The aim of this work was to compare the oxidative processes occurring in myofibrillar proteins during meat maturation and after an in vitro exposure to different enzymic and nonenzymic oxidative systems. Myofibrils were prepared from bovine Longissimus lumborum and Diaphragma pedialis at day 1 and day 10 post-mortem. Myofibrillar protein oxidation was measured by the carbonyl content, with the 2,4-dinitrophenylhydrazine (DNPH) method, and by (thiol group) SH content with the 2,2‘-dithiobis(5-nitropyridine) (DTNP) method. Polymerization and/or fragmentation of oxidized proteins were estimated by SDS-PAGE and Western blot analysis using a polyclonal antibody to myosin. Oxidation of myofibrillar proteins is dependent upon the different metal-catalyzed oxidation (MCO) systems. The increase in carbonyl content and also the decrease in SH content of myofibrillar proteins, after maturation of 10 days, were similar to those obtained after a 1 h incubation of myofibrillar proteins in the presence of several MCO systems. Electrophoretic studies showed that myosin was the protein the most sensitive to oxidation, and to a lesser extent, troponin T. Myosin-oxidative products were also detected by Western blot analysis. Keywords: Beef; myofibrillar proteins; protein oxidation; carbonyl content; SH-content; metal-ion catalyzed oxidation system; SDS−PAGE; Western blotting

For important food crops such as wheat and rice, grain yield depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for yield and encodes an E3 RING ligase that negatively regulates grain size. Wheat (Triticum aestivum) has TaGW2 homologues in the A, B, and D genomes, and polymorphisms in TaGW2-A were associated with grain width. Here, to investigate TaGW2 function, RNA interference (RNAi) was used to down-regulate TaGW2 transcript levels. Transgenic wheat lines showed significantly decreased grain size-related dimensions compared with controls. Furthermore, TaGW2 knockdown also caused a significant reduction in endosperm cell number. These results indicate that TaGW2 regulates grain size in wheat, possibly by controlling endosperm cell number. Wheat and rice GW2 genes thus seem to have divergent functions, with rice GW2 negatively regulating grain size and TaGW2 positively regulating grain size. Analysis of transcription of TaGW2 homoeologues in developing grains suggested that TaGW2-A and -D act in both the division and late grain-filling phases. Furthermore, biochemical and molecular analyses revealed that TaGW2-A is a functional E3 RING ubiquitin ligase with nucleocytoplasmic subcellular partitioning. A functional nuclear export sequence responsible for TaGW2-A export from the nucleus to the cytosol and retention in the nucleolus was identified. Therefore, these results show that TaGW2 acts in the regulation of grain size and may provide an important tool for enhancement of grain yield.

SummaryResistance (R) genes protect plants very effectively from disease, but many of them are rapidly overcome when present in widely grown cultivars. To overcome this lack of durability, strategies that increase host resistance diversity have been proposed. Among them is the use of multilines composed of near-isogenic lines (NILs) containing different disease resistance genes. In contrast to classical R-gene introgression by recurrent backcrossing, a transgenic approach allows the development of lines with identical genetic background, differing only in a single R gene. We have used alleles of the resistance locus Pm3 in wheat, conferring race-specific resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici), to develop transgenic wheat lines overexpressing Pm3a, Pm3c, Pm3d, Pm3f or Pm3g.In field experiments, all tested transgenic lines were significantly more resistant than their respective nontransformed sister lines. The resistance level of the transgenic Pm3 lines was determined mainly by the frequency of virulence to the particular Pm3 allele in the powdery mildew population, Pm3 expression levels and most likely also allele-specific properties. We created six two-way multilines by mixing seeds of the parental line Bobwhite and transgenic Pm3a, Pm3b and Pm3d lines. The Pm3 multilines were more resistant than their components when tested in the field. This demonstrates that the difference in a single R gene is sufficient to cause host-diversity effects and that multilines of transgenic Pm3 wheat lines represent a promising strategy for an effective and sustainable use of Pm3 alleles.

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than alpha-actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.