Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Evidence of adoption,monozygotic twinning,and low inbreeding rates in a large genetic pedigree of polar bears

Multigenerational pedigrees have been developed for free-ranging populations of many species, are frequently used to describe mating systems, and are used in studies of quantitative genetics. Here, we document the development of a 4449-individual pedigree for the Western Hudson Bay subpopulation of polar bears (Ursus maritimus), created from relationships inferred from field and genetic data collected over six generations of bears sampled between 1966 and 2011. Microsatellite genotypes for 22–25 loci were obtained for 2945 individuals, and parentage analysis was performed using the program FRANz, including additional offspring–dam associations known only from capture data. Parentage assignments for a subset of 859 individuals were confirmed using an independent medium-density set of single nucleotide polymorphisms. To account for unsampled males in our population, we performed half-sib–full-sib analysis to reconstruct males using the program COLONY, resulting in a final pedigree containing 2957 assigned maternities and 1861 assigned paternities with only one observed case of inbreeding between close relatives. During genotyping, we identified two independently captured 2-year-old males with identical genotypes at all 25 loci, showing—for the first time—a case of monozygotic twinning among polar bears. In addition, we documented six new cases of cub adoption, which we attribute to cub misidentification or misdirected maternal care by a female bereaved of her young. Importantly, none of these adoptions could be attributed to reduced female vigilance caused by immobilization to facilitate scientific handling, as has previously been suggested.     

Amino-substituted thalidomide analogs: potent inhibitors of TNF-alpha production.

Thalidomide, (1), is a known inhibitor of TNF-alpha release in LPS stimulated human PBMC. Herein we describe the TNF-alpha inhibitory activity of amino substituted analogs of thalidomide (1) and its isoindolin-1-one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors of TNF-alpha release in LPS stimulated human PBMC.     

The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I.

BACKGROUND: The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised. The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction. What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS: We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons. Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria. Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p. Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus. Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome. CONCLUSIONS: CDC9 encodes two distinct forms of DNA ligase I. The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.     

Effect of ante- and postmortem hide clipping on the microbiological quality and safety and ultimate pH value of beef carcasses in an EC-approved abattoir

Aims:  Effect of ante- and postmortem hide clipping on the microbiological quality of beef carcasses. Methods and Results:  Bovine carcasses (362) were tested for indicator micro-organisms and the presence of pathogens. Prior to slaughter, hide cleanliness of each animal was categorized on a scale of 1–5 (clean to dirty). Lowest mean aerobic colony counts (ACC) (log₁₀ 3·0 CFU cm⁻²) came from carcasses where clipping had been performed in lairage, antemortem. ACC from animals clipped online (log₁₀ 3·2 CFU cm⁻²) were significantly higher (P < 0·05) than those clipped in lairage, but comparable to those carcasses from Category 1 and 2 animals. There were no significant differences in the detection of pathogens from any of the carcass groups. Ultimate pH values for carcasses from Category 3 and 4 animals showed clipping animals in lairage, as opposed to online, resulted in a small, but significant increase (P < 0·05) in pH value (mean pH 5·66 and 5·59, respectively). Conclusions:  Hide clipping does not adversely affect microbiological quality of carcasses, although higher ultimate pH values indicate increases in antemortem stress. Significance and Impact of the Study:  Hide clipping carcasses both ante- and postmortem appears to be an effective intervention to minimize transfer of hide microflora to carcasses during slaughtering operations. Online clipping offers advantages for animal welfare and improves safety for operatives.     

Species distribution and <Emphasis Type="Italic">in vitro</Emphasis> antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

Background  

In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.