Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.     

Metabolism of carbohydrates during the development of seeds of the brazilian rubber tree [Hevea brasiliensis (Willd. Ex Adr. de Juss) Muell.-Arg.]

This work aimed at the assessment of the metabolism of carbohydrate during the development of the seeds of Brazilian rubber trees. The enzymatic activity of the acid invertase, neutral invertase and sucrose synthase (SuSy) and the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose were evaluated separately in each part of the fruit and seed—pericarp, seed coat, embryo and endosperm—on different days after the pollination (DAP). Based on the results obtained in this study, it is possible to conclude that in the beginning of the development of the rubber tree seeds, until 95 DAP, the endosperm presents high concentration of RS and low concentration of sucrose. After this period, the endosperm of the seed initiates starch accumulation and the concentration of RS decreases followed by the increase in the concentration of sucrose, presenting, after 120 DAP, an inversion of concentration of these two sugars. In the embryo, the levels of TSS, RS and sucrose show significant increase with the progress of the seed development. In the endosperm, the transition of the division phase and cell expansion for the storage of reserve material seem to occur around 120 DAP and is to be controlled mainly by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and is to be controlled mainly by the enzymes acid and neutral invertases.     

IFABP portal region insertion during membrane interaction depends on phospholipid composition

Intestinal fatty acid-binding protein (IFABP) is highly expressed in the intestinal epithelium and it belongs to the family of soluble lipid binding proteins. These proteins are thought to participate in most aspects of the biology of lipids, regulating its availability for specific metabolic pathways, targeting and vectorial trafficking of lipids to specific subcellular compartments. The present study is based on the ability of IFABP to interact with phospholipid membranes, and we characterized its immersion into the bilayer's hydrophobic central region occupied by the acyl-chains. We constructed a series of Trp-mutants of IFABP to selectively probe the interaction of different regions of the protein, particularly the elements forming the portal domain that is proposed to regulate the exit and entry of ligands to/from the binding cavity. We employed several fluorescent techniques based on selective quenching induced by soluble or membrane confined agents. The results indicate that the portal region of IFABP penetrates deeply into the phospholipid bilayer, especially when CL-containing vesicles are employed. The orientation of the protein and the degree of penetration were highly dependent on the lipid composition, the superficial net charge and the ionic strength of the medium. These results may be relevant to understand the mechanism of ligand transfer and the specificity responsible for the unique functions of each member of the FABP family.     

Cytoprotective Effect of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> Extract on an In Vitro Experimental Model of Parkinson Disease

Parkinson′s disease (PD) is one of the most important neurodegenerative worldwide disorders. The potential cytoprotective effects of aqueous extract of Valeriana officinalis on rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells were demonstrated. The cytotoxicity, cell viability and analysis of cellular morphology were performed by MTT-tetrazole (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and phase contrast microscopy, respectively. Significant changes in the cellular morphology, and condensation of the cell body could be observed when cells were treated with 300 nM rotenone for 48 h. Three different concentrations of Valeriana officinalis extract were used (0.049, 0.098 and 0.195 mg/mL). These extracts brought about an increase of 7.0 ± 1.3%, 14.5 ± 1.3% and 14.5 ± 3.2% in cell viability. Our results indicated that neuroprotector action of the Valeriana officinalis extract provides support for later studies as they help understanding this drug for the development of cytoprotective various therapies in PD.     

Spatial relationships between polychaete assemblages and environmental variables over broad geographical scales

This study examined spatial relationships between rocky shore polychaete assemblages and environmental variables over broad geographical scales, using a database compiled within the Census of Marine Life NaGISA (Natural Geography In Shore Areas) research program. The database consisted of abundance measures of polychaetes classified at the genus and family levels for 74 and 93 sites, respectively, from nine geographic regions. We tested the general hypothesis that the set of environmental variables emerging as potentially important drivers of variation in polychaete assemblages depend on the spatial scale considered. Through Moran's eigenvector maps we indentified three submodels reflecting spatial relationships among sampling sites at intercontinental (>10,000 km), continental (1000-5000 km) and regional (20-500 km) scales. Using redundancy analysis we found that most environmental variables contributed to explain a large and significant proportion of variation of the intercontinental submodel both for genera and families (54% and 53%, respectively). A subset of these variables, organic pollution, inorganic pollution, primary productivity and nutrient contamination was also significantly related to spatial variation at the continental scale, explaining 25% and 32% of the variance at the genus and family levels, respectively. These variables should therefore be preferably considered when forecasting large-scale spatial patterns of polychaete assemblages in relation to ongoing or predicted changes in environmental conditions. None of the variables considered in this study were significantly related to the regional submodel.     

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca²⁺]_i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X₃ subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg₂₊-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X₇ receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca²⁺]_i measurements in single synaptic terminals. Additionally, the P2X₇ receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.     

Variability in patterns of growth and morphology of Posidonia oceanica exposed to urban and industrial wastes: contrasts with two reference locations

Urban and industrial wastes have been claimed to negatively affect Posidonia oceanica meadows, but few studies have addressed this issue by comparing disturbed locations with replicated reference locations. Here, we examined the general proposition that patterns of growth and morphology of P. oceanica exposed to urban and industrial effluents were different from those observed in reference meadows. Hypotheses were both on differences in mean values of response variables and on variation of these measures at a hierarchy of spatial scales (from centimetres to hundreds of metres). Results indicated a significant reduction in mean number of intermediate leaves at the outfall compared to reference locations, whereas the opposite pattern occurred for juvenile leaves. There were significant, though temporally variable differences in growth of rhizomes between disturbed and reference locations, with reduced growth at the outfall in 2 out of 3 years analysed. Measures of spatial variance in number of juvenile leaves and length of adult leaves at the scale of shoots were significantly larger at the outfall compared to reference locations. At the same scale, measures of spatial variance in length of juvenile and intermediate leaves were significantly lower at the putatively impacted location. Spatial variance in number of intermediate leaves was reduced at the outfall compared to reference locations at the scale of quadrats. Past values of spatial variance in number of leaves per shoot were lower at the outfall than at the two reference locations at the scale of shoots, whereas the opposite occurred at the scale of areas. None of the structural variables examined showed any difference between the putatively impacted location and the two reference locations, either in terms of mean response or as changes in spatial variance. These results indicated that integrating methods to examine present and past events of disturbance, including analyses to detect changes in spatial variance of response variables, may provide a powerful approach to the analysis of environmental impacts on P. oceanica.