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W. R. DIXON and G. W. PENNINGTON

SUMMARY

Three highly polar unconjugated Δ4-3-oxosteroids containing a 6β-hydroxyl group have been isolated from human pregnancy urine. They have been identified as 6β,17α,20β,21-tetrahydroxypregn-4-ene-3,11-dione (compound 3), 6β,11β,17α,20α,21-pentahydroxypregn-4-en-3-one (Compound 4c) and 6β,11β,17α,20β,21-pentahydroxypregn-4-en-3-one (Compound 4d). Two further steroidal compounds were found, but have not as yet been identified. The isolation procedure involved the use of thin-layer chromatography for purification of the crude urinary extracts and paper chromatography for the isolation of the individual steroids. Chemical transformations and spectroscopic analyses have been used for the identification of the individual compounds.

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G Dixon, J Nolan, N McClenaghan, PR Flatt, and P Newsholme

Evidence has been published that L -alanine may, under appropriate conditions, promote insulin secretion in normal rodent islets and various beta cell lines. Previous results utilising the clonal beta-cell line BRIN-BD11, demonstrated that alanine dramatically elevated insulin release by a mechanism requiring oxidative metabolism. We demonstrate in this paper that addition ofL -alanine had an insulinotropic effect in dispersed primary islet cells. Addition of D -glucose increasedL -alanine consumption in both BRIN-BD11 cells and primary islet cells.L -glutamine consumption in the BRIN-BD11 cell line and primary rat islets was also determined. The consumption rate was in line with that previously reported for cells of the immune system and other glutamine-utilising cells or tIssues. However,L -alanine consumption was at least an order of magnitude higher thanL -glutamine consumption. The metabolism ofL -alanine in the beta-cell may result in stimulation of insulin secretion via generation of metabolic stimulus secretion coupling factors such asL -glutamate.

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H. M. Docherty, M. J. Dixon-Lewis, P. G. Milton, A. Blight, and D. A. Heath

ABSTRACT

The distribution and molecular characteristics of parathyroid hormone-related protein (PTH-rP) in conditioned media and cell extracts of cultured human keratinocytes, and in media from a variety of both normal and transformed epithelial and non-epithelial cell cultures were studied. PTH-rP of M r 20 000 was observed in keratinocyte-conditioned media, and a larger form, M r 29 000, in the keratinocyte cell extract. PTH-like bioactivity was also detected in media from 12 out of 17 epithelial cell cultures, but was not present in media from 14 cell cultures of non-epithelial origin. The molecular size of the PTH-like protein present in the epithelial cell media was approximately 20 000, corresponding with the PTH-rP in keratinocyte-conditioned medium. These observations may explain why hypercalcaemia is most commonly associated with tumours of epithelial origin.

Journal of Endocrinology (1989) 123, 487–493

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KATHLEEN PEASE, HIRAM SHEN, G. S. ACRES, J. H. RUPNOW, and J. E. DIXON

SUMMARY

Developmental changes in levels of hypothalamic thyrotrophin releasing hormone (TRH) and the role that hypothalamic degrading enzymes may play in these alterations were investigated. Levels of TRH in male and female fetal rats and in male neonatal rats were measured by radioimmunoassay. The hormone content of the hypothalamus was shown to increase from less than 1 ng at 1–4 days of age to approximately 10 ng at 20 days of age. Thereafter, the content of TRH declined to the adult level of about 5 ng. The ability of fractionated hypothalamic homogenates to degrade TRH was measured over the same time. The 27 000 g supernatant fraction contained a degrading activity that yielded only radiolabelled deamido-TRH upon incubation with [l-proline 2,3-3H]TRH. The corresponding particulate fraction contained at least two distinct TRH degrading activities as determined by the number of metabolites present. Changes in rates of degradation were not large enough to account for the differences observed in levels of TRH.

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SJ Conroy, I Green, G Dixon, PM Byrne, J Nolan, YH Abdel-Wahab, N McClenaghan, PR Flatt, and P Newsholme

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).