Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein.

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

Diagnosis of bovine spongiform encephalopathy: a review.

Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post-mortem diagnostic examination of brains from BSE-suspected cattle as currently performed at ID-Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrP(BSE), in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days. Recently, at ID-Lelystad a new post-mortem test has been developed that enables screening of larger volumes of brain samples for PrP(BSE) within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.

Applicability of three anti-PrP peptide sera including staining of tonsils and brainstem of sheep with scrapie.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94-105, 100-111, and 223-234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized other small epitopes. ELISA and radio-immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrP(c)). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrP(Sc) and PrP27-30, extracted from the brain stem of a scrapie-affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrP(Sc) and PrP27-30 isoforms, only R521 recognised PrP(c) from a healthy sheep. The usefulness of all three anti-peptide sera in the immunohistochemical detection of PrP(Sc) in brain stem and tonsils of scrapie-affected sheep was demonstrated and compared with an established rabbit anti-PrP serum.

mRNA levels of the calpain system in longissimus muscle of young pigs during prolonged feeding of a protein-free diet.

This experiment was conducted to investigate the effects of feeding a protein-free diet on mRNA levels of the calpain system in skeletal muscle of growing pigs during a 15-d feeding trial. Twenty crossbred barrows were divided into two dietary treatments: control or protein-free diet (mean initial weight for both groups: 38.3 kg). Daily diets were provided at 2.5 times energy for maintenance (twice a day). On d 0, 3, and 14, biopsies were taken from longissimus muscle between the third and fourth ribs (d 0 and 3) and between the fourth and fifth rib (d 14). On d 15, animals were slaughtered and longissimus muscles were dissected and analyzed for calpastatin, and mu- and m-calpain activity. From biopsies, mRNA level of skeletal muscle calpain, mu- and m-calpain, and calpastatin were measured using reversed transcription PCR. Subsequently, PCR products were quantified using ELISA. Feeding the protein-free diet lowered growth rate to almost zero. Only total level of mRNA of mu-calpain on d 14 was influenced by dietary treatments, being lower for the protein-free group than for the control group (P < .05). However, proteolytic activities were not different between treatments. Total RNA concentration in longissimus muscle decreased during the experiment for both treatments, but on d 14 this was more pronounced for the protein-free than for the control group (P < .05). If mRNA levels were corrected for this change, specific mRNA level on d 14 of skeletal muscle calpain and mu-calpain were lower (P < .05) for the protein-free than for the control group. These data suggest that activity of the components of the calpain system are differentially regulated.

Comparison between 3-methylhistidine production and proteinase activity as measures of skeletal muscle breakdown in protein-deficient growing barrows.

This experiment was conducted to determine the relationship between 3-methylhistidine (3MH) production and proteinase activity in skeletal muscles of growing barrows. Barrows at 13 wk of age were randomly assigned to either control diet available on an ad libitum basis (21% of ME consisted of protein; control group), control diet fed restricted (pair-fed with barrows in protein-free group; intake-restricted group), or protein-free diet available on an ad libitum basis (protein-free group) for 14 d. During the last 3 d, blood samples were collected for determination of 3MH production rate, which is a measure of myofibrillar protein breakdown. At slaughter, two muscles were taken: masseter (M) and longissimus (L) muscles. The muscle samples were analyzed for calpastatin, mu-calpain, m-calpain, multicatalytic proteinase (MCP), cathepsin B, cathepsins B+L, and cystatins activities. Both muscles were also analyzed for amounts of DNA, RNA, total protein, and myofibrillar and sarcoplasmic proteins. Growth rate (kilograms/day) was influenced by dietary treatments (P < .05). Fractional breakdown rate (FBR, percentage/day) of skeletal muscle, as calculated from 3MH production rate (micromoles.kilogram-1.day-1), was 27% higher for the protein-free group than for the control group. However, no differences in proteinase activities were observed, except for lower MCP activity in the M muscle of the protein-free group than in that of the other groups (P < .05). In the present study, no direct relation was observed between myofibrillar protein degradation rate and proteinase activities in skeletal muscle during a protein-free feeding strategy.

Effects of dietary clenbuterol and salbutamol on meat quality in veal calves.

Sixty male calves were assigned to one of four treatments, including (1) an untreated control, (2) clenbuterol; 1·6 mg/(calf.day), (3) and (4) salbutamol: 60 and 100 mg/(calf.day). Animals were milk-fed during 24 weeks, and ß(2)-agonists (BA) treatment occurred during the last 4 weeks including a 3-day withdrawal period before slaughter. Under identical feed intake amongst groups, treatments with BA had significantly positive effects on carcass weights and dressing percentages. BA induced muscle hypertrophy as evidenced by an improved EUROP carcass classification, 1 19-24% higher m. longissimus loin eye area and higher muscle protein/DNA and protein/RNA ratios. Through a dilution effect, this might explain the lower haem content of the m. semimembranosus and m. longissimus lumborum (LL) and diminished Hunter a(∗) value (redness) of the latter muscle. A higher ultimate pH was found in the m. semitendinosus and m. psoas major of BA-treated calves. The m. longissimus thoracis glycogen content appeared to be lowered by salbutamol. No effects of BA were encountered for water-holding properties of this muscle. Significantly higher shear force values (31-45%) for treated animals were measured for the LL which were parallelled by higher initial values for the calpain-inhibiting activity of calpastatin and a decrease of the µ-calpain/calpastatin ratio. EUROP fatness of carcasses and LL intramuscular fat content tended to be lowered by both BA. Effects of clenbuterol and of the higher salbutamol dose, which on a molecular base was administered at a 70 times higher level, were roughly of a comparable order. The lower dose mostly had intermediate effects. This means that in veal calves, clenbuterol is biologically more potent than salbutamol.

Effects of treatment of dairy cows with recombinant bovine somatotropin over three or four lactations.

Jersey, Dutch Red and White, and Friesian cows were subcutaneously injected with 640 mg of recombinant bST at 28-d intervals from 87 to 115 d after calving through four successive lactations. A TMR (6.72 MJ of NEL) and 168 g of CP/kg of DM) was fed for ad libitum consumption. The bST effects per day were 3.3 kg for milk yield, 189 g for fat yield, 109 g for protein yield, 157 g for lactose yield, 4 MJ of NEL for feed intake, and -4 kg for body weight. Responses in blood parameters measured 7 d after injection were -.007 mmol/L for glucose, -1.3 mg of N/100 ml for urea, 221 mumol/L for 3-hydroxybutyrate, 59 mumol/L for NEFA, 65 ng/L for insulin, 2.8 micrograms/L for thyroxine, and 26.7 micrograms/L for somatotropin. Somatic cell count in milk was 75,000 cells/ml higher in treated cows. Concentrations of NEFA, Ca, Mg, and phosphorus were unaffected. Repeatability of the maximum response in milk yield after bST treatment was low: .2 within and .5 between lactations. Cows treated in the previous lactation had slightly more retained placentas, and birth weight of their calves was 2 kg less. No differences were observed between treated and control cows in disease incidence. Six treated cows were culled in third and fourth lactations. No indications for tissue damage, inflammation, or stress after bST injections were detected.

Marbling, intramuscular fat and meat colour of Dutch pork.

The amounts of intramuscular (i.m.) fat in the lean meat of the lumbar region of 21 slaughterpigs have been determined according to the Soxhlet method as a reference, and compared with those determined with the faster Foss-let procedure. These methods were highly correlated, over the lower range of fat concentrations (0·30-4·35%) studied. Although the Foss-Let values were on average slightly lower than the Soxhlet values, the Foss-Let procedure can be recommended for the determination of i.m. fat in pork. Furthermore, chemically determined i.m. fat was found to be significantly correlated with marbling. The sensory evaluations of pork colour were significantly correlated with each other and with the instrumentally determined colour lightness.

[Effects of long-term BST application in dairy cows]. / De effecten van langdurige BST-toediening bij melkkoeien.

The effects of administration of BST on milk production of dairy cows are summarised on the basis of a number of review papers. Then the results of a trial with forty control cows and thirty-seven cows, in which six doses of 640 mg of BST were subcutaneously injected at twenty-eight day intervals from the fourth month after calving in a third of fourth lactation are discussed. The response to administration of BST was a 3.2 kg of milk yield, 165 g for milk fat, 105 g of milk protein and 155 g lactose. Efficiency of milk production during the period of treatment was seven per cent higher in the treated cows. During the successive lactations, the response to BST in milk fat slightly declined, but similar responses were recorded for milk protein and lactose. Plasma levels of 3-hydroxybutyrate and NEFA were higher after treatment with BST. Fertility and health were not affected by this treatment. Calves from treated cows showed five per cent lower birth weight, which had no effects on viability and postnatal weight gain. Treatment with BST did not have any effect on the concentrations of calcium, magnesium, phosphorus and NEFA in milk. In the successive lactations, the somatic cell count in the milk of treated cows increased slightly more than that in controls. During the third and fourth lactation, in the period of treatment, the number and percentage of polymorphonuclear neutrophils and number of macrophages were higher in milk showing higher cell counts. More quarters of the udders of treated cows were bacteriologically infected compared to the controls before and after treatment. Small injection sites were detected on 50-60 per cent of the treated cows, which disappeared rapidly. In the third and fourth lactation, no marked differences between controls and treated cows were recorded in frequencies of diseases and culling.

[Effect of BST administration on milk production and metabolism of dairy cows in 3 consecutive years of IVO trials(Institute for Animal Husbandry Research)]. / Het effect van BST-toediening op de melkproduktie en de stofwisseling van melkkoeien gedurende drie achtereenvolgende jaren in IVO-proeven.

The effect of subcutaneous administration of recombinantly derived bovine somatotropin (somidobove) in a sustained delivery vehicle was studied in three successive lactations. After a preliminary period of three months, cows of different breeds and parities were treated during six periods of treatment of 28 days. The efficacy of BST application was equal in Jerseys (n = 35), Red and Whites (n = 54) and Friesians (n = 149); was slightly higher in cows (n = 211) than in heifers (n = 32); was higher on concentrates- (n = 105) than on roughage (n = 138) based diets and was slightly lower in a third successive lactation. After the start of treatment body tissues were mobilised for the increased milk production, however after some weeks of treatment, energy intake increased and compensated for the higher food requirements. Somatic cell counts in milk were higher in treated than they were in control cows. Calves of treated cows (n = 80) showed a five per cent lower birth weight than those of control cows (n = 80). No differences were found between treated and control cows in disease frequency and in reasons for culling. Administration of BST improved efficiency of milk production in three lactations, without adverse effects on health or culling rates of cows and without negative effects on the quality of the milk.

Lack of effect of exogenous insulin-like growth factor-I (IGF-I) on chick embryo growth rate.

Direct evidence that IGF-I has any significant effect on embryo growth is lacking. We therefore studied the effect of administration of IGF-I on the chick embryo in ovo. Five hundred ng pure IGF-I (purified from human plasma) were given to chick embryos on 2 occasions (7 and 14 d) by injection directly into the allantoic sac. Treated and control (saline injected) chicks hatched on the same day and were killed. IGF-I appeared to reach the tissues as the [35S]-sulphate uptake of treated sternal cartilage was significantly greater than that of control (P less than 0.02). However, there were no significant effects of treatment on total body weight, bone length measurements, organ (lung, liver, heart) weights, muscle DNA, RNA or protein levels. From these results we conclude that administration of exogenous IGF-I to the chick embryo at 7 and 14 d does not stimulate further growth of the chick embryo.

Somatostatin inhibits insulin-stimulated amino acid uptake into cultured rat myoblasts.

The effects of somatostatin on the acute metabolic actions of insulin on newborn rat myoblasts in culture has been examined during monolayer culture. Somatostatin significantly inhibited the insulin-stimulated uptake of [3H]leucine and [3H]amino-isobutyric acid into myoblasts but had no effect on basal (unstimulated) uptake of these two substances. The lowest concentration of somatostatin to have a significant effect was 10 micrograms/l, and this was apparent in all the experiments undertaken. The inhibitory effect of somatostatin was seen at all effective concentrations of insulin used (0.3-1 U/l). These findings lend support to the concept of an endocrine role for somatostatin in vivo and suggest that a peripheral antagonism may exist between circulating insulin and somatostatin on anabolic processes such as nutrient uptake into cells.

Effect of somatostatin and thyrotrophin-releasing hormone on the levels of growth hormone in the circulation of the chronically catheterized pig fetus in utero.

The effects of i.v. administration of thyrotrophin-releasing hormone (TRH) and of somatostatin on circulating plasma levels of porcine GH in the chronically catheterized pig fetus have been examined. Growth hormone levels increased markedly (P less than 0.01) following TRH administration, but there was no change in thyroxine levels by 1 h after treatment. Administration of somatostatin caused a significant (P less than 0.05) decrease in mean GH levels, but the response was variable between pigs. Saline administration had no significant effect on GH levels. These results suggest that the mechanisms regulating postnatal GH release are present in the fetal pig, but may not be fully developed 8-12 days before delivery.

Glucose, growth hormone, somatomedin, cortisol and ACTH changes in the plasma of unanaesthetised pig foetuses following intravenous insulin administration in utero.

Insulin tolerance tests were carried out in chronically catheterised foetal pigs. The experiment was carried out 7 days after catheterisation. The foetuses were, therefore, considered to be free from the effects of anaesthetics and stress. Under these conditions growth hormone levels were high compared with post-natal growth hormone levels but, under the favourable conditions in this study, both growth hormone and ACTH levels were lower than those found in anaesthetised or stressed foetuses. By contrast, cortisol levels were somewhat higher than those mentioned in previous reports. Somatomedin activity measured by post-natal cartilage bioassay was low. Following iv insulin administration there was a marked depression in plasma glucose (P less than 0.01), an elevation in growth hormone (P less than 0.05) and an increase in ACTH (P less than 0.01). Levels of cortisol and somatomedins did not change significantly. From these data it is concluded that insulin is an hypoglycaemic factor in the foetal pig and that, for the most part, the foetal pig pituitary responds to an insulin challenge in a similar way to the post-natal pig.

Somatomedin activity and growth hormone levels in body fluids of the fetal pig: effect of chronic hyperinsulinaemia.

Chronic hyperinsulinaemia in the presence of euglycaemia was obtained in pig fetuses between 90 and 104 days gestational age (term is 114 days) by the implantation of insulin-filled osmotic minipumps. At 104 days these fetuses were compared with both saline-implanted controls and with unoperated fetuses from the same sows. Mean plasma GH levels were the same in all three treatment groups and were much greater than in the maternal peripheral venous circulation. Levels of GH in amniotic fluid were low, and even lower levels were measured in lung fluid. Glucose and protein levels were also lower in amniotic fluid than in plasma and lower still in lung fluid. In contrast, somatomedin activity was higher in amniotic and lung fluids than in fetal plasma and, when expressed relative to protein content, was highest in lung fluid. Insulin-treated fetuses had significantly (P less than 0.05) higher levels of somatomedin activity than control fetuses, but despite this were neither longer nor heavier than control fetuses. From these data it is concluded that neither insulin nor somatomedin directly affect fetal growth.

Lack of effect of chronic hyperinsulinaemia on growth and body composition in the fetal pig.

Chronic hyperinsulinaemia in the presence of euglycaemia was obtained in pig fetuses using implanted osmotic minipumps to deliver 3 U of insulin per day over 14 days (90-104 days gestational age); term is 114 days. Total body growth (length and weight) was unaffected by insulin administration, although some changes in organ weights were observed. There was a significant retardation of lung growth (apparently as a result of operative stress) and, in the insulin-treated fetuses, an increase in liver weight which was attributable to enhanced glycogen deposition. Two of the insulin-treated fetuses appeared to have an increase in subcutaneous fat at delivery, but overall there was no statistically significant change in body fat, water, or protein content of the carcases. Growth hormone levels in all the fetuses were high, but there were no differences between treatment groups. Bioassayable plasma somatomedin activity was increased in the insulin-treated fetuses. It is proposed that this increase may be a result of insulin-induced enhancement of liver growth hormone receptors. From these data we conclude that a 14-day period of hyperinsulinaemia towards the end of gestation does not stimulate growth of the pig fetus despite producing an increase in somatomedin activity. The growth enhancement seen in offspring from diabetic mothers is probably due to increased nutrient availability rather than a direct effect of fetal insulin. Insulin does, however, significantly increase glycogen deposition in the fetus.

NMR studies of the interaction of gene-V protein of bacteriophage M13 with oligonucleotides.

This paper describes the preparation of deuterated phenylalanine ([2H7]-phenylalanine) and the isolation of phage M13 encoded gene-V protein in which this deuterated amino acid was incorporated. Using this protein spectral assignments of resonances in the aromatic region of the 1H-NMR spectrum of the gene-V protein have been made. Furthermore the interaction of the gene-V protein with the tetranucleotide d(pC-G-C-G) and the hexanucleotide d(pC-G-C-G-C-G) was investigated. From the changes in the aromatic region of the NMR spectrum occurring after binding, it is concluded that at least one phenylalanine and one tyrosine is involved in the interaction with the oligonucleotides via stacking.

A method for the determination of glutamate oxaloacetate transaminase in sperm plasma from boars and bulls.

A method is described for the determination of glutamate oxaloacetate transaminase (GOT) activity in sperm plasma from boars and bulls as a test for fertilizing ability.

A photo-CIDNP study of the interaction of oligonucleotides with gene-5 protein of bacteriophage M13.

It is shown that photo-CIDNP effects (CIDNP, chemically induced dynamic nuclear polarization) can be generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to determine the number of tyrosyl residues at the surface of the protein and to assign the resonances from the 3,5-ring protons of these residues. The DNA-binding site of the protein is investigated by formation of complexes with oligonucleotides. Complex formation leads to shifting and/or quenching of the photo-CIDNP emission signals of the surface tyrosines, implying that they are involved in DNA-protein interaction. These experiments are complemented by studying the complex formation of Lys-Tyr-Lys to poly(A).

Studies on DNA unwinding. Proton and phosphorus nuclear-magnetic-resonance studies of gene V protein from bacteriophage M13, interacting with d(pC-G-C-G).

The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C. Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction. From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast. Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure.