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D A Belford, M-L Rogers, G L Francis, C Payne, F J Ballard and C Goddard

Abstract

Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80–100 kDa. In contrast, acid gelfiltration chromatography resolved two peaks of cell growth activity. A peak at 15–25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bioactivity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15–25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor β (TGF-β), and all inhibitory activity for Mv1Lu cells was immuno-neutralised by an antibody against TGF-β. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-β and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-β; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-β account for the 15–25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.

Journal of Endocrinology (1997) 154, 45–55

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C. P. SIBLEY, B. J. WHITEHOUSE, G. P. VINSON, C. GODDARD and E. McCREDIE

A technique for the perfusion of the rat adrenal cortex is described. With tissue culture Medium 199 the preparation was responsive in terms of steroid production to both ACTH and K+ ions. Production of corticosterone and 18-hydroxydeoxycorticosterone (18-hydroxy-DOC) was stimulated by ACTH when it was administered at rates between 5 μu./min and 5 mu./min. Increasing the K+ ion concentration of the perfusate from 3·6 to 5·4 and 8·9 mmol/l stimulated the production of aldosterone, 18-hydroxycorticosterone and deoxycorticosterone, although not of corticosterone or 18-hydroxy-DOC.

This preparation has been used to study further the mechanism of secretion of corticosterone and 18-hydroxy-DOC. Thus, production of these two steroids was measured at different perfusion flows, varying between 0·1 and 0·6 ml/min, with different levels of ACTH stimulation. Corticosterone production was significantly (P < 0·001) increased by increasing flows both under control conditions and when ACTH was administered at constant rates of 50 μu./min or 1 mu./min. Production of 18-hydroxy-DOC was not affected by flow either under control conditions or with 50 μu. ACTH/min. However, when ACTH was administered at 1 mu./min, 18-hydroxy-DOC production was also significantly (P < 0·001) increased by flow.

The results are consistent with those obtained in previous in-vitro studies and have been interpreted as suggesting that the main mechanism of corticosterone secretion is simple diffusion. In contrast, 18-hydroxy-DOC secretion, at least at sub-maximal levels of stimulation, appears to require a more complex process.

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B. Houston, I. E. O'Neill, M. A. Mitchell and C. Goddard

ABSTRACT

The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chicken GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6·86 and the major peak (cGH P2) an isomer with an isoelectric point of 7·52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (M r = 23 500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence.

This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l. The output of insulin-like growth factor-I by hepatocytes in culture was increased from a basal rate of 50·4±11·6 (mean ± s.e.m.) to 787·9 ± 98·6 pg/6 × 106 cells per 48 h by two separate pulses of 1 μg cGH P2/ml. An i.v. injection of cGH P2 (15 μg/kg body weight) decreased the thyroxine:tri-iodothyronine ratio in serum of adult hens from 15·71 to 4·44, indicating an increase in 5′-monodeiodinase activity. These results demonstrate that the single most abundant charge isomer of chicken pituitary GH is likely to contain all the biological activity ascribed to the hormone.

Journal of Endocrinology (1990) 125, 207–215

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M. J. Duclos, B. Chevalier, Y. Le Marchand-Brustel, J. F. Tanti, C. Goddard and J. Simon

ABSTRACT

The effects of insulin and insulin-like growth factor-I (IGF-I) on glucose transport were compared in myotubes derived from chicken breast muscle satellite cells in vitro. Myotubes were incubated (for 0·5 or 4 h) with or without glucose in the presence or absence of insulin or IGF-I. Glucose uptake was subsequently measured by the incorporation of 2-[1,2-3H(N)] deoxy-d-glucose ([3H]2DG) in glucose-free medium (10 min at 20 °C). Glucose uptake was almost completely abolished by the addition of cytochalasin B or phloretin. It was increased by a decrease in glucose concentration in the incubation medium. Insulin (5 mg/l) stimulated [3H]2DG uptake to a maximum of 43 ± 10% above basal after 30-min incubation and 101 ± 15% after 4-h incubation. IGF-I and insulin at equimolar concentrations (25 μg/l and 20 μg/l respectively) were almost equipotent after 0·5 h but after 4-h incubation IGF-I was 17-fold more potent, suggesting that this 'late' effect was mediated through the IGF-I receptor. Incubation with cycloheximide suggested that the effect of IGF-I involved increased protein synthesis. The results suggest that chicken myotubes express a glucose transporter which is regulated by IGF-I and glucose concentration. However, they do not appear to express a typical insulin-responsive transport system.

Journal of Endocrinology (1993) 137, 465–472

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C. Goddard, Y. S. Davidson, B. B. Moser, I. Davies and E. B. Faragher

ABSTRACT

The effect of age on the cyclic AMP (cAMP) response to increases in the concentration of arginine vasopressin in the presence of isobutyl methylxanthine (100 μmol/l) was studied in an in-vitro renal cell suspension prepared from C57BL/Icrfat mice at 6, 12, 18, 24, 29 and 35 months of age. Comparison of the response of the preparation to vasopressin, calcitonin and parathyroid hormone suggested that it was enriched with renal medullary cells. Basal cAMP output was similar throughout but the threshold dose of vasopressin increased from 1 × 10−11 mol/l (6, 12 and 18 months of age) to 1 × 10−10 mol/l (24, 29 and 35 months of age). The dose–response curve in 35-month-old mice was shifted to the right with the concentration of vasopressin required to give half maximal cAMP increased from 9·4 ± 0·37 × 10−11 mol/l (6 months) to 3·5±1·6 × 10−10 mol/l (35 months). Maximum cAMP output at 1 × 10 −9 mol/l was also reduced in the same animals (stimulated:basal ratio, 51·22±19·12 at 6 months; 11·50 ± 6·02 at 35 months). The results suggest that the lack of renal response to vasopressin in terms of cAMP metabolism may play a role in the well-documented age-related decline in urine-concentrating ability in experimental animals and elderly people.

J. Endocr. (1984) 103, 133–139

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M-L Rogers, C Goddard, G O Regester, F J Ballard and D A Belford

Abstract

Transforming growth factor β (TGF-β) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-β in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a mink lung epithelial cell line. Most of the TGF-β bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-β concentration was 4·3 ± 0·8 ng and 3·7 ± 0·7 ng TGF-β per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6·5 was used to concentrate latent whey-derived TGF-β, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-β. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-β with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-β from both high molecular mass peaks. Whey-derived TGF-β was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-β2, which accounted for over 85% of the TGF-β bioactivity in whey. All TGF-β activity in whey could be neutralised by a monoclonal antibody directed against TGF-β1, -β2 and -β3. The results suggest that the majority of TGF-β in bovine milk is present in a small latent complex.

Journal of Endocrinology (1996) 151, 77–86