Animal board invited review: advances in proteomics for animal and food sciences.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Changes in protein abundance between tender and tough meat from bovine longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis.

The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4 tough samples, based on shear force measurements at 7 d postmortem, from young Norwegian red (NRF) bulls, taken at 1 h postmortem. A number of the proteins which have previously been related to tenderness were found to change in abundance between tender and tough samples, both in iTRAQ (P < 0.1) and 2-DE analysis (P < 0.05). Furthermore, 3 proteins that have not previously been related to tenderness were found to change significantly in abundance between tender and tough meat samples in the present study. These include proteins related to control of flux through the tricarboxylate cycle [2-oxoglutarate dehydrogenase complex component E2 (OGDC-E2)], apoptosis (galectin-1) and regulatory role in the release of Ca(2+) from intracellular stores (annexin A6). Even though the overlap in significantly changing proteins was relatively low between iTRAQ and 2-DE analysis, certain proteins predicted to have the same function were found in both analyses and showed similar changes between the groups, such as structural proteins and proteins related to apoptosis and energy metabolism.

Relationship between muscle microstructure, the calpain system, and shear force in bovine longissimus dorsi muscle.

The objective of this study was to examine the occurrence of microstructural changes in aged LM from Norwegian Red cattle, and to investigate how these changes relate to pH decline, calpain and calpastatin activities, and tenderness (Warner-Bratzler shear force; WBSF). Samples of the LM from 403 Norwegian Red dual-purpose bulls were collected over a 4-yr period and analyzed for muscle pH, protease activity, and WBSF. Microstructural analysis of fiber-fiber detachment, muscle fiber-perimysium detachment, contracted muscle fibers, and fractured muscle fibers were performed on a subset of 50 animals. The occurrence of fractured muscle fibers was negatively correlated with WBSF (r = -0.33, P < 0.05) and calpastatin activity (r = -0.51, P < 0.01) and was positively correlated with the ratio of µ-calpain:calpastatin activity (r = 0.66, P < 0.001), strongly indicating that these fractures are important in the development of meat tenderness and that they are a result of calpain-mediated proteolysis. In contrast, detachments between individual muscle fibers and between muscle fibers and the perimysium did not play an important role in determining variation in LM tenderness in these animals. Moreover, both pH decline and what is considered a normal ultimate pH range in beef were positively correlated with calpastatin activity and WBSF and were negatively correlated with fractured muscle fibers. Thus, an accelerated muscle pH decline and a low ultimate pH seem to increase calpain-mediated proteolysis, causing fractures in muscle fibers and thereby giving rise to more tender meat.

Peroxiredoxin-6--a potential protein marker for meat tenderness in bovine longissimus thoracis muscle.

The muscle sarcoplasmic proteins from bovine M. longissimus thoracis muscle were studied using proteomics to identify possible protein markers for meat tenderness. This study included 3 experiments: A1, A2, and B. From a collection of biopsies from the bovine M. longissimus thoracis muscle, excised 4 d before slaughter from 178 Norwegian Red young bulls, 26 biopsies were studied in Exp. A1. Based on Warner-Bratzler shear force (WBSF) values at 7 d postmortem, the biopsies were separated into a tender and a tough group of 13 bulls each and analyzed by 2-dimensional gel electrophoresis (2-DE) and Western blotting. The 2-DE experiments identified 4 different proteins: stress-70 protein, protein DJ-1, peroxiredoxin-6, and malate dehydrogenase, which were different in abundance in the tender and tough groups. However, only peroxiredoxin-6 was confirmed by quantification from Western blots. Peroxiredoxin-6 is an antioxidant enzyme that plays a role in protecting cells from oxidative stress. Peroxiredoxin-6 was identified through 3 spots of the same molecular weight, but with different pI on the Western blots. Only one of the spots was more abundant in the biopsies from the tender group. In Exp. A2, samples collected 1 h postmortem from the same animals and muscles as in Exp. A1 were analyzed by Western blotting. In these postmortem samples, the same spot from peroxiredoxin-6 as in Exp. A1 was more abundant in the tender group. In addition, one of the other peroxiredoxin-6 spots was also more abundant in the tender group. To verify the results from Exp. A, biopsies from 14 additional animals were analyzed in Exp. B by Western blotting against stress-70 protein, protein DJ-1, peroxiredoxin-6, and malate dehydrogenase. No significant differences between the tough and tender groups could be observed in these biopsies. However, for peroxiredoxin-6, the tendencies pointed in the same direction as in Exp. A. In conclusion, peroxiredoxin-6 might be a potential protein marker for meat tenderness detectable in biopsies and in samples collected shortly after slaughter. However, more animals are needed to verify the findings in the present study.

Reduction of leptin gene expression by dietary polyunsaturated fatty acids.

Supplementation with n-3 polyunsaturated fatty acids (PUFA) for 6 weeks did not alter plasma leptin concentrations in male smokers. Changes in dietary intake of saturated fatty acids (FA) correlated positively, whereas changes in the intake of PUFA correlated negatively to changes in plasma leptin levels. A 3-week n-3 PUFA-enriched diet, as compared with a 3-week lard-enriched diet, induced lower plasma leptin concentration and reduced leptin mRNA expression in rat epididymal adipose tissue. In the human trophoblast cell line (BeWo), n-3 PUFA had a dose- and time-dependent effect on leptin expression. One mM of eicosapentaenoic acid or docosahexaenoic acid (DHA) reduced leptin expression by 71% and 78%, respectively, as compared with control, after 72 h. There was no effect on expression of the signal transducing form of the leptin receptor. In BeWo cells transfected with the human leptin promoter, we found that n-3 PUFA reduced leptin promoter activity; in contrast saturated and monounsaturated FA had no effect on leptin promoter activity. The transcription factors peroxysomal proliferator activated receptor gamma and sterol regulatory element binding protein-1 mRNAs were reduced after incubation with n-3 PUFA, whereas the expression of CCAAT/enhancer binding protein alpha was unchanged. DHA-reduced leptin expression was abolished in BeWo cells grown in cholesterol-free medium. In conclusion, n-3 FA decreased leptin gene expression both in vivo and in vitro. The direct effects of PUFA on leptin promoter activity indicate a specific regulatory action of FA on leptin expression.

[Leptin--a fatty tissue hormone with many functions]. / Leptin--fettvevshormon med mange funksjoner.

The importance of genetic factors in obesity is under investigation. During the last few years, our understanding of the regulation of body weight in mammals has broadened to include several new proteins based on the cloning of genes from rodent mutants and characterization of their effects on energy stores and metabolism. Since its discovery in 1994, leptin has been acknowledged as an adipocyte-derived signal molecule, able to limit food intake and increase energy expenditure by interacting with specific leptin receptors located in the central nervous system and in peripheral tissues. Leptin is also important for growth, reproduction and neuroendocrine signalling. Although leptin is mainly synthesized in adipocytes, it is also expressed in the placenta, epithelium of the stomach and breast glands and, under certain conditions, in skeletal muscles. The importance of leptin is demonstrated by the discovery of mutations in the genes encoding leptin and its receptor among some subjects with morbid obesity and infertility. Plasma concentration of leptin should be measured in obese infertile adolescents since replacement could be curative among individuals with leptin deficiency.

Developmental, stress and ABA modulation of mRNA levels for bZip transcription factors and Vp1 in barley embryos and embryo-derived suspension cultures.

The transcription factors VP1 (Viviparous-1), EmBP-1 (Em-binding protein 1) and OSBZ8, originally cloned and analysed in various monocot species, have been implicated in the regulation of the Lea (late embryogenesis-abundant) group 1 genes. We have investigated the modulation of the levels of these mRNAs in barley during embryogenesis, and in developing embryos subjected to various kinds of osmotic stress. The accumulation of mRNA for VP1 and EmBP-1 transcription factors, using cDNAs cloned from barley, starts at 10 and 15 days after anthesis, respectively, whereas Lea B19 mRNA levels are very low or undetectable until 25 days after anthesis during normal development. The EmBP-1 mRNA is predominantly induced in mannitol-stressed immature embryos. Vp1 mRNA was not significantly modulated by ABA, salt or mannitol. Inhibition of ABA biosynthesis by norflurazon showed that the induction of both Vp1 and EmBP-1 mRNAs was ABA-independent. In embryo-derived suspension-cultured cells, neither of the two transcripts would be induced by ABA or osmotic stress, although both OSBZ8 and one member of the Lea B19 family was up-regulated by ABA. Electrophoretic mobility shift assays using a Lea B19.1 probe with an ABRE (abscisic acid-responsive element) similar to that which binds EmBP-1 and OSBZ8 in the wheat and rice Em promoters show that the binding activity is increased by ABA and osmotic stress. Taken together, these data show that both VP1 and EmBP-1 are involved in embryo-specific signal transduction pathways, that they are differentially regulated at the mRNA level, and that EmBP-1 can be induced by osmotic stress independently of any increase in endogenous ABA. The difference in mRNA regulation patterns of OSBZ8 and EmBP-1 may suggest that they are involved in different signal transduction pathways in connection with osmotic stress/ABA regulation of Lea genes.

Evolution of the Group 1 late embryogenesis abundant (Lea) genes: analysis of the Lea B19 gene family in barley.

The highly conserved Group 1 late embryogenesis abundant (Lea) genes are present in the genome of most plants as a gene family. Family members are conserved along the entire coding region, especially within the extremely hydrophilic internal 20 amino acid motif, which may be repeated. Cloning of Lea Group 1 genes from barley resulted in the characterization of four family members named B19.1, B19.1b, B19.3 and B19.4 after the presence of this motif 1, 1, 3 and 4 times in each gene, respectively. We present here the results of comparative and evolutionary analyses of the barley Group 1 Lea gene family (B19). The most important findings resulting from this work are (1) the tandem clustering of B19.3 and B19.4, (2) the spatial conservation of putative regulatory elements between the four B19 gene promoters, (3) the determination of the relative 'age' of the gene family members and (4) the 'chimeric' nature of B19.3 and B19.4, reflecting a cross-over or gene-conversion event in their common ancestor. We also show evidence for the presence of one or two additional expressed B19 genes in the barley genome. Based on our results, we present a model for the evolution of the family in barley, including the 20 amino acid motif. Comparisons of the relatedness between the barley family and all other known Group 1 Lea genes using maximum parsimony (PAUP) analysis provide evidence for the time of divergence between the barley genes containing the internal motif as a single copy and as a repeat. The PAUP analyses also provide evidence for independent duplications of Group 1 genes containing the internal motif as a repeat in both monocots and dicots.

Another Lea B19 gene (Group1 Lea) from barley containing a single 20 amino acid hydrophilic motif.

A new member of the Lea B19 gene family from barley, termed B19.1b, has been isolated and characterized. The coding region of B19.1b is highly similar to the other members of the B19 family (Espelund et al., Plant J 2 (1992) 241-252) and contains only one copy of the hydrophilic sequence found as a repeat in two other B19 genes. Like the other B19 genes, B19.1b is only expressed in embryos. The transcript appears during development at 25 days after anthesis and remains at a high level throughout embryogenesis. In immature embryos the B19.1b mRNA can be induced by abscisic acid, salt and mannitol.