Annie Vénien

Antioxidant Supplementation Restores Defective Leucine Stimulation of Protein Synthesis in Skeletal Muscle from Old Rats

Aging is characterized by a progressive loss of muscle mass that could be partly explained by a defect in the anabolic effect of food intake. We previously reported that this defect resulted from a decrease in the protein synthesis response to leucine in muscles from old rats. Because aging is associated with changes in oxidative status, we hypothesized that reactive oxygen species-induced oxidative damage may be involved in the impairment of the anabolic effect of leucine with age. The present study assessed the effect of antioxidant supplementation on leucine-regulated protein metabolism in muscles from adult and old rats. Four groups of 8- and 20-mo-old male rats were supplemented or not for 7 wk with an antioxidant mixture containing rutin, vitamin E, vitamin A, zinc, and selenium. At the end of supplementation, muscle protein metabolism was examined in vitro using epitrochlearis muscles incubated with increasing leucine concentrations. In old rats, the ability of leucine to stimulate muscle protein synthesis was significantly decreased compared with adults. This defect was reversed when old rats were supplemented with antioxidants. It was not related to increased oxidative damage to 70-kDa ribosomal protein S6 kinase that is involved in amino acid signaling. These effects could be mediated through a reduction in the inflammatory state, which decreased with antioxidant supplementation. Antioxidant supplementation could benefit muscle protein metabolism during aging, but further studies are needed to determine the mechanism involved and to establish if it could be a useful nutritional tool to slow down sarcopenia with longer supplementation.

Early immunodiagnosis of bovine fascioliasis using the specific antigen f2 in a passive hemagglutination test

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for early diagnosis of infection. On experimental infection of beef calves 6-8 months of age, f2-specific antibodies were detected 2-4 weeks after inoculation and persisted at high levels during the 28 week experimental period. On natural infection of dairy calves, 6-9 months of age, allowed to graze in infected fields from April to June, 48% of the animals were positive in July although coproscopic analyses were negative. In November all the calves were HA positive but only 24% of them excreted F. hepatica eggs. Beef calves 1-3 months of age allowed to graze with their dams in infected fields continued to suckle their dams and were weakly infected according to HA results. This weak infection was not detected by coproscopical analysis. Colostral f2-specific antibodies persisted for approximately 6 months in the serum of dairy calves allowed to suckle their dams immediately after birth.

In situ thermal denaturation of myofibre sub-type proteins studied by immunohistofluorescence and synchrotron radiation FT-IR microspectroscopy.

The thermal denaturation of proteins in skeletal muscle was studied and characterised for the first time taking into account the in situ metabolic and contractile fibre types. From serial histological sections, collagen, elastin, various type I, IIa and IIx fibres and type I-IIa and IIa-IIx hybrids were identified by immunohistofluorescence. Histological sections were incubated in buffer solutions at increasing temperatures (40, 50, 60, 70 and 80°C). Protein secondary structure was investigated by synchrotron radiation FT-IR microspectroscopy on connective tissue and in muscle fibres rigorously identified for sub-type. Whatever the target protein components, increasing temperature resulted in a decrease in α-helix secondary structure and an increase in β-sheet structure. This phenomenon was more pronounced for intracellular proteins than for connective tissue. Although hybrid fibres were generally somewhat less sensitive to unfolding than the pure types, the amplitude of the thermal denaturation of intracellular proteins was practically independent of fibre type.

Differentiation of gelatins using polyclonal antibodies raised against tyrosylated bovine and porcine gelatins.

Abstract Gelatin is obtained from bones and hides/skin, mainly from cows and pigs using alkaline or acidic processes. The use of bovine gelatin in feed, food, and pharmaceutical products has been restricted by regulatory authorities as a consequence of the outbreak of bovine spongiform encephalopathy (BSE). On the other hand, some religions ban the porcine gelatin for human consumption. Thus, there is a need for methods able to control the species origin of gelatins. The large similarity in structure of gelatins from different origins has made unsuccessful their differentiation by physicochemical methods. Moreover, the development of immunochemical methods has been hampered by the poor immunogenicity of gelatins. We obtained high titers antibodies upon immunization of rabbits with tyrosylated bovine and porcine gelatins. Using indirect and competitive indirect ELISAs we observed large differences in titers and specificity among rabbits and during the course of immunization. Some of the antisera were not sensitive to the species origin of raw material or to the process used for gelatin production and could be used for gelatin quantitation in food. Other antisera detected the porcine acidic gelatins with 10‐ to 30‐fold higher sensitivity than their bovine counterparts and could be used for the differentiation of the species origin of gelatins. Lastly, other antisera were highly sensitive to subtle changes in conformation of gelatins obtained by alkaline or acidic processes such as a 1,000‐fold higher reactivity of bovine acid hide gelatin compared to that of its limed counterpart or a 30,000‐fold higher reactivity of porcine acid bone compared to that of its limed counterpart; such antisera could be used to monitor the process induced structural changes of collagen during its transformation to gelatin.

Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle.

Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12-70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of approximately 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80 degrees C and pH 5-12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.

An improved passive hemagglutination test for the serological diagnosis of bovine fascioliasis using the specific antigen f2.

Optimum conditions for coupling the specific antigen f2 of Fasciola hepatica to sheep red cells and for the preparation of control cells coated with an unrelated protein are described. With a careful selection of donor sheep for erythrocytes, the problem of anti-species antibodies in bovine sera has been overcome and results may be obtained within 1 h as only one step is required in the assay system. Incorporation of a concentration of 25 mM CaCl2 in the diluent achieved increased stability of the agglutinates and enabled a more precise estimation of the end-point to be made. Analyses performed on bovine sera obtained at slaughter provided results in good agreement with the presence of flukes or fascioliasis lesions in livers at routine slaughter inspection. The developed assay is simpler, faster, more sensitive (P less than 0.01) and has a cut-off between populations of negative sera more easy to define than a recently marketed enzyme-linked immunosorbent assay.

Protein Matrix Involved in the Lipid Retention of Foie Gras during Cooking: A Multimodal Hyperspectral Imaging Study

Denaturation of the protein matrix during heat treatment of duck foie gras was studied in relationship to the amount of fat loss during cooking. A low fat loss group was compared with a high fat loss group by histochemistry, FT-IR, and synchrotron UV microspectroscopy combination to characterize their protein matrix at different scales. After cooking, the high fat loss group showed higher densification of its matrix, higher ultraviolet tyrosine autofluorescence, and an infrared shift of the amide I band. These results revealed a higher level of protein denaturation and aggregation during cooking in high fat loss than in low fat loss foie gras. In addition, the fluorescence and infrared responses of the raw tissue revealed differences according to the level of fat losses after cooking. These findings highlight the importance of the supramolecular state of the protein matrix in determining the fat loss of foie gras.

Hyperspectral deep ultraviolet autofluorescence of muscle fibers is affected by postmortem changes.

After slaughter, muscle cells undergo biochemical and physicochemical changes that may affect their autofluorescence characteristics. The autofluorescent response of different rat extensor digitorum longus (EDL) and soleus muscle fiber types was investigated by deep ultraviolet (UV) synchrotron microspectroscopy immediately after animal sacrifice and after 24 h of storage in a moist chamber at 20 °C. The glycogen content decreased from 23 to 18 μmol/g of fresh muscle in 24 h postmortem. Following a 275 nm excitation wavelength, the spectral muscle fiber autofluorescence response showed discrimination depending upon postmortem time (t0 versus t24 h) on both muscles at 346 and 302 nm and, to a lesser extent, at 408 and 325 nm. Taken individually, all fiber types were discriminated but with variable accuracy, with type IIA showing better separation of t0/t24 h than other fiber types. These results suggest the usefulness of the autofluorescent response of muscle cells for rapid meat-aging characterization.

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Relationship between histochemical, structural characteristics and oxidative stability of rhea limb muscles

Histochemical and structural characteristics were investigated in Gastrocnemius pars interna (GN) and Iliofiburalis (IF) limb muscles of Rhea americana. The average myofibre area cross-section was greater in GN than IF muscle (p<0.001), whereas the fibre density per section was higher in IF than GN muscle. The only type of myofibre found in both the rhea limb muscles analysed in this study was fast-twitch oxidative-glycolytic fibres (FOG). Immunolabelling analysis and ultrastructural observation of myofibres confirmed the contractile and metabolic characteristics of rhea myofibres, revealing the absolute fast isoform of myosin heavy chain and the abundance of glycogen and mitochondria inside the cells, mainly in IF muscle. These findings converged with previous results on the biochemical and physicochemical characteristics of rhea meat to provide further evidence that myofibre composition substantially influences the oxidative reactions of the muscle and therefore the meat quality, but more in-depth examination is needed to establish the links between myofibre characteristics, myofibre glycogen concentration and meat stability during storage.