Evaluation of Soil Contamination by Polycyclic Aromatic Hydrocarbons in Gipuzkoa (Northern Spain)

Representative polycyclic aromatic hydrocarbons (PAHs) of low-medium molecular weight were determined using headspace solid-phase microextraction and gas chromatography with a flame ionization detector (HS-SPME-GC-FID) in ten surface soil samples from Gipuzkoa (Northern Spain). The sum of the PAHs ranged from 0.21 to 136.26 mg kg^?1. Pyrene and chrysene were the most abundant detected PAHs with an average concentration around 3.1 mg kg^?1. Pearson's correlation and PAH isomer ratios were applied to study the different origins of contamination. The results indicated that the PAH contamination in the studied area was a mixed pattern of pyrolytic and petrogenic inputs. Multivariate exploratory techniques, principal component analysis (PCA), and cluster analysis (CA) were also applied corroborating the PAH compounds patterns in the soils.     

Polymorphisms in the α4 integrin of neotropical primates: insights for binding of natural ligands and HIV-1 gp120 to the human α4β7

The α4 integrin subunit associates with β7 and β1 and plays important roles in immune function and cell trafficking. The gut-homing receptor α4β7 has been recently described as a new receptor for HIV. Here, we describe polymorphisms of ITGA4 gene in New World primates (NWP), and tested their impact on the binding to monoclonal antibodies, natural ligands (MAdCAM and VCAM), and several gp120 HIV-1 envelope proteins. Genomic DNA of NWP specimens comprising all genera of the group had their exons 5 and 6 (encoding the region of binding to the ligands studied) analyzed. The polymorphisms found were introduced into an ITGA4 cDNA clone encoding the human α4 subunit. Mutant α4 proteins were co-expressed with β7 and were tested for binding of mAbs, MAdCAM, VCAM and gp120 of HIV-1, which was compared to the wild-type (human) α4. Mutant α4 proteins harboring the K201E/I/N substitution had reduced binding of all ligands tested, including HIV-1 gp120 envelopes. The mAbs found with reduced biding included one from which a clinically-approved drug for the treatment of neurological disorders has been derived. α4 polymorphisms in other primate species may influence outcomes in the development and treatment of infectious and autoimmune diseases in humans and in non-human primates.     

Protection against tuberculosis in Eurasian wild boar vaccinated with heat-inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines.     

TGF β-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.     

Age-dependent alteration of TGF-β signalling in osteoarthritis

Osteoarthritis (OA) is a disease of articular cartilage, with aging as the main risk factor. In OA, changes in chondrocytes lead to the autolytic destruction of cartilage. Transforming growth factor-β has recently been demonstrated to signal not only via activin receptor-like kinase 5 (ALK5)-induced Smad2/3 phosphorylation, but also via ALK1-induced Smad1/5/8 phosphorylation in articular cartilage. In aging cartilage and experimental OA, the ratio ALK1/ALK5 has been found to be increased, and the expression of ALK1 is correlated with matrix metalloproteinase-13 expression. The age-dependent shift towards Smad1/5/8 signalling might trigger the differentiation of articular chondrocytes with an autolytic phenotype.     

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

A steady-state model of spreading depression predicts the importance of an unknown conductance in specific dendritic domains

Spreading depression (SD) is a pathological wave of transient neuronal inactivation. We recently reported that the characteristic sustained complete depolarization is restricted to specific cell domains where the input resistance (R(in)) first becomes negligible before achieving partial recovery, whereas in adjacent, more polarized membranes it drops by much less. The experimental study of the participating membrane channels is hindered by their mixed contribution and heterogeneous distribution. Therefore, we derived a biophysical model to analyze the conductances that replicate the subcellular profile of R(in) during SD. Systematic variation of conductance densities far beyond the ranges reported failed to fit the experimental values. Besides standard potassium, sodium, and Glu-mediated conductances, the initial opening and gradual closing of an as yet undetermined large conductance is required to account for the evolution of R(in). Potassium conductances follow in the relative contribution and their closing during the late phase is also predicted. Large intracellular potential gradients from zero to rest are readily sustained between shunted and adjacent SD-spared membranes, which remain electroregenerative. The gradients are achieved by a combination of high-conductance subcellular domains and transmembrane ion redistribution in extended but discrete dendritic domains. We conclude that the heterogeneous subcellular behavior is due to local membrane properties, some of which may be specifically activated under extreme SD conditions.