Physiological role of phospholipid hydroperoxide glutathione peroxidase in mammals

The redox enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as one of the most significant selenoenzymes in mammals, corroborated by early embryonic lethality of PHGPx null mice. PHGPx is one of five selenium-dependent glutathione peroxidases and the second glutathione peroxidase to be discovered in 1982. PHGPx has a particular position within this family owing to its peculiar structural and catalytic properties, its multifaceted roles during male gametogenesis, and its necessity for early mouse development. Interestingly, mice devoid of endogenous glutathione die at the same embryonic stage as PHGPx-deficient mice compatible with the hypothesis that a similar phenotype of embryonic lethality may be provoked by PHGPx deficiency and lack of its reducing substrate glutathione. Various gain- and loss-of-function approaches in mice have provided some insights into the physiological functions of PHGPx. These include a protective role for PHGPx in response to irradiation, increased resistance of transgenic PHGPx mice to toxin-induced liver damage, a putative role in various steps of embryogenesis, and a contribution to sperm chromatin condensation. The expression of three forms of PHGPx and early embryonic lethality call for more specific studies, such as tissue-specific disruption of PHGPx, to precisely understand the contribution of PHGPx to mammalian physiology and under pathological conditions.     

Evidence for panmixia despite barriers to gene flow in the southern African endemic, <Emphasis Type="Italic">Caffrogobius caffer</Emphasis> (Teleostei: Gobiidae)

Background  

Oceanography and life-history characteristics are known to influence the genetic structure of marine species, however the relative role that these factors play in shaping phylogeographic patterns remains unresolved. The population genetic structure of the endemic, rocky shore dwelling Caffrogobius caffer was investigated across a known major oceanographic barrier, Cape Agulhas, which has previously been shown to strongly influence genetic structuring of South African rocky shore and intertidal marine organisms. Given the variable and dynamic oceanographical features of the region, we further sought to test how the pattern of gene flow between C. caffer populations is affected by the dominant Agulhas and Benguela current systems of the southern oceans.     

Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy

Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Previously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single (13)C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5-7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only (1)H, but for the first time also partial (13)C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC- and gHMQC-NMR experiments.     

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.     

Extensive genetic diversity, unique population structure and evidence of genetic exchange in the sexually transmitted parasite Trichomonas vaginalis

Background

Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite''s genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes.

Methodology/Principal Findings

Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages.

Conclusions/Significance

Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.     

Campylobacter insulaenigrae isolates from northern elephant seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42 degrees C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

Natural selection and the evolution of neutralism

The efficiency of evolutionary search increases as the density of acceptable proteins in a protein space increases. Populations caught in regions whose density is too low to support evolution can be pulled into high density regions by hitchhiking selection. As they move into such regions, the action of natural selection becomes more effective, yet these populations will satisfy conditions which lead to predictions made by neutral, so-called non-Darwinian models.     

Microbial production and uptake of nitric oxide in soil

Abstract Fluxes of NO from three different soils have been studied by a flow-through system in the laboratory as a function of gas flow rate, of NO mixing ratio, and of incubation conditions. The dependence of net NO fluxes on gas flow rates and on NO mixing ratios could be described by a simple model of simultaneous NO production and NO uptake. By using this model, rates of gross NO production, rate constants of NO uptake, and NO compensation mixing ratios could be determined as function of the soil type and the incubation condition. Gross NO production rates were one to two orders of magnitude larger under anaerobic than under aerobic conditions. NO uptake rate constants, on the other hand, were only 5–8 times larger so that the compensation mixing ratios of NO were in a range of about 1600–2200 ppbv under anaerobic and of about 50–600 ppbv under aerobic conditions. The different soils exhibited similar NO uptake rate constants, but the gross NO production rate and compensation mixing ratio was significantly higher in an acidic (pH 4.7) sandy clay loam than in other less acidic soils. Experiments with autoclaved soil samples showed that both NO production and NO uptake was mainly due to microbial metabolism.