Genetic structure and signature of population decrease in the critically endangered freshwater cyprinid <Emphasis Type="Italic">Chondrostoma lusitanicum</Emphasis>

The endemic and critically endangered cyprinid Chondrostoma lusitanicum has a very restricted distribution range. In order to estimate genetic diversity, characterize population structure and infer the demographic history, we examined six microsatellite loci and cytochrome b (mtDNA) sequences from samples taken throughout C. lusitanicum’s geographical range. Estimates of genetic diversity were low in all samples (average He < 0.35). The microsatellite data pointed to a major difference between northern (Samarra and Tejo drainages) and southern (Sado and Sines drainages) samples. This separation was not so clear with mtDNA, since one sample from the Tejo drainage grouped with the southern samples. This could be related with ancestral polymorphism or with admixture events between northern and southern sites during the late Pleistocene. Nevertheless, both markers indicate high levels of population differentiation in the north (for microsatellites F _ST >  0.23; and for mtDNA Φ_ST > 0.74) and lower levels in the south (F _ST < 0.05; Φ_ST < 0.40). With microsatellites we detected strong signals of a recent population decrease in effective size, by more than one order of magnitude, starting in the last centuries. This is consistent with field observations reporting a severe anthropogenic-driven population decline in the last decades. On the contrary mtDNA suggested a much older expansion. Overall, these results suggest that the distribution of genetic diversity in C. lusitanicum is the result of both ancient events related with drainage system formation, and recent human activities. The potential effect of population substructure generating genetic patterns similar to a population decrease is discussed, as well as the implications of these results for the conservation of C. lusitanicum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mitochondrial function in Parkinson's disease cybrids containing an nt2 neuron-like nuclear background

Mitochondria likely play a role in Parkinson's disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP+ exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD.     

The Gammaherpesvirus m2 protein manipulates the Fyn/Vav pathway through a multidocking mechanism of assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.     

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the carotenoid biosynthesis pathway in photosynthetic eukaryotes and provide information about how they have diversified and acquired new functions in the diatoms.     

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2R)-2-(α-d-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2S)-2-(1-O-α-d-mannopyranosyl)propionate, 2-(1-O-α-d-mannopyranosyl)acetate, (2R)-2-(1-O-α-d-glucopyranosyl)glycerate and 1-O-(2-glyceryl)-α-d-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di-myo-inositol-1,3′-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.     

Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Weight gain and cardiovascular risk factors during smoking cessation with bupropion or nicotine.

Weight gain is frequent after smoking cessation, and may limit patient's will to quit and long-term success. Nicotine and bupropion are effective drugs for smoking withdrawal. However, their influence on weight gain, insulin resistance and other cardiovascular risk factors, as well as possible differences in obese and lean subjects, have not been fully evaluated. We randomised 25 lean and 25 obese smokers to receive either bupropion or nicotine patches. Clinical evaluation and lipid profile were performed at baseline and after treatment. Insulin resistance was also assessed at the end. Weight, BMI, waist-to-hip ratio, and diastolic blood pressure increased (p < 0.005), whereas lipid profile improved (p < 0.001) after smoking cessation independently of obesity at baseline or drug used. Obese patients had higher insulin resistance at the end (p < 0.05) regardless of drug used. Weight gain was inversely related to age (beta= - 0.125, R = 0.38, p = 0.046), and insulin resistance was related to obesity at baseline (beta = 0.85, R = 0.46, p = 0.02). In conclusion, weight gain after smoking cessation is not dependent on obesity or drug taken. A beneficial lipid profile is achieved after quitting smoking with either bupropion or nicotine patch in both obese and lean subjects.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Seasonal variations in upwelling and in the grazing impact of copepods on phytoplankton off A Coruña (Galicia, NW Spain)

The impact of grazing by copepods on phytoplankton was studied during a seasonal cycle on the Galician shelf off A Coruña (NW Spain). Grazing was estimated by measuring the chlorophyll gut content and the evacuation rates of copepods from three mesh-size classes: 200-500 (small), 500-1000 (medium), and 1000-2000 μm (large). Between February 1996 and June 1997, monthly measurements of water temperature, chlorophyll concentration, primary production rates, and copepod abundance, chlorophyll gut content, and evacuation rates were taken at an 80-m-deep, fixed shelf station. Additionally, the same measurements were collected daily during two bloom events in March and in July 1996. Small copepods were the most abundant through the seasonal cycle. The highest grazing impact, however, was due to the medium and large size classes. Grazing by small copepods exceeded grazing by medium and large copepods only during phytoplankton spring blooms. The impact of copepod grazing (considering all size fractions) was generally low. On average, 2% of the phytoplankton biomass and 6% of the primary production were removed daily by the copepod community. Maximum grazing impact values (9% of the phytoplankton biomass and 39% of the primary production) were found in mid-summer. These results suggest that most of the phytoplankton biomass would escape direct copepod grazing in this upwelling area.     

Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.