Polymorphisms in the NQO1, GSTT and GSTM genes are associated with coronary heart disease and biomarkers of oxidative stress

It was hypothesized that the presence of genetic polymorphisms that decrease activity of the detoxification enzymes glutathione-S-transferase (GST) and quinone oxido-reductase (NQO1) may contribute to heart disease and affect biomarkers of coronary health and oxidative stress. Sixty-seven patients with angiographically confirmed coronary heart disease (CHD) and 63 healthy controls were genotyped for polymorphisms in the GST isoforms Mu and Theta (GSTM and GSTT respectively) and NQO1. Participants' blood levels of homocysteine (Hcy), C-reactive protein (CRP), oxidized low density lipoprotein (LDL) and total antioxidant capacity (TAOX) were measured. TAOX levels were significantly lower in women than men (P < or = 0.001) and this finding was more marked in the control group (P < or = 0.001). Hcy levels were higher in CHD patients (P=0.003 vs. control) which was mostly attributed to female patients (P=0.034 case vs. control). GSTM polymorphisms were present with greater frequencies in CHD cases with the odds ratio (OR) for GSTM equal to 3.77 vs. control. CHD patients also have an increased incidence of both GSTM and GSTT null polymorphisms (OR=5.13). In contrast, NQO1 polymorphisms were protective in CHD patients (OR=0.18 vs. control), which when stratified for genotype was due to heterozygous individuals. Significantly higher C-reactive protein levels occurred in CHD patients with lower NQO1 activity (P=0.001), however, due to the large variations in CRP levels seen in CHD patients; the clinical importance of this difference is unclear. Smokers with the GSTM null polymorphism were more likely to have CHD than non-smokers expressing the GSTM null polymorphism (OR=3.54, P=0.079). We conclude that a lack of activity in the detoxification enzymes NQO1 and GSTM, and biomarker levels are strongly associated with coronary heart disease with sex as a mitigating factor.     

Preparation of high-capacity supports containing protein G immobilized to porous silica

This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 Å pore size silica, followed by 33 mg/g for 50 Å silica and 9.3-24 mg/g for 300-4000 Å silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 μmol/m² being obtained for 4000 Å silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 Å silica.     

Early penguin fossils, plus mitochondrial genomes, calibrate avian evolution

Testing models of macroevolution, and especially the sufficiency of microevolutionary processes, requires good collaboration between molecular biologists and paleontologists. We report such a test for events around the Late Cretaceous by describing the earliest penguin fossils, analyzing complete mitochondrial genomes from an albatross, a petrel, and a loon, and describe the gradual decline of pterosaurs at the same time modern birds radiate. The penguin fossils comprise four naturally associated skeletons from the New Zealand Waipara Greensand, a Paleocene (early Tertiary) formation just above a well-known Cretaceous/Tertiary boundary site. The fossils, in a new genus (Waimanu), provide a lower estimate of 61-62 Ma for the divergence between penguins and other birds and thus establish a reliable calibration point for avian evolution. Combining fossil calibration points, DNA sequences, maximum likelihood, and Bayesian analysis, the penguin calibrations imply a radiation of modern (crown group) birds in the Late Cretaceous. This includes a conservative estimate that modern sea and shorebird lineages diverged at least by the Late Cretaceous about 74 +/- 3 Ma (Campanian). It is clear that modern birds from at least the latest Cretaceous lived at the same time as archaic birds including Hesperornis, Ichthyornis, and the diverse Enantiornithiformes. Pterosaurs, which also coexisted with early crown birds, show notable changes through the Late Cretaceous. There was a decrease in taxonomic diversity, and small- to medium-sized species disappeared well before the end of the Cretaceous. A simple reading of the fossil record might suggest competitive interactions with birds, but much more needs to be understood about pterosaur life histories. Additional fossils and molecular data are still required to help understand the role of biotic interactions in the evolution of Late Cretaceous birds and thus to test that the mechanisms of microevolution are sufficient to explain macroevolution.     

Vannella epipetala n. sp. isolated from the leaf surface of Spondias mombin (Anacardiaceae) growing in the dry forest of Costa Rica

As part of a Microbial Observatory of Caterpillars located in the Area de Conservacíon Guanacaste (ACG) in northwestern Costa Rica, we isolated a novel species of the genus Vannella associated with the food of the caterpillars of the saturniid moth Rothschildia lebeau, namely the leaves of the dry forest deciduous tree Spondias mombin (Anacardiaceae). The new species can be distinguished from other described species of the genus by the presence of a plasmalemma coated with a thickened, osmiophilic lamina containing glycostyles, and by its unusual habitat, the leaf surfaces or phylosphere of S. mombin. We further established the novelty of our isolate by sequencing its nuclear small-subunit (SSU) rRNA gene and inferring its phylogenetic position among all other currently sequenced members of the genera Vannella and Platyamoeba. Our results reveal that our isolate shares most recent common ancestry with three strains of Platyamoeba placida, the type species of the genus Platyamoeba. Despite this placement, the isolate clearly possesses glycostyles that are the hallmark of the genus Vannella. In addition to the cultured isolate, we also present a closely related sequence from a SSU rRNA gene clone library constructed from a DNA extract of leaf-wash of S. mombin with sterile water.     

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P_s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS³ in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1–3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ297631","term_id":"9931190","term_text":"AJ297631"}}AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6–8). Investigations showed that treatment of sperm with α-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9–11), sperm motility (9–13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15–17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18, 19), providing the specificity required for a potential contraceptive. Questions surrounding these particular compounds were raised when a number of side effects were evident from in vivo trials (7, 20–22); however, the design of small molecule inhibitors of GAPDS may provide a viable alternative. Its potential as a contraceptive target was supported by data from mice where GAPDS^−/− males (23) were infertile because of defects in sperm motility.Glyceraldehyde-3-phosphate dehydrogenases are tetrameric enzymes that catalyze the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (Glc-3-P) into 1,3-diphosphoglycerate in the presence of an NAD cofactor via a two-step chemical mechanism (24). The first models of substrate binding were proposed on the basis of crystal structures of the holoenzyme from lobster (25) and Bacillus stearothermophilus (26), and Moras and co-workers (25) identified two anion-binding sites postulated to correspond to those binding the C-3 phosphate group of d-Glc-3-P (P_s site) and the inorganic phosphate ion (P_i site).Structure-based design of small molecules to inhibit GAPDH is not unprecedented. GAPDH has been targeted from protozoan parasites (27–30), as the bloodstream forms rely solely on glycolysis for energy production (31, 32). A number of mammalian GAPDH structures have also been solved, including rabbit muscle (33, 34), human liver (35), and human placenta (36); however, no structures are available for sperm-specific isoforms of this enzyme.Active heterotetramers of GAPDH between different species have been reported and biochemically characterized previously, both in ratios of 2:2 and 3:1 (37–40). In this study we have successfully obtained crystals of rat recombinant GAPDS as a heterotetramer with Escherichia coli GAPDH in a 1:3 ratio. To understand the basis of inhibition of the sperm isoform by substrate analogue 3-chlorolactaldehyde, a metabolite of α-chlorohydrin, a structure was also determined in the presence of the substrate glyceraldehyde 3-phosphate. The sperm-specific structure was compared with the human placental GAPDH structure (PDB entry 1U8F; Ref. 36) to identify differences that may provide a target for the design of inhibitors specific to the GAPDS protein. The unique structural features identified offer potential candidates for further investigation as inhibitor targets.     

Bayesian Linkage Analysis of Categorical Traits for Arbitrary Pedigree Designs

Background

Pedigree studies of complex heritable diseases often feature nominal or ordinal phenotypic measurements and missing genetic marker or phenotype data.

Methodology

We have developed a Bayesian method for Linkage analysis of Ordinal and Categorical traits (LOCate) that can analyze complex genealogical structure for family groups and incorporate missing data. LOCate uses a Gibbs sampling approach to assess linkage, incorporating a simulated tempering algorithm for fast mixing. While our treatment is Bayesian, we develop a LOD (log of odds) score estimator for assessing linkage from Gibbs sampling that is highly accurate for simulated data. LOCate is applicable to linkage analysis for ordinal or nominal traits, a versatility which we demonstrate by analyzing simulated data with a nominal trait, on which LOCate outperforms LOT, an existing method which is designed for ordinal traits. We additionally demonstrate our method''s versatility by analyzing a candidate locus (D2S1788) for panic disorder in humans, in a dataset with a large amount of missing data, which LOT was unable to handle.

Conclusion

LOCate''s accuracy and applicability to both ordinal and nominal traits will prove useful to researchers interested in mapping loci for categorical traits.     

DNA transposon dynamics in populations of Daphnia pulex with and without sex

We investigate the role of recombination in transposable element (TE) proliferation in the cyclical parthenogen, Daphnia pulex. Recombination provides a mechanism by which the rate of both TE gain and loss can be accelerated, a duality that has long intrigued many biologists interested in the influence of sex on mutation accumulation. We compared TE loads among populations of D. pulex where sex occurs regularly (cyclical parthenogens or ‘sexuals’) with those in which the ability to reproduce sexually has been completely lost (obligate ‘asexuals’) for six different families of DNA transposons. Transposon display assays showed that sexuals have more TEs than asexuals, contrary to the expectations under Muller''s ratchet but consistent with the idea that sex facilitates TE spread. Sexuals also exhibit higher insertion site polymorphism among lineages, as predicted because recombination accelerates rates of loss and gain. Asexuals, however, have proportionally more singletons (loci occupied in a single isolate), which differs from previous studies where selfing and outcrossing were used as a proxy for high and low recombination. Our multi-element survey reveals that the impact of sex on TE proliferation is consistent among different Class II TE families and we discuss the genomic consequences of different reproductive strategies over long time periods.