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.     

Behavioral and Psychological Characteristics of Canine Victims of Abuse

Abuse is an intentional act that causes harm to an individual. Dogs (Canis familiaris) with a known or suspected history of abuse were solicited for the study. A panel of 5 experts in canine behavior and abuse selected the dogs judged as having a certain or near certain history of being abused for inclusion in the study. Behavioral evaluations of the dogs were obtained using the Canine Behavioral Assessment and Research Questionnaire, which utilizes ordinal scales to rate either the intensity or frequency of the dog's behaviors. Sixty-nine dogs ultimately met the criteria for inclusion in the study. When compared with a convenience sample of 5,239 companion dogs, abused dogs were reported as displaying significantly higher rates of aggression and fear directed toward unfamiliar humans and dogs, excitability, hyperactivity, attachment and attention-seeking behaviors, persistent barking, and miscellaneous strange or repetitive behaviors. Delineating the behavioral and psychological characteristics of abused dogs provides the first step in identifying and distinguishing the risk factors and sequelae associated with abuse, which may inform the development of preventive and therapeutic programs for nonhuman animal abuse.     

136 Dendritic Alkyl Chains on DNA Cages: A Geometry-Dependent Inter- or Intramolecular “Handshake”

The selective association of hydrophobic sidechains is a strong determinant of protein organization. We have observed a parallel mode of assembly in DNA nanotechnology. Firstly, dendritic DNA amphiphiles (D-DNA) were synthesized (Carneiro, Aldaye, & Sleiman, 2009) comprising an addressable oligonucleotide portion and a hydrophobic alkyl dendron at the 5’ terminus. DNA amphiphiles have gathered interest recently as they can self-assemble in aqueous media to form well defined micelles while also retaining the ability to hybridize to their complement (Kwak & Herrmann, 2011; Patwa, et al. 2011) Two variations of alkyl D-DNA were hybridized to the single-stranded edges of a DNA cube (McLaughlin, et al., 2012). It was found that anisotropic organization of these hydrophobic domains on the 3D scaffold results in a new set of assembly rules, dependent on spatial orientation, number, and chemical identity of the D-DNA on the cubic structure (Edwardson. et al. 2012). When four amphiphiles are organized on one cube face, the hydrophobic residues engage in an intermolecular “handshake” between two cubes, resulting in a dimer. When eight amphiphiles are organized on the top and bottom faces of the cube, they engage in a “handshake” inside the cube. Combining the highly specific recognition of the oligonucleotide sequence with the orthogonal association of hydrophobic moieties can lead to a variety of structures with such diverse applications as membrane anchoring, cell uptake, directed hydrophobic assembly, and encapsulation and release of small molecules.     

Length variation and secondary structure of introns in the Mlc1 gene in six species of Drosophila

A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.      

A simple algorithm locates beta-strands in the amyloid fibril core of alpha-synuclein, Abeta, and tau using the amino acid sequence alone

Fibrillar inclusions are a characteristic feature of the neuropathology found in the alpha-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Familial forms of alpha-synucleinopathies have also been linked with missense mutations or gene multiplications that result in higher protein expression levels. In order to form these fibrils, the protein, alpha-synuclein (alpha-syn), must undergo a process of self-assembly in which its native state is converted from a disordered conformer into a beta-sheet-dominated form. Here, we have developed a novel polypeptide property calculator to locate and quantify relative propensities for beta-strand structure in the sequence of alpha-syn. The output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of the beta-sheet core in alpha-syn fibrils. In particular, the plot features three peaks, the largest of which is completely absent for the nonfibrillogenic protein, beta-syn. We also report similar significant correlations for the Alzheimer's disease-related proteins, Abeta and tau. A substantial region of alpha-syn is capable [corrected] of converting from its disordered conformation into a long [corrected] alpha-helical protein. We have developed the aforementioned algorithm to locate and quantify the alpha-helical hydrophobic moment in the amino acid sequence of alpha-syn. As before, the output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of alpha-helical structure in membrane bilayer-associated alpha-syn.     

Role of cryptic genes in microbial evolution

Cryptic genes are phenotypically silent DNA sequences, not normally expressed during the life cycle of an individual. They may, however, be activated in a few individuals of a large population by mutation, recombination, insertion elements, or other genetic mechanisms. A consideration of the microbial literature concerning biochemical evolution, physiology, and taxonomy provides the basis for a hypothesis of microbial adaptation and evolution by mutational activation of cryptic genes. Evidence is presented, and a mathematical model is derived, indicating that powerful and biologically important mechanisms exist to prevent the loss of cryptic genes. We propose that cryptic genes persist as a vital element of the genetic repertoire, ready for recall by mutational activation in future generations. Cryptic genes provide a versatile endogenous genetic reservoir that enhances the adaptive potential of a species by a mechanism that is independent of genetic exchange.      

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Sequence evolution and phylogenetic signal in control-region and cytochrome b sequences of rainbow fishes (Melanotaeniidae)

The nucleotide sequences of segments of the cytochrome b gene (351 bp), the tRNA(Pro) gene (49 bp), and the control region (approximately 313 bp) of mitochondrial DNA were obtained from 26 fish representing different populations and species of Melanotaenia and one species of Glossolepis, freshwater rainbow fishes confined to Australia and New Guinea. The purpose was to investigate relative rates and patterns of sequence evolution. Overall levels of divergence were similar for the cytochrome b and tRNA control-region sequences, both ranging from < 1% within subspecies to 15%-19% between genera. However, the patterns of sequence evolution differed. For the cytochrome b gene, transitions consistently exceeded transversions, the bias ranging from 4.2:1 to 2:1, depending on the level of sequence divergence. However, in the control-region sequence, a bias toward transitions (2:1) was observed only in comparisons between very similar sequences, and transversions outnumbered transitions in comparisons of divergent sequences. Graphic comparisons suggested that the control region was saturated for transitions at relatively low levels of sequence divergence but accumulated transversions at a greater rate than did the cytochrome b sequence. These distinct patterns of base substitution are associated with differences in A+T content, which is 70% for the tRNA control- region segment versus 50% for cytochrome b. A test for skewness in the distribution of lengths of random trees indicated that both segments contained phylogenetic signal. Parsimony analyses of the data from the two regions, with or without weighting schemes appropriate to the respective patterns of sequence evolution, identified the same five groupings of sequences, but the relationships among the groups differed. However, in most cases the branches uniting different combinations of groups were poorly supported, and the differences among topologies were insignificant. Considering the observed patterns of base substitution and the results of the phylogenetic analyses, we deduce that both the control region and cytochrome b are appropriate for population genetic studies but that the control region is less effective than cytochrome b for resolving relationships among divergent lineages of rainbow fishes.      

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.