Modelling of full-length human alpha4beta2 nicotinic receptor by fragmental approach and analysis of its binding modes

The objective of the study was to generate a full-length model for the heteropentameric structure of human α4β2 nicotinic receptor. The monomers structure was derived using a fragmental approach and the pentamer was assembled by protein-protein docking. The reliability of the model was assessed docking a representative set of known nicotinic ligands. Docking results unveiled that the ligand affinity depends on key interactions that the ligand’s charged moiety realizes with conserved apolar residues of α4 monomer, whereas the H-bond acceptor group interacts with a less conserved and more heterogeneous subpocket, involving polar residues of β2 subunit. The consistency of docking results and the agreement with the experimental data afford an encouraging validation for the proposed model and emphasize the soundness of such a fragmental approach to model any transmembrane protein.     

Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

Respiratory syndrome very similar to extrinsic allergic alveolitis due to Penicillium verrucosum in workers in a cheese factory

A respiratory syndrome very similar to extrinsic allergic alveolitis due to Penicillium verrucosum was recognized in 4 workers employed in a Gorgonzola cheese factory. A mycogen allergy to P. verrucosum, used as starter in the production, was demonstrated by positive sputum culture and detection of specific antibodies in the blood. Intense and prolonged exposure to inhalation of fungal spores could have lead to the development of this allergic response. The fact that 2 of the subjects are siblings seems to indicate host susceptibility or immunological constitution in the pathogenesis of the respiratory allergy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aminoacylation of Plasmodium falciparum tRNAAsn and Insights in the Synthesis of Asparagine Repeats

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA^Asn. However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA^Asn accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA^Asn in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as “tRNA sponges” and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.     

Severe South American Ocular Toxoplasmosis Is Associated with Decreased Ifn-γ/Il-17a and Increased Il-6/Il-13 Intraocular Levels

In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ.     

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 ± 0.3 × 10⁸ and 8.35 ± 0.6 × 10⁸, respectively, vs 9.2 ± 0.3 × 10⁷). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.     

Positioning the Red Deer (Cervus elaphus) Hunted by the Tyrolean Iceman into a Mitochondrial DNA Phylogeny

In the last years several phylogeographic studies of both extant and extinct red deer populations have been conducted. Three distinct mitochondrial lineages (western, eastern and North-African/Sardinian) have been identified reflecting different glacial refugia and postglacial recolonisation processes. However, little is known about the genetics of the Alpine populations and no mitochondrial DNA sequences from Alpine archaeological specimens are available. Here we provide the first mitochondrial sequences of an Alpine Copper Age Cervus elaphus. DNA was extracted from hair shafts which were part of the remains of the clothes of the glacier mummy known as the Tyrolean Iceman or Ötzi (5,350–5,100 years before present). A 2,297 base pairs long fragment was sequenced using a mixed sequencing procedure based on PCR amplifications and 454 sequencing of pooled amplification products. We analyzed the phylogenetic relationships of the Alpine Copper Age red deer''s haplotype with haplotypes of modern and ancient European red deer. The phylogenetic analyses showed that the haplotype of the Alpine Copper Age red deer falls within the western European mitochondrial lineage in contrast with the current populations from the Italian Alps belonging to the eastern lineage. We also discussed the phylogenetic relationships of the Alpine Copper Age red deer with the populations from Mesola Wood (northern Italy) and Sardinia.     

Electrophysiological correlates of action observation treatment in children with cerebral palsy: A pilot study

Action Observation Treatment (AOT) has been shown to be effective in the functional recovery of several clinical populations. However, little is known about the neural underpinnings of the clinical efficacy of AOT in children with Cerebral Palsy (CP). Using electroencephalography (EEG), we recorded µ rhythm desynchronization as an index of sensorimotor cortex modulation during a passive action observation task before and after AOT. The relationship between sensorimotor modulation and clinical outcomes was also assessed. Eight children with CP entered the present randomized controlled crossover pilot study in which the experimental AOT preceded or followed a control Videogame Observation Treatment (VOT). Results provide further evidence of the clinical efficacy of AOT for improving hand motor function in CP, as assessed with the Assisting Hand Assessment (AHA) and Melbourne Assessment of Unilateral Upper Limb Function Scale (MUUL). The novel finding is that AOT increases µ rhythm desynchronization at scalp locations corresponding to the hand representation areas. This effect is associated to functional improvement assessed with the MUUL. These preliminary findings, although referred to as a small sample, suggest that AOT may affect upper limb motor recovery in children with CP and modulate the activation of sensorimotor areas, offering a potential neurophysiological correlate to support the clinical utility of AOT.     

Molecular characterization of peroxidase (EC 1.11.1.7) from the rat intestine

Peroxidase partially purified from rat intestine exhibited a tendency to aggregate which was inversely related to I values of the medium. This enzyme preparation showed an optimum pH 7.5-9.0 and was inhibited by excess H2O2 and Fe complexing agents.