Genes with expression levels correlating to drip loss prove association of their polymorphism with water holding capacity of pork

Six genes that were known to exhibit expression levels that are correlated to drip loss BVES, SLC3A2, ZDHHC5, CS, COQ9, and EGFR have been for candidate gene analysis. Based on in silico analysis SNPs were detected, confirmed by sequencing, and used for genotyping. The SNPs were genotyped in about 1,800 animals from six pig populations including commercial herds of Pietrain (PI) and German Landrace (DL), different commercial herds of Pietrain × (German Large White × German Landrace) (PIF1(a/b/c)), and one experimental F₂-population Duroc × Pietrain (DUPI). Comparative and genetic mapping established the location of BVES on SSC1, of SLC3A2 and ZDHHC5 on SSC2, of CS on SSC5, of COQ9 on SSC6 and of EGFR on SSC9, respectively, coinciding with QTL regions for carcass and meat quality traits. BVES, SLC3A2, and CS revealed association at least with drip loss and with several other measures of water holding capacity (WHC). Moreover, COQ9 and EGFR were associated with several meat quality traits such as meat color and/or thawing loss. This study reveals statistic evidence in addition to the functional relationship of these genes to WHC previously evidenced by expression analysis. This study reveals positional and genetic statistical evidence for a link of genetic variation at these loci or close to them and promotes those six candidate genes as functional and/or positional candidate genes for meat quality traits.     

Properties of several protein kinases that copurify with rat spinal cord neurofilaments

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.     

In vitro renin inhibition to prevent generation of angiotensins during determination of angiotensin I and II

To prevent in vitro generation of angiotensins, the renin inhibitor CGP 29287 (CGP) was added to blood sampling tubes. Plasma immunoreactive angiotensin (ir-ANG) I and II were simultaneously measured by radioimmunoassay after rapid and quantitative extraction from a single plasma sample on phenylsilylsilica (Bondelut PH). True plasma ANG-(1-8)octapeptide was determined after additional separation of the different angiotensins by high performance liquid chromatography. Ir-ANG II/CGP showed the known linear relationship with ANG-(1-8)octapeptide (r = 0.87, n = 23), but - in contrast to studies without addition of CGP - the y-axis intercept which presumably represents cross-reacting angiotensins other than ANG II was very small. Ir-ANG II/CGP concentrations fell below 1 fmol/ml after converting enzyme inhibition. The results suggest that CGP 29287 prevents in vitro generation of ANG I and ANG II as well as the ANG-metabolites. Ir-ANG I/CGP measured after Bondelut PH extraction of the plasma was strongly correlated with ir-ANG I obtained after blood ethanol extraction (r = 0.97, n = 23). Thus, it is now possible to measure reliably both ANG I and ANG II within the same plasma extract after a simple extraction procedure.     

Deaggregation and proteolytic modification of a galactosyltransferase of Poterioochromonas malhamensis

We isolated hybridomas that produced monoclonal antibodies specific for the UDP-galactose: sn -glycerol-3-phosphate α-D-galactosyltransferase (IFP synthase, EC 2.4.1.96), an enzyme involved in the volume regulation of Poterioochromonas malhamensis Peterfi. Western blotting of native gradient gels with the most reactive antibody S 162 revealed several immunoreactive proteins in crude homogenates suggesting the occurrence of multiple molecular mass species of the galactosyltransferase. The amount of the presumed enzyme monomer (64 kDa under native conditions) was strongly increased by a pH shift of crude homogenates from pH 8 to 6. During activation of the galactosyltransferase in the cell homogenate and also by shrinking the cells, the presumed enzyme monomer appeared to be proteolytically degraded generating stepwise products of 52 and 40 kDa. We assume that the proteolytically processed enzyme becomes highly active, but is very susceptible to further proteolytic degradation.     

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.