The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function.

BACKGROUND: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. AIM: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. METHODS/RESULTS: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. CONCLUSIONS: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Growth, body composition, hormonal and metabolic status in lambs treated long-term with growth hormone.

The effect of long-term (10 weeks) treatment with growth hormone (GH) was investigated in twin lambs, one sibling being a control and the other treated with GH (0.1 mg/kg live weight per d). The lambs were fed on a concentrate-grass cube (9:1 w/w) diet at a daily rate of 40 g fresh weight/kg live weight. The average daily live-weight gain of the GH-treated lambs was 36% greater than that of the controls (307 v. 225 g/d, P less than 0.01). The carcass composition of the GH-treated lambs changed: fat content was decreased (P less than 0.01) and protein content was increased (P less than 0.05) when expressed relative to carcass dry matter. The absolute weights and the weights when expressed relative to fleece-free empty body of some muscles were significantly increased in GH-treated lambs. The mean retention times of both particulate- and liquid-phase components of the digesta were unchanged by GH treatment, when calculated for the rumen or for the entire gastrointestinal tract. The feed conversion ratio was significantly greater (P less than 0.01) in GH-treated lambs compared with controls. Nitrogen retained per g N intake was also significantly increased (P less than 0.05) by GH treatment. Plasma urea concentrations were decreased (P less than 0.05) and glucose concentrations were increased (P less than 0.01) in GH-treated lambs, whereas non-esterified fatty acid concentrations were unchanged. Plasma insulin and total insulin-like growth factor-1 concentrations progressively increased in GH-treated lambs as treatment time continued. They were significantly correlated after week 4 of treatment. Two types of hepatic GH-binding site were detected, with high and low affinities for GH. The capacities of both binding sites were significantly increased (P less than 0.05) in GH-treated lambs when expressed per unit microsomal protein but, when expressed per liver, only the capacity of the high-affinity site was increased.

Ostertagia ostertagi infection in the calf: effects of a trickle challenge on the hormonal control of digestive and metabolic function.

Metabolic effects of a trickle challenge with the equivalent of 10,000 infective Ostertagia ostertagi larvae per day were investigated in 12 calves allocated to infected, pair-fed control or ad libitum-fed control groups. Changes in hormone levels reflecting abomasal, pituitary and pancreatic function were monitored using radioimmunoassay techniques previously validated for use in cattle. A range of metabolic profile parameters and blood metabolites was also measured. Feed intake of the infected calves began to decline as blood gastrin and pepsinogen levels reached a peak. The depression in appetite recorded in this group was responsible for significant increases in plasma urea and non-esterified fatty acid levels and associated with an increase in growth hormone/insulin ratio. No significant difference in glucagon levels was recorded between groups. A decline in blood albumin values was also shown in the infected group and associated with a drop in nitrogen digestibility. A significant depression in circulating calcium levels was related to either the hypoalbuminaemia or impaired mineral absorption in the intestine. A decrease in plasma cholesterol values in the infected group was associated with changes in digestive function.

Feeding frequency for lactating cows: diurnal patterns of hormones and metabolites in peripheral blood in relation to milk-fat concentration.

1. The present paper reports the effects of dietary modifications on the diurnal pattern of concentrations of certain metabolites and hormones in the peripheral blood of lactating dairy cows. The cows were given fixed rations of hay and high-cereal concentrates in the proportions of 30:70 or 10:90 (w/w). The concentrates were given in either two or six equal meals daily; the hay was given twice daily. 2. Previous reports of the same experiment had shown that milk-fat yield and concentration were reduced by increasing the proportion of concentrates in the diet and increased by more frequent feeding of the concentrates. These changes could be explained in part by changes in rumen volatile fatty acid (VFA) proportions and mean daily concentrations of VFA, particularly propionic acid, and insulin in the peripheral blood, but these factors failed to explain all the increase in milk-fat concentration caused by more frequent feeding. 3. Analysis of blood samples taken at hourly intervals for 24 h at two stages of lactation showed that, in the cows fed six times daily, the concentrations of metabolites and hormones remained relatively constant over the day. In the cows fed twice daily, the concentrations of VFA, 3-hydroxybutyric acid and insulin all increased after both meals whereas the concentrations of glucose and growth hormone tended to fall. The concentration of non-esterified fatty acids tended to increase overnight and fall rapidly after the morning feed. The concentrations of glucagon, thyroxine and prolactin showed no clear pattern in relation to meals. The postprandial responses of propionate, insulin and growth hormone were greater with the higher concentrate diet. 4. The maximum concentration and the diurnal range of concentrations were reduced by more frequent feeding of both diets in the case of propionic acid and of the higher concentrate diet in the case of insulin, but the effects on insulin concentrations of more frequent feeding of the lower concentrate diet were smaller and not significant. The maximum concentration and the diurnal range of concentrations of growth hormone were unaffected by meal frequency. 5. It is concluded that the severity of milk-fat depression in cows fed twice daily is increased by the rapid rise in propionic acid concentration in the peripheral blood after a meal, which in turn increases insulin secretion and may be accompanied by a suppression of growth hormone release. This causes lipogenesis to be diverted towards adipose tissue at the expense of the mammary gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of intravenous bovine growth hormone or human pancreatic growth hormone-releasing factor on milk production and plasma hormones and metabolites in sheep.

Although it is well known that exogenous bovine GH (bGH) increases milk yield in ruminants it has not been possible to determine whether an increase in endogenous GH secretion has the same effect. The recent isolation of human pancreatic GH-releasing factor (hpGRF-44) has enabled this comparison of the effects of bGH and hpGRF-44 on milk production in sheep. Three pairs of Dorset ewes underwent three 4-day treatments according to a Latin square design. Treatment 1 involved: 2-hourly i.v. injections (approximately 3.0 ml) of bGH (15 micrograms/kg; 1.8 units/mg); treatment 2: 2-hourly i.v. injections (approximately 3.0 ml) of hpGRF-44 (0.6 microgram/kg); treatment 3: 2-hourly i.v. injections (3.0 ml) of the vehicle. Treatment periods were separated by 10 days. Sheep were milked twice daily and the milk was analysed for fat, protein and lactose. Blood samples (5.0 ml) were taken before and at 15, 45, 75 and 100 min after every third injection throughout the 4 days. Plasma was analysed for insulin, glucose, urea and non-esterified fatty acids (NEFA). The changes in plasma GH stimulated by hpGRF-44 were consistent and repeatable throughout the 4 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of different growth hormone-releasing factors on the concentrations of growth hormone, insulin and metabolites in the plasma of sheep maintained in positive and negative energy balance.

Three experiments were conducted to compare the ability of different preparations of growth hormone-releasing factor (GRF) to stimulate GH secretion in sheep maintained in positive and negative energy balance. In experiment 1 five sheep were injected (i.v.) with three preparations of human pancreatic GRF (hpGRF-44, hpGRF-40, hpGRF-29-NH2) and one preparation of rat hypothalamic GRF (rhGRF-29-NH2) all at 98.0 pmol/kg, or control vehicle, in a Latin square design when the animals either had free access to food or were fed half their maintenance requirements. Analysis of plasma samples, obtained before and for 150 min after injection, revealed that the reduced food intake resulted in the expected changes in body weight and circulating GH, insulin, glucose, urea and non-esterified fatty acids. The maximum post-injection concentrations of GH did not differ between either the two levels of feeding or the four GRF preparations but the mean post-injection concentration of GH was significantly higher for all GRF treatments on the restricted ration (P less than 0.001). The mean post-injection response to rhGRF-29-NH2 was less than that obtained with hpGRF-44 for sheep with food available ad libitum (P less than 0.05) and was clearly more persistent for all GRF treatments in animals fed the reduced diet (P less than 0.001). In experiment 2 the same five sheep were injected i.v. with rhGRF-29-NH2 (98.0 pmol/kg) when they had free access to food and after food had been withdrawn for 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of growth hormone release by rumen distension in female goats.

Six rumen-fistulated, overnight-fasted, female British Saanen goats were used. In preliminary experiments on two of the goats, evidence was obtained that inflation of a balloon with water in the cranial sac of the rumen was accompanied by inhibition of GH release. In a definitive series of experiments on the other four goats (one goat on 1 day = one experiment), a total of 96 experiments were carried out, 48 of which were balloon inflations with 2.5 litres water at 37 degrees C in the cranial sac of the rumen for 40 min and 48 were control experiments on alternate days. It was found that the mean plasma GH concentration of the samples taken at the end of the 40-min inflation period was significantly (P less than 0.05) lower than the mean value of the samples taken at the start of inflation. Mean control values at the start and finish of inflation were not significantly different. There were no significant differences between mean plasma insulin concentrations at the start and finish of inflation in experimental and control groups. It is concluded that distension of the cranial rumen in the goat is a signal which can inhibit the release of GH.

Effect of intravenous administration of growth hormone-releasing factor (hpGRF-44) and Tyr-D-Trp-Ala-Trp-D-Phe-NH2 on plasma hormones and metabolites in goats.

It has recently been demonstrated that human pancreatic GH-releasing factor (hpGRF-44) and Tyr-D-Trp-Ala-Trp-D-Phe-NH2 (subsequently referred to as 'the peptide') release GH from rat pituitary glands maintained in vitro and, in the former case, increase circulating GH in rats and man. The commercial importance of discovering an agent capable of specifically enhancing GH secretion in ruminants stimulated the present study which examined: the intravenous administration of both peptides on plasma GH, prolactin, insulin, glucose, urea and non-esterified fatty acids in goats and the effect of the peptide on the release of GH from sheep pituitary glands maintained in vitro. The peptide was injected into the jugular vein of goats in three different forms and at several concentrations (dispersal by shaking, 0.07 microgram/kg; 0.7 microgram/kg; ball-milled, 7.0 micrograms/kg, 70 micrograms/kg; dimethyl sulphoxide (5%), 7.0 micrograms/kg, 70 micrograms/kg). None of the treatments stimulated a significant increase in circulating GH. Nevertheless the peptide (20 micrograms/ml medium) was found to stimulate a 50-60% increase in the production of GH from sheep pituitary glands maintained in vitro. The effect of intravenously injecting hpGRF-44 (1.0 microgram/kg) was investigated in the present and absence of passive immunization with sheep anti-somatostatin immunoglobulin G (IgG) (a bolus of 600 mg, 3 h before treatment with hpGRF-44). Plasma GH was increased (P less than 0.001) within 15 min of treatment and the magnitude of the response was the same for both the immunized and non-immunized goats. A second peak was measured after approximately 75 min which was only significant (P less than 0.05) in the immunized group.(ABSTRACT TRUNCATED AT 250 WORDS)

The heterogeneity of bovine growth hormone. Extraction from the pituitary of components with different biological and immunological properties.

Bovine growth hormone (somatotropin) was extracted from anterior pituitaries and fractionated into four protein peaks (A-D) by chromatography on DEAE-Sephacel. Analysis by high-pressure liquid chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the homogeneity of the material increased from fraction A through to D. The properties of the fractions were examined in the following manner: immunological activity (radioimmunoassays for ruminant growth hormone and prolactin); growth-promoting activity (rat tibia test); lipolytic activity (release of glycerol from rat epididymal fat in the presence of dexamethasone); diabetogenic activity (rate of glucose transport in epididymal fat of hypophysectomized rats and intravenous insulin-tolerance tests in goats). None of the fractions contained immunoreactive prolactin and all were equally lipolytic. Although fraction A contained a small quantity of immunoreactive growth hormone it had no growth-promoting or diabetogenic activities. Both fractions B and C were diabetogenic and contained high concentrations of immunoreactive growth hormone, consistent with their growth-promoting activity. Although the growth-promoting activity of fraction D was higher than that of the other three fractions, it was not diabetogenic and was only weakly immunoreactive. These results for bovine growth hormone support the contention that growth hormone, as commonly extracted, is a mixture of different molecular forms and that these different metabolic properties of the hormone might be explained in terms of this heterogeneity.

Control of growth hormone release in goats: effect of vagal cooling, feeding and artificial distension of the rumen.

It was confirmed that, as in overnight-fasted castrate male sheep, plasma growth hormone (GH) levels also fell in overnight-fasted castrate male goats shortly after they were given hay the following morning and that GH levels could also fall in anticipation of being fed. A further observation was that plasma GH levels usually remained low for 2--3 hours if the goats were allowed to continue eating but that if the hay was removed after only one hour of eating the plasma GH level rose 20--50 min later. Although initial attempts to determine the role of visceral stimuli in the regulation of GH release in castrate male goats by bilateral cooling of exteriorized vagi were inconclusive because of side-effects, they drew attention to the role of rumen distension and activation of stretch receptors in the modulation of GH release. This was investigated by artificial distension of the cranial sac of the rumen with a balloon in a castrate male, overnight-fasted goat prepared with a rumen fistula. Inflation of the balloon with warm water was followed by a fall, and deflation of the balloon by a rise, in plasma GH levels. It is concluded that rumen distension and hence activation of ruminal stretch receptors is a sufficient stimulus to explain the immediate inhibition of GH release by feeding in the goat. As a result of these, and other, observations during this work, it is also suggested that unrestrained release of GH, and hence large oscillations in plasma GH level, may occur in the goat unless it is suppressed by any of several factors, which include activation of rumen stretch receptors by feeding or inflation, anticipation of being fed, stress and metabolic satiety.

A comparison of the ability of beta-galactosidase and horseradish peroxidase enzyme-antibody conjugates to detect specific antibodies.

The ability of two different enzyme-antibody conjugates to detect specific antibodies has been compared. beta-Galactosidase was conjugated to antibodies raised against rabbit Fc fragments using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Horseradish peroxidase (HRP) was conjugated to part of the same batch of antibodies using the periodate method. The beta-galactosidase and HRP labels enabled detection of approximately 8 fmoles and 4 fmoles respectively of human growth hormone (HGH) antibodies, when their enzyme activities were measured spectrophotometrically. The detection limit of the beta-galactosidase label was increased 4-fold when a fluorimetric detection system was employed.

The influence of antigen properties on the conditions required to elute antibodies from immunoadsorbents.

Immunoadsorbents were used to purify a number of antibodies. Using pH 2.0 acid conditions alone it was possible to elute antibodies raised against human growth hormone (HGH) and Fc fragments from such immunoadsorbents with 50% or better recovery of antibody activity. However, to elute antibodies raised against triiodothyronine and cortisol required 6 M guanidine HCl, pH 2.0. The avidities of the purified antibodies were similar to those of the non-purified antibodies. The purified antibodies were stable in solution at 4 degrees C for at least six months.