G L Francis

Natural and synthetic forms of insulin-like growth factor-1 (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding.

Insulin-like growth factor-1 (IGF-1), whether recombinant, chemically-synthesised or purified from bovine colostrum, was equipotent in radioreceptor assays with IGF-1 or insulin-like growth factor-2 (IGF-2) as radioligand as well as in its ability to stimulate protein synthesis in L6 myoblasts. The N-terminal truncated, destripeptide derivative of IGF-1 was approximately 7 times more potent than IGF-1 in the protein synthesis bioassay. This increased activity occurred equally with the peptide purified from bovine colostrum as with chemically-synthesised material. The higher potency of the truncated form was not associated with an increased ability to compete for IGF-1 binding to L6 myoblasts.

Influence of whey and purified whey proteins on neutrophil functions in sheep

The effects of ruminant whey and its purified fractions on neutrophil chemotaxis and superoxide production in sheep were studied. Both colostral whey and milk whey were found to inhibit chemotaxis regardless of whether they were autologous or homologous, but the inhibitory effects were abolished by washing neutrophils with culture medium before their use in the chemotaxis assay. Colostral whey and milk whey also inhibited the chemotactic activity of zymosan-activated serum. Whey fractions of various degrees of purity such as lactoferrin, lactoperoxidase, lactoferrin-lactoperoxidase, alpha-lactalbumin, bovine serum albumin and whey protein concentrate were then studied. While none of these proteins showed any effects on chemotaxis, lactoferrin-lactoperoxidase and whey protein concentrate were found to have an enhancing effect on superoxide production in a dose-dependent manner. Our results provide information on the modulatory role of ruminant milk proteins in inflammatory responses and warrant future investigation.

Factors in ruminant colostrum that influence cell growth and murine IgE antibody responses

Bovine colostrum was investigated as a source of biologically active molecules capable of stimulating the growth of mammalian cells in culture and modifying the immune response in a murine model. An extract prepared from bovine colostral whey by cation exchange and reversed-phase chromatography stimulated the growth of L6 rat myoblasts, Balb/c-3T3 mouse fibroblasts and BHK-21 baby hamster kidney cells with equal or greater potency than fetal bovine serum. Fractionation of the bovine colostral extract by gel-permeation chromatography in M-acetic acid identified a number of cell-growth factors for each cell type. Bovine colostral extract was compared with an ovine colostral whey preparation for its ability to modulate IgE antibody responses in mice. Doses of 8 and 4 mg/d of ovine colostral whey or bovine colostral extract specifically suppressed IgE antibody responses, whereas at lower doses suppression did not occur. We conclude that bovine colostrum contains cell-growth factors as well as immunomodulatory factors that are able to regulate the IgE response in a heterologous species.

Secondary structure determination of 15N-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy

Insulin‐like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long‐[Arg‐3]‐IGF‐I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213–223), a potent analogue of IGF‐I, which has a Glu‐3 to Arg‐3 substitution and a hydrophobic, thirteen amino acid N‐terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity‐enhanced 15N‐NOESY‐HSQC and sensitivity‐enhanced 15N‐HSQC‐NOESY‐HSQC spectra. The IGF‐I domain of Long‐[Arg‐3]‐IGF‐I was found to have an almost identical structure to IGF‐I. The N‐terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn‐like structure that is spatially close to the beginning of helix 1 in the IGF‐I domain. Hydrogen‐deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF‐I domain are either in the helical or the extended structural elements. Many of the amide protons in the N‐terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long‐[Arg‐3]‐IGF‐I and the decreased binding to insulin‐like growth factor binding proteins.

Inactivation of Cytosol Enzymes by a Liver Membrane Protein

Non-proteolytic inactivation reactions have been suggested to play an important role in determining the relative rates of enzyme degradation within cells. An inactivation factor is present at high specific activity in hepatocyte plasma membrane which inactivates cytosol enzymes at rates roughly proportional to their rates of degradation in vivo. This factor has been purified about 100-fold and has similar catalytic selectivity to the crude factor. The inactivation reaction is accelerated by disulphide compounds and can be partially reversed by thiols. Furthermore, inactivation of enzymes is accompanied by a loss of measurable thiols in the enzymes. From these experiments it is concluded that the inactivation factor carries out a disulphide attack on surface thiol groups in cytosol enzymes leading to the formation of mixed disulphides which have lost catalytic activity. The inactive enzymes may be substrates for lysosomal or non-lysosomal proteolysis.

A Potent Growth Factor Supplement For Cell Culture Purified From Whey

We developed a single-step cation-exchange process for enriching growth factors present in cheese whey. Whey-derived growth factor extract (WGFE) contained only 0.5% whey protein, but all the growth factor activity. It stimulated the growth of mesodermal-derived cells and inhibited epithelial cells. Maximum growth of fibroblasts exceeded the response to serum, whereas other lines, such as CHO cells and L6 myoblasts, required the presence of low serum concentrations (1% v/v) to achieve similar growth rates. Specific assays detected IGF-I and -II, FGF-1 and -2, PDGF, and TGF-s1 and -s2 at concentrations that partially account for the growth-promoting activity. A combination of these growth factors could not reproduce the maximum effect of WGFE, suggesting the presence of other growth factors. Membrane Ultrafiltration of WGFE partitioned and concentrated growth factors, producing fractions enriched in IGF or TGF-s peptides, with enhancement of potency by up to 30-fold. WGFE as a source of growth factors, has application as a serum supplement or extender, particularly for fibroblasts and other mesodermal-derived commercially important cell lines.