New synthesis of (+/-)-alpha-CMBHC and its confirmation as a metabolite of alpha-tocopherol (vitamin E)

There is currently interest in the metabolism of the various compounds which make up the vitamin E family, especially with regards to the possible use of vitamin E metabolites as markers of oxidative stress and adequate vitamin E supply. A number of vitamin E metabolites have been described to date and we have recently developed a method to extract and quantitate a range of vitamin E metabolites in human urine. During the development of this method a new metabolite of alpha-tocopherol was identified, which we tentatively characterised as 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid (alpha-CMBHC).(1) Here we describe the synthesis of alpha-CMBHC as a standard and confirm that it is a metabolite of alpha-tocopherol.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Revisiting the phylogeography and demography of European badgers (Meles meles) based on broad sampling,multiple markers and simulations

Although the phylogeography of European mammals has been extensively investigated since the 1990s, many studies were limited in terms of sampling distribution, the number of molecular markers used and the analytical techniques employed, frequently leading to incomplete postglacial recolonisation scenarios. The broad-scale genetic structure of the European badger (Meles meles) is of interest as it may result from historic restriction to glacial refugia and/or recent anthropogenic impact. However, previous studies were based mostly on samples from western Europe, making it difficult to draw robust conclusions about the location of refugia, patterns of postglacial expansion and recent demography. In the present study, continent-wide sampling and analyses with multiple markers provided evidence for two glacial refugia (Iberia and southeast Europe) that contributed to the genetic variation observed in badgers in Europe today. Approximate Bayesian computation provided support for a colonisation of Scandinavia from both Iberian and southeastern refugia. In the whole of Europe, we observed a decline in genetic diversity with increasing latitude, suggesting that the reduced diversity in the peripheral populations resulted from a postglacial expansion processes. Although MSVAR v.1.3 also provided evidence for recent genetic bottlenecks in some of these peripheral populations, the simulations performed to estimate the method''s power to correctly infer the past demography of our empirical populations suggested that the timing and severity of bottlenecks could not be established with certainty. We urge caution against trying to relate demographic declines inferred using MSVAR with particular historic or climatological events.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.     

Electron crystallography - the waking beauty of structural biology

Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.