Atypical spI interferon binds on porcine cells to a major component of type I interferon receptor.

The short porcine type I interferon (spI IFN), encoded by a gene physiologically expressed by the pig embryonic trophoblast during implantation, represents the first member of a novel family type I IFN. Binding and cross-linking experiments were carried out to characterize its cellular receptor. On porcine kidney cells, specific binding of 125I-spI IFN could be displaced significantly by spI IFN, rpIFN-alpha 1, and rhIFN-alpha 1, but not by rhIFN-alpha 2a or by rpIFN-gamma. On the other hand, all these type I IFNs but not rpIFN-gamma were capable of displacing bound 32P-hIFN-alpha A-P1 on these cells. Cross-linking data show that the specific 120 kD complex formed with these two radiolabeled ligands was displaceable by an excess of both spI IFN and rpIFN-alpha 1. These results provide primary evidence that spI IFN shares at least the major binding subunit of type I IFN receptor on porcine cells. On human WISH cells, 125I-spI IFN did not form any complex, nor did spI IFN affect cross-linking complexes of 32P-hIFN-alpha A-P1 on these cells, unlike rpIFN-alpha 1. The lack of antiviral and antiproliferative effects of spI IFN on human cells is primarily a result of its inability to recognize human type I IFN receptor.

Atypical porcine type I interferon. Biochemical and biological characterization of the recombinant protein expressed in insect cells.

A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.

Development of a protein binding assay for teleost insulin-like growth factor (IGF)-like: relationships between growth hormone (GH) and IGF-like in the blood of rainbow trout (Oncorhynchus mykiss).

Using rainbow trout plasma protein (IGF-BP) which specifically binds human insulin-like growth factor (IGF) (Niu and Le Bail 1993), we have developed an assay to measure plasma IGF-like levels in different teleost species. Before the assay and to prevent interference by IGF-BP, IGF-like was extracted from all samples, using SP Sephadex C-25 in acidic conditions. After this treatment, contamination of the IGF fraction by IGF-BP which was estimated by binding assay, was approximately 5%, and was not detectable by western ligand blot.Human IGF-I was used as standard and labelled hormone. Sensitivity of the assay was 0.15-0.40 ng/ml (ED90) and ED50 was 1-3 ng/ml. hIGF-II crossreaction was partial and no significant displacement was observed with Insulin from different species or with other hormones. Inhibition curves were obtained with plasma IGF fractions (but not with tissue extracts) from teleost and mammals and are parallel to the standard curve. These results suggest that the protein binding assay can quantify an IGF-like factor in the plasma of teleost and that the binding sites of IGF are well conserved during vertebrate evolution.Using this IGF binding assay, IGF-like was measured in parallel with growth hormone (GH) in plasma from young rainbow trout killed every 1.5h throughout one day. The daily profiles for both hormones, which appear pulsatile, are similar. A significant correlation was observed between GH levels and IGF-like levels with a 1.5h delay. Analogous observations were obtained in individual catheterized adult rainbow trout. Although plasma GH levels differ greatly between fish, less variability is found with IGF-like. In a third experiment, rainbow trout were starved or submitted to bovine GH treatment for four weeks. Starved fish, in which plasma GH levels increased, had plasma IGF-like level significantly lower than in fed fish. In bGH injected fish, plasma IGF-like level was significantly higher than in non-injected fish. These results suggest that, as in mammals, IGF-like secretion depends on plasma GH level and could be modulated by the nutritional status of fish.

In vivo and in vitro studies on sex steroid binding protein (SBP) regulation in rainbow trout (Oncorhynchus mykiss): influence of sex steroid hormones and of factors linked to growth and metabolism.

The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout. In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration. Testosterone or cortisol injections have no effect. In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP. Its production is increased by addition of E2 (maximum: +300%). This effect develops slowly over several days of culture and is dose dependent; as little as 1-10 nM E2 is effective. Recombinant rainbow trout GH (rtGH)--0.01 to 1 microgram/ml--also increases SBP accumulation as compared to control cells and seems to maintain SBP production over culture duration. In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF1 (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory. Other hormones tested in vitro: triiodothyronine (10-1000 nM), thyroxine (100 nM), 17 alpha, 20 beta-dihydroprogesterone (10-2000 nM), and testosterone (1-1000 nM) did not influence SBP concentration in hepatic cells culture media.

Development and validation of a highly sensitive radioimmunoassay for chinook salmon (Oncorhynchus tshawytscha) growth hormone.

This study describes the development of a highly specific and very sensitive radioimmunoassay for salmonid growth hormone. Antiserum raised against chinook (Oncorhynchus tshawytscha) GH2, which did not recognize 125I-sPRL and 125I-sGTH (at 1:1000 initial dilution), was able to inhibit growth when injected into rainbow trout (Oncorhynchus mykiss). 125I-sGH2, used as tracer, was not recognized by anti-sGTH or by anti-sPRL. Mammalian GH and ACTH and salmonid GTH, TSH, and PRL did not cross-react in the sGH assay. The inhibition curves for pituitary extracts and plasma from salmonids were parallel to the salmon GH standard, whereas those from carp, tilapia, and catfish showed no significant cross reactivity. The RIA ED90 and ED50 values were 0.2 and 1.5 ng/ml, respectively. Using this RIA for measuring GH release by cultured pituitary cell we observed a strong inhibiting effect of SRIF (10(-6) M) and a stimulatory effect of hGRF (10(-6) M). This RIA allowed us also to detect daily fluctuations in the plasma GH concentration in cannulated rainbow trout.

Presence of specific growth hormone binding sites in rainbow trout (Oncorhynchus mykiss) tissues: characterization of the hepatic receptor.

The present work outlines the presence of specific binding for chinook salmon growth hormone (sGH) in different tissue preparations of rainbow trout. Optimal incubation conditions (pH, Tris, MgCl2) were determined. Specific binding was very sensitive to salt concentration during incubation. The specific binding reached a plateau after 15 and 25 hr of incubation at 12 and 4 degrees. At 20 degrees, specific and nonspecific binding were not stable. Specific binding dissociation was slower than association and was only partial. The binding was saturable (Bmax = 187 +/- 167 pmol), of high affinity (Ka = 2.4 +/- 0.8 10(9) M-1), and very specific for GH, properties which are in agreement with the characteristics of hormonal receptors. Sea bream and mammalian GH appeared 2- and 30-fold, respectively, less potent than cold sGH2 for displacing 125I-sGH2. Tissue preparations from ovary, testis, fat, skin, cartilage, gill, blood pellet, brain, spleen, kidney, and muscle showed significant saturable binding.