Effect of phytase supplementation on phosphorus digestibility in low-phytate barley fed to finishing pigs

Forty crossbred barrows (Camborough 15 Line female×Canabred sire) weighing an average of 79.6±8.0?kg were used in a factorial design experiment (5 barleys×2 enzyme levels) conducted to determine the effects of phytase supplementation on nutrient digestibility in low-phytate barleys fed to finishing pigs. The pigs were assigned to one of 10 dietary treatments comprised of a normal 2-rowed, hulled variety of barley (CDC Fleet, 0.26% phytate) or 2 low-phytate hulled genotypes designated as LP422 (0.14% phytate) and LP635 (0.09% phytate). A normal, hulless barley (CDC Dawn, 0.26% phytate) and a hulless genotype designated as LP422H (0.14% phytate) were also included. All barleys were fed with and without phytase (Natuphos 5000 FTU/kg). The diets fed contained 98% barley, 0.5% vitamin premix, 0.5% trace mineral premix, 0.5% NaCl and 0.5% chromic oxide but no supplemental phosphorus. The marked feed was provided for a 7-day acclimatization period, followed by a 3-day faecal collection. In the absence of phytase, phosphorus digestibility increased substantially (P<0.05) as the level of phytate in the barley declined. For the hulled varieties, phosphorus digestibility increased from 12.9% for the normal barley (0.26% phytate) to 35.3 and 39.8% for the two low-phytate genotypes (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 9.2% for the normal barley (0.26% phytate) to 34.7% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In contrast, when phytase was added to the diet, there was little difference in phosphorus digestibility between pigs fed normal barley and those fed the low-phytate genotypes (significant barley×enzyme interaction, P=0.01). For the hulled varieties, phosphorus digestibility was 50.1% for the barley with the normal level of phytate (0.26% phytate) compared with 51.1 and 52.4% for the varieties with 54 and 35% of the normal level of phytate (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 47.1% for the normal barley (0.26% phytate) to 54.4% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In conclusion, both supplementation with phytase and selection for low-phytate genotypes of barley were successful in increasing the digestibility of phosphorus for pigs. Unfortunately, the effects did not appear to be additive. Whether or not swine producers will choose low-phytate barley or supplementation with phytase as a means to improve phosphorus utilization, will likely depend on the yield potential of low-phytate barley and the additional costs associated with supplementation with phytase.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

WD repeat domains target dictyostelium myosin heavy chain kinases by binding directly to myosin filaments

Myosin heavy chain kinase (MHCK) A phosphorylates mapped sites at the C-terminal tail of Dictyostelium myosin II heavy chain, driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an N-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD repeat domain at the C terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD repeat domains. In the current study, we explored the role of the WD repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially expressed truncations of these kinases either with or without their WD repeat domains. We demonstrate that substrate targeting is a conserved function of the WD repeat domains of both MHCK A and MHCK B and that this targeting is specific for Dictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct binding of the WD repeat domains to the myosin substrate. To our knowledge, this is the first report of WD repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for enzyme targeting in other systems.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.     

Asp563 of the horizontal helix of subunit NuoL is involved in proton translocation by the respiratory complex I

The NADH:ubiquinone oxidoreductase couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. It contains a 110 Å long helix running parallel to the membrane part of the complex. Deletion of the helix resulted in a reduced H⁺/e^? stoichiometry indicating its direct involvement in proton translocation. Here, we show that the mutation of the conserved amino acid D563^L, which is part of the horizontal helix of the Escherichia coli complex I, leads to a reduced H⁺/e^? stoichiometry. It is discussed that this residue is involved in transferring protons to the membranous proton translocation site.