lower fiducial limit of 26% for P = 0·05.

It is concluded that LATS is less stable to heat than is TSH. LATS did not differ from the antibody in heat stability.

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R. J. HEITZMAN
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T. C. ADAMS
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G. D. HUNTER
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17α-Hydroxycorticosterone (F) and corticosterone (B) are the two most abundant steroids in bovine adrenal venous blood (Bush, 1953). The competitive protein-binding assay of Bassett & Hinks (1969) is equally sensitive for both these steroids. This fortuitous advantage, together with the need for only small volumes of blood (< 5 ml.), makes this method of assay ideal for the determination of bovine peripheral plasma corticosteroids.

Ayrshire or Friesian cows, kept under cover and accustomed to being handled, were fed on a diet normal for the Institute herd. A list of the different groups of cows is given in Table 1. All blood samples were taken between 14.00 and 15.00 hr. The sample (about 5 ml.) was taken, on to heparin, from the jugular vein within 30 sec. of approaching the animal. After centrifugation, plasma was stored at −15° until required.

The assay of plasma corticosteroids was essentially as described by Bassett

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T. A. Bramley
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G. S. Menzies
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R. J. Williams
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O. S. Kinsman
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D. J. Adams
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ABSTRACT

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16 000–21 000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125 I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for β-core protein, a cleavage product of the β subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified β-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites.

Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to β-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.

Journal of Endocrinology (1991) 128, 139–151

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T. A. Bramley
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G. S. Menzies
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R. J. Williams
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O. S. Kinsman
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D. J. Adams
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ABSTRACT

We have described recently the presence of binding sites for human LH (hLH) and human chorionic gonadotrophin (hCG) in microsomal and cytosol fractions prepared from the dimorphic, pathogenic fungus, Candida albicans. We have now compared the properties ofCandida LH/hCG-binding sites with those of the ovine luteal LH receptor. Sheep luteal LH-binding sites were associated with luteal membranes, and little or no binding activity was present in cytosol fractions. In contrast, significant LH/hCG-binding activity was present in Candida cytosol. Moreover, there were marked differences in sensitivity to inhibition by metal ions, association and dissociation rates, and affinity constants between sheep and Candida LH-binding sites. Scatchard plots of 125I-labelled hLH binding to sheep luteal receptors demonstrated a single high-affinity component (association constant (K a) 0·3 litres/pmol) which was displaceable by hCG. In contrast, Scatchard plots of binding to Candida microsomes and cytosol fractions demonstrated two components, one with high affinity (K a 0·18 litres/pmol) and low capacity and a second site with lower affinity (K a 4 litres/nmol) and high capacity, both of which were displaceable by unlabelled hCG. Gel permeation chromatography of cytosol demonstrated two distinct peaks of LHbinding activity with approximate molecular weights of > 1 000 000 and 30 000–50 000. Scatchard plots of 125I-labelled hLH binding to the higher molecular weight peak demonstrated a single, high-affinity LH-binding site (K a 0·18 litres/pmol), whereas the lower molecular weight fraction contained both high- (K a 0·17 litres/pmol) and low-affinity (K a 4 litres/nmol) LH-binding sites.

Both partially purified and highly purified hCG and hLH preparations displaced binding of 125I-labelled hLH and hCG to sheep luteal LH receptors at similar concentrations. 125I-Labelled hLH/hCG binding to Candida membranes was also displaceable by low levels (ng) of partially purified hCG preparations, but much higher levels (μg) of highly purified hLH and hCG were required. This paradoxical observation suggested the presence of radiolabelled contaminants, or damaged forms induced during radioiodination of hormone tracers, which can bind more strongly to Candida membranes than unlabelled hCG and hLH but which do not bind to sheep LH receptors. However, no evidence for hLH tracer contaminants with differential binding to Candida and sheep luteal receptors was obtained following gel exclusion chromatography or fractionation on Concanavalin A–Sepharose. (Although three distinct 125I-labelled hLH fractions were resolved on Concanavalin A–Sepharose, presumably reflecting differences in their carbohydrate compositions, all three tracer peaks bound equivalently to both Candida membranes and ovine luteal LH receptors.) Moreover, iodination of highly purified hLH and hCG with 127I failed to generate substances with differential binding to sheep luteal and Candida LH-binding sites. Furthermore, preincubation of 125I-labelled hLH tracer with Candida membranes at different temperatures with or without protease inhibitors did not affect the ability of the hormone tracer to bind to fresh ovine luteal or Candida LH-binding sites. Thus, differences between ovine luteal and Candida LH-binding sites cannot be attributed to differential binding of contaminants present in the hormone tracer preparations, nor to (proteolytic) modification of tracer by Candida membranes during incubation.

Journal of Endocrinology (1991) 130, 177–190

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P. ADAMS
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T. M. CHALMERS
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B. L. RIGGS JR.
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J. D. JONES
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SUMMARY

Calcium and phosphorus metabolism were studied in 22 patients with spontaneous primary hypothyroidism. Two patients were found to have hypercalcaemia but the mean serum calcium concentration of the group was significantly less than that of control subjects. The renal tubular reabsorption of phosphate was decreased and could be increased to normal with small calcium infusions. The response to calcium deprivation and to infusions of EDTA was abnormal and suggested an impaired ability to mobilize calcium from bone. There was a significant correlation between the defect in calcium mobilization, as judged from the response to EDTA, and the renal tubular reabsorption of phosphate.

In three patients serum parathyroid hormone concentrations, measured by radioimmunoassay, were in the upper part of the normal range.

It is suggested that in patients with hypothyroidism the target cells in bone are less responsive to the effects of parathyroid hormone than normal; as a consequence parathyroid hormone secretion may be increased.

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CJ Newton
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G Ran
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YX Xie
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D Bilko
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CH Burgoyne
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I Adams
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A Abidia
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PT McCollum
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SL Atkin
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Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.

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C J Newton Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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G Ran Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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Y X Xie Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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D Bilko Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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C H Burgoyne Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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I Adams Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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A Abidia Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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P T McCollum Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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S L Atkin Jacob’s Well Medical Research Laboratory,
Diabetes and Endocrinology and
Vascular Surgery, Hull and York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK

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