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The objective of this study was to investigate the response of sarcoplasmic proteins in bovine LM to low-voltage electrical stimulation (ES; 80 V, 35 s) after dressing and its contribution to meat tenderization at an early postmortem time. Proteome analysis showed that ES resulted in decreased (P < 0.05) phosphorylation of creatine kinase M chain, fructose bisphosphate aldolase C-A, β-enolase, and pyruvate kinase at 3 h postmortem. Zymography indicated an earlier (P < 0.05) activation of μ-calpain in ES muscles. Free lysosomal cathepsin B and L activity increased faster (P < 0.05) in ES muscles up to 24 h. Immunohistochemistry and transmission electron microscopy further indicated that lysosomal enzymes were released at an early postmortem time. Electrical stimulation also induced ultrastructural disruption of sarcomeres. In addition, ES accelerated (P < 0.05) the depletion of ATP, creatine phosphate, and glycogen, as well as a pH decline and the more preferred pH/temperature decline mode. Finally, ES accelerated meat tenderization, resulting in lesser (P < 0.05) shear force values than the control over the testing time. A possible relationship was suggested between a change in the phosphorylation of energy metabolic enzymes and the postmortem tenderization of beef. Our results suggested the possible importance of the activation of μ-calpain, phosphorylation of sarcoplasmic proteins, and release of lysosomal enzymes for ES-induced tenderization of beef muscle.

Abstract: Quick clay has been involved in most serious, large clay slides in Sweden, Norway, and Canada. This paper describes geotechnical and geophysical methods that can be used to locate and map the extent of quick clay formations. Surface resistivity measurements and four different sounding methods have been tested. The results have been compared with sensitivities determined using fall-cone tests. The investigation shows that there is a correlation between sensitivity and electrical resistivity, which can be used to discriminate between marine clays that have been leached sufficiently to possibly form quick clay and those for which the salt content remains sufficiently high to prevent this. Although the most reliable evaluation of the variation in sensitivity was obtained by the CPT with additional measurement of total penetration force, this investigation suggests that any sounding method that uses a constant rate of advance into the ground and in which the penetration force applied on the top of the rods is measured may be used for quick clay mapping.

Androstenone is one of the main compounds responsible for boar taint, and 3β-hydroxysteroid dehydrogenase (3βHSD) might be involved in its metabolism. In this study, the gene expression of 3βHSD and 17β-hydroxysteroid dehydrogenase (17βHSD) were determined by real-time PCR analysis and related to the concentrations of androstenone, testosterone, and estrone sulphate (E1S). The experiments were performed on gonadally intact male pigs classified based on high or low fat androstenone concentrations, as predetermined by HPLC, as well as on immunocastrated and surgically castrated male pigs. The male pigs with high androstenone concentrations in fat had low 3βHSD gene expression in liver and testis. Moreover, the 17βHSD gene expression in liver, but not in testis, varied negatively with fat androstenone concentrations. Immunocastrated and surgically castrated male pigs had nondetectable concentrations of fat androstenone and plasma testosterone and E1S, and the castration procedure induced a significant increase of 3βHSD and 17βHSD gene expression. The mRNA expression was generally much greater from the 3βHSD than from the 17βHSD gene. Furthermore, fat androstenone was negatively correlated with liver 3βHSD gene expression (Pearson correlation, r = −0.69; P< 0.05), and the 17βHSD gene expression in liver was negatively correlated with plasma E1S (r = −0.95; P< 0.001), indicating an important role of liver 17βHSD in the estrogen metabolism of gonadally intact male pigs. Another strong correlation was found between 3βHSD and 17βHSD gene expression in liver of the gonadally intact male pigs (r = 0.86; P< 0.01), possibly reflecting similar regulation mechanisms of these genes.

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT3receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivopackaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A VibromixTMagitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 106cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT3receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.

A study was conducted to elucidate the effects of social factors on the concentrations of boar taint substances, androstenone and skatole, in boars. The factors included dominance (social rank) and the effects of strongly tainted animals on other members of the group. Four successive replicates of 100 pigs (50 boars + 50 gilts) with an average live weight of 24 kg were randomly allocated to 10 pens of 10. Data for this study were collected during the period of 67 to 114 kg of live weight and included the repetitive recording of agonistic behavior during competitive feeding; blood sampling for determination of plasma androstenone, skatole and testosterone in boars; feces sampling for determination of skatole content; and collection of bulbourethral glands in boars, and uteri plus ovaries in gilts at slaughter, for the assessment of sexual maturity. Results show an influence of social rank on plasma concentrations of androstenone (P= .0001) and testosterone (P= .0001), the weight of the bulbourethral glands (P= .0001), and plasma skatole (P= .02). Pens were classified according to the pig with the highest concentration of androstenone in the pen into high, medium, and low maximum pens. In pens with high maximum concentrations of androstenone, the second-highest androstenone concentration (P= .0001), and the average concentration (P= .0003) in the pen were higher than those in pens with medium or low maximum concentrations of androstenone. Mean aggression level was also higher (P= .02), but pens with high maximum aggression level did not have higher mean androstenone concentration. Rank effect on androstenone was more important than aggression effect. Neither maximum androstenone concentration nor maximum aggression level in a pen was related to the pen mean stage of sexual maturity in either sex. No influences of rank, aggression, or aggression received were found on the feces skatole level, and no pheromonal communicative function was demonstrated for skatole. High androstenone concentrations did not have a suppressive effect on androstenone concentrations in other males of the group; on the contrary, the levels were increased. This may be due to a stimulating effect of androstenone and, possibly, mating activity. Consequently, in the production of boars for slaughter, strongly tainted animals should be avoided or removed and mating activity minimized. This could be facilitated by, for instance, slaughtering before sexual maturity or separate rearing of the sexes.

An intercross between wild boar and a domestic Large White pig population was used to map quantitative trait loci (QTL) for body proportions, weight of internal organs, carcass composition, and meat quality. The results concerning growth traits and fat deposition traits have been reported elsewhere. In the present study, all 200 F2animals, their parents, and their grandparents were genotyped for 236 markers. The marker genotypes were used to calculate the additive and dominance coefficients at fixed positions in the genome of each F2animal, and the trait values were regressed onto these coefficients in intervals of 1 cM. In addition, the effect of proportion of wild boar alleles was tested for each chromosome. Significant QTL effects were found for percentage lean meat and percentage lean meat plus bone in various cuts, proportion of bone in relation to lean meat in ham, muscle area, and carcass length. The significant QTL were located on chromosomes 2, 3, 4, and 8. Each QTL explained 9 to 16% of the residual variance of the traits. Gene action for most QTL was largely additive. For meat quality traits, there were no QTL that reached the significance threshold. However, the average proportion of wild boar alleles across the genome had highly significant effects on reflectance and drip loss. The results show that there are several chromosome regions with a considerable effect on carcass traits in pigs.

Two lines of pigs selected for rate of gain and backfat thickness in opposite directions were used to study the possible role of some steroid hormones for meat vs fat accretion. Both growth rate and sidefat thickness differed between lines (P⩽.001). The concentration of cytosolic protein was lower (P⩽.05) and the DNA content higher (P⩽.01) in muscle samples (M. rectus femoris) from the lean line. The glucocorticoid receptor concentration was influenced by pH and was higher in the lean line. The Cortisol concentration in blood plasma and in cytosol was lower in the lean line (P⩽.05), as was the corticosteroid binding globulin (CBG) concentration (P⩽.001). On the whole, this indicates a more pronounced glucocorticoid action in the fat line. A high positive correlation was found between CBG and Cortisol in blood. The glucocorticoid receptor concentration was correlated to meat color; a paler muscle having a lower concentration of free receptor. Muscle growth vs fat accretion in pigs may thus be partly related to the steroid hormonal status of the animals, which in turn, may well be genetically determined.

The presence of free androgen and glucocorticoid receptors was demonstrated in the rectus femorismuscle of the pig. No detectable free androgen receptors were observed in intact male pigs. Androgen receptor characteristics in castrates and gilts were related to the growth rate of the lean meat in the ham. The amount of free androgen receptor was less (P<.05) and the androgen was more tightly bound to its receptor (P<.05) in pigs with above-average ham growth. Moreover, a higher DNA content (P<.01) was also found in these pigs, giving a lower (P<.05) protein to DNA ratio. The anabolic action of androgens is likely to be mediated via androgen receptors in skeletal muscle cytosol. The amounts of free glucocorticoid receptors found were influenced by the pH of the cytosolic fraction. The relationships between cytosolic pH and both the amount of glucocorticoid receptor (positive; P<.01) and plasma levels of Cortisol (negative; P<.05) might reflect the stress to which the pigs are exposed at slaughter.

Effects of stress on concentrations of cortisol and corticosteroid-binding globulin (CBG) in blood plasma and on glucocorticoid receptor concentrations in muscle cytosol were studied in pigs representing three Halothane (Hal) genotypes (NN, Nn, nn). At 12 wk of age, animals were divided into four groups: pigs subjected to transport (5 h in a truck), pigs treated with amperozide prior to transport, pigs not transported but treated with amperozide and pigs neither transported nor given amperozide. Animals were slaughtered the week they reached 100 kg live weight (3 mo later). The Halgene showed no major influence on the variables studied except for cortisol concentrations (P= .06) measured directly after transport at 12 wk of age (NN= 66.8 nM, Nn= 61.4 nM, nn= 69.4 nM). However, the response in each Halgenotype differed, depending on whether or not the pigs had been exposed to transport. Two weeks after transport, NNpigs developed higher cortisol concentrations than untransported animals, whereas the response was reversed in nnanimals; Nnpigs showed no difference in this regard. At slaughter, the effect of transport (12 wk of age) on cortisol and CBG was still apparent. In NNpigs cortisol and CBG concentrations were elevated (P⩽ .05, P= .08, respectively), whereas concentrations tended to be lower in nnpigs (P= .17, P= .07, respectively) when compared with untransported pigs. Transported pigs had lower receptor concentrations at slaughter (P⩽ .01) than untransported pigs. However, pigs given amperozide in connection with transport had a receptor concentration comparable to that in untransported pigs. Our study shows conclusively that transport stress had long-term effects on cortisol, CBG and glucocorticoid receptor concentrations. In addition, amperozide had long-term effects on cortisol and receptor concentrations.

It is suggested that the physiological response caused by an excited membrane-bound receptor can be either local, i.e. localized close to the receptor site, or nonlocal, spreading over a large volume in the cell. A simple model is presented which shows how locality and non-locality are related to the intrinsic "efficacy" of the drug used in classical receptor theory. It is further demonstrated how the shape of the physiological response versus receptor occupancy is determined by the number of receptor sites which contribute to the physiological response at a given location in the cell. Copyright 1993, 1999 Academic Press

The binding of a new calcium sensitizer, levosimendan, to human cardiac troponin C (cTnC) is described. Fluorescence studies done on dansylated recombinant human cTnC and a site-directed mutant showed that levosimendan modulated the calcium-induced conformational change in cTnC, and revealed the role of Asp-88 in the binding of the drug to the NH2-terminal domain of cTnC. Furthermore, NMR studies performed on the NH2-terminal fragment of cTnC showed a spatial proximity between levosimendan and Met81, Met85, and Phe77 in the drug-protein complex. These data were used to build an optimized model of the drug-protein complex, in which levosimendan binds cTnC at the hydrophobic pocket of the NH2-terminal domain. The role of the binding of levosimendan to cTnC in the pharmacological action of this drug in vivo is discussed.

We have been able to assign the human catechol-O-methyltransferase gene (COMT) to chromosome 22q11.2 by using Southern blot analysis of panels of somatic cell hybrids and chromosomal in situ hybridization. Furthermore, Southern blot analysis of DNA from blood and bone marrow samples of a patient with chronic myeloid leukemia (CML), having an extra Philadelphia chromosome (Ph1) in addition to the one produced by the reciprocal translocation between chromosomes 9 and 22, showed increased COMT and BCR gene dosage as compared to DNAs originating from CML patients with only one Ph1 chromosome or from chromosomally normal individuals. Control hybridizations of the same blot with TCRG- and TCRA-specific probes showed corresponding signal intensities in all samples. A relatively frequent two-allele COMT gene RFLP (PIC = 0.37) was recognized in DNAs digested with BglI. Our gene mapping result is in concordance with that previously reported by Brahe et al. (1986), who used an autoradiozymogram assay on different somatic cell hybrids to map this gene to chromosome 22.

Abstract: We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial β-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 109 cells−1) with an average specific activity of 3942 ± 765 pg PGE2 μg−1 microsomal protein 5 min−1.

Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

The purpose of this study was to describe the temporal changes in peripheral plasma levels of testosterone and Cortisol in boars during and after heat stress. A total of 8 boars were utilized, 4 of them were exposed to 35°C, for 100 h in a climatic room, and 4 served as controls and were kept at 20 °C for 100 h in the climatic room.

Blood levels of calcitonin (CT), calcium (Ca), inorg. phosphate (P) and alkaline phosphatase (ALP) in 27 bulls of Swedish Red and White Breed (SRB) and 21 Swedish Friesian Breed (SLB) were studied. There was a significant lowering of Ca, P, ALP and CT with increasing age. The fact that the osteopetrotic syndrome is increasing with age in bulls and that the CT level in blood is decreasing with age does not support the idea that an increased CT discharge could be responsible for the osteopetrotic syndrome in bulls.

Summary Serum specimens from 161 pregnant women or newborn children and from 280 females of different age groups, obtained in the cities of Stockholm and Eskilstuna, Sweden, were tested for neutralizing antibody to rubella virus, some of them also for hemagglutination-inhibiting antibody. Lack of NT antibody in undiluted serum suggested susceptibility to rubella in 15–20% of women of child-bearing age. There was some correlation — most evident in the younger age groups — between reported history of overt rubella and presence of NT antibody. The distribution of serum titers, NT as well as HI, and the actual rarity of reports on remote attacks of the disease suggested that ignorance of childhood rubella was the prime reason for the poorer correlation in adults.

The RNlocus in pigs has a major effect on the amount of stored glycogen in white muscle and affects meat quality. The fully dominant RN−allele, associated with high glycogen content, occurs in the Hampshire breed. We have mapped the RNlocus using a large half-sib family comprising one heterozygous RN−/ rn+Hampshire boar mated to homozygous rn+/rn+Swedish Landrace × Swedish Yorkshire sows. The segregation at the RNlocus was inferred from data on glycolytic potential and residual glycogen in white muscle which both showed clear bimodal distributions. Highly significant evidence for genetic linkage was obtained against microsatellite markers on Chromosome (Chr) 15. Multipoint analysis revealed the order Sw1111-8.0 -S0088-10.6 -RN-4.8 -Sw936,Sw906(recombination estimates are given as Kosambi cM). Comparative mapping data imply that the human homolog of RNis located on Chr 2q.