Molecular phylogeny of the subgenus Drosophila (Diptera, Drosophilidae) with an emphasis on Neotropical species and groups: a nuclear versus mitochondrial gene approach

The genus Drosophila has played an essential role in many biological studies during the last 100 years but much controversy and many incompletely addressed issues still remain to be elucidated regarding the phylogeny of this genus. Because information on the Neotropical species contained in the subgenus Drosophila is particularly incomplete, with this taxonomic group being underrepresented in many studies, we designed a study to answer some evolutionary questions related to these species. We subjected at least 41 Drosophilidae taxa to a phylogenetic analysis using a 516-base pair (bp) fragment of the alpha-methyldopa (Amd) nuclear gene and a 672 bp fragment of the mitochondrial cytochrome oxidase subunit II (COII) gene both individually and in combination. We found that the subgenus Drosophila is paraphyletic and subdivided into two main clusters: the first containing species traditionally placed in the virilis-repleta radiation and the second assembling species of the immigrans-Hirtodrosophila radiation. Inside the first of these clusters we could detect the monophyly of both the flavopilosa (the sister-clade of the annulimana group) and the mesophragmatica (closely related to the repleta group) species groups. Concerning the immigrans-Hirtodrosophila lineage, Zaprionus, Liodrosophila, Samoaia, and Hirtodrosophila were the early offshoots, followed by the immigrans, quinaria, testacea, and funebris species groups. The tripunctata radiation appears to be a derived clade, composed of a paraphyletic tripunctata group, intimately interposed with members of the cardini, guarani, and guaramunu species groups. Overall, the COII gene yielded a poor phylogenetic performance when compared to the Amd gene, the evolutionary hypothesis of which agreed with the total evidence tree. This phenomenon can be explained by the fast saturation of transitional substitutions in COII, due to strong biases in both base composition and substitution patterns, as also by its great among-site rate variation heterogeneity.     

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.     

CXCR3/CXCL10 expression in the synovium of children with juvenile idiopathic arthritis

The accumulation of T cells in the synovial membrane is the crucial step in the pathophysiology of the inflammatory processes characterizing juvenile idiopathic arthritis (JIA). In this study, we evaluated the expression and the pathogenetic role in oligoarticular JIA of a CXC chemokine involved in the directional migration of activated T cells, i.e. IFNγ-inducible protein 10 (CXCL10) and its receptor, CXCR3. Immunochemistry with an antihuman CXCL10 showed that synovial macrophages, epithelial cells, and endothelial cells bear the chemokine. By flow cytometry and immunochemistry, it has been shown that CXCR3 is expressed at high density by virtually all T lymphocytes isolated from synovial fluid (SF) and infiltrating the synovial membrane. Particularly strongly stained CXCR3⁺ T cells can be observed close to the luminal space and in the perivascular area. Furthermore, densitometric analysis has revealed that the mRNA levels for CXCR3 are significantly higher in JIA patients than in controls. T cells purified from SF exhibit a definite migratory capability in response to CXCL10. Furthermore, SF exerts significant chemotactic activity on the CXCR3⁺ T-cell line, and this activity is inhibited by the addition of an anti-CXCL10 neutralizing antibody. Taken together, these data suggest that CXCR3/CXCL10 interactions are involved in the pathophysiology of JIA-associated inflammatory processes, regulating both the activation of T cells and their recruitment into the inflamed synovium.     

Regioselective glycosylation: synthesis of alpha-indoline nucleosides

Novel indoline ribonucleosides with the alpha-N-glycoside configuration are synthesized with very high regioselectivity in 90-96%yield, using TMS protected indolines and 2,3-O-(1-methylethylidene)-5-O-(triphenylmethyl)-alpha/beta-D-ribofuranose. The structures of these ribonucleosides were elucidated with X-ray crystallography as well as 2D (NOESY, COSY, and HMQC) NMR spectroscopy.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

New synthesis of PNA-3'DNA linker monomers, useful building blocks to obtain PNA/DNA chimeras

A new synthetic strategy to get the PNA-3'DNA linker with the monomethoxytrityl (Mmt) group as temporary protection of the backbone to be used for the synthesis of PNA/DNA chimeras was employed and a convenient strategy to obtain Mmt PNA monomers was developed. The synthetic strategies take advantage of the introduction of the acid-labile Mmt-protecting group in the first step.     

Chronic pain as expression of neural substrates. Issues from the neuronal dynamics and mutual relations

The Thalamo-Cortical somatosensory loop shows important synaptic re-organization in cases of chronic pain. Animal models exhibit severe functional distortions, potentially related to the anatomic rearrangements. Connectivity and information theoretic measurement represent important tools to quantify the functional disarrays. We performed electrophysiological experiments with multisite, multielectrode simultaneous recordings in the Thalamus and in the Somatosensory Cortex. The recurrent anomalies in the analytic estimates induce to hypothesize a potential neurodynamical explanation of the sensory context.