Structural modeling of ataxin-3 reveals distant homology to adaptins

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3.     

Chemical constituents from the infusion of Zollernia ilicifolia Vog. and comparison with Maytenus species

The new flavonoid glycoside kaempferol-3-O-alpha-L-rhamnopyranosyl(1-->2)-O-[alpha-L-rhamnopyranosyl(1-->6)]-O-beta-D-galactopyranoside-7-O-alpha-L-rhamnopyranoside was isolated together with (S)-zierin from the leaves of Zollernia ilicifolia (Fabaceae), a medicinal plant used as analgesic and antiulcerogenic effects in Brazilian Tropical Atlantic Rain Forest. The structures were established on the basis of 1H, 13C NMR and 2D NMR (COSY, HMBC, HMQC), UV, MS and IV spectra. The infusion of Zollernia ilicifolia was qualitatively compared to the infusion of the espinheiras-santas (Maytenus aquifolium and Maytenus ilicifolia) by HPLC-DAD.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.     

The Futile Cycling of Hexose Phosphates Could Account for the Fact That Hexokinase Exerts a High Control on Glucose Phosphorylation but Not on Glycolytic Rate in Transgenic Potato (Solanum tuberosum) Roots

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O₂ uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using ¹⁴C-glucose as precursor showed the formation of ¹⁴C-fructose and ¹⁴C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.     

Mechanisms of adrenergic control of sino-atrial node discharge

Among the mechanisms proposed for the increase in discharge of sino-atrial node (SAN) by norepinephrine (NE) are an increase in the hyperpolarization-activated current I(f) and in the slow inward current I(Ca,L). If I(f) is the primary mechanism, cesium (a blocker of I(f)) should eliminate the positive chronotropic effect of NE. If I(Ca,L), is involved, [Ca(2+)](o) should condition NE effects. We studied the electrophysiological changes induced by NE in isolated guinea pig SAN superfused in vitro with Tyrode solution (both SAN dominant and subsidiary pacemaker mechanisms are present) as well as with high [K(+)](o), higher Cs(+) or Ba(2+) (only the dominant pacemaker mechanism is present). In Tyrode solution, NE (0.5-1microM) increased the SAN rate and adding Cs(+) (approximately 12 mM) caused a decaying voltage tail during diastole in subsidiary pacemakers. NE enhanced the Cs(+)-induced tail, and increased the rate but less than in Tyrode solution. In higher [Cs(+)](o) (15- 18 mM), Ba(2+) (1 mM) or Ba(2+) plus Cs(+) (10 mM) dominant action potentials (not followed by a tail) were present and NE accelerated them as in Tyrode solution. In high [K(+)](o), NE increased the rate in the absence and presence of Cs(+), Ba(2+) or Ba(2+) plus Cs(+). In these solutions, NE increased the overshoot and maximum diastolic potential of dominant action potentials (APs) and increased the rate by steepening diastolic depolarization and shifting the threshold for upstroke to more negative values. High [Ca(2+)](o) alone increased the rate and NE enhanced this action, whereas low [Ca(2+)](o) reduced or abolished the increase in rate by NE. In SAN quiescent in high [K(+)](o) plus indapamide, NE induced spontaneous discharge by decreasing the resting potential and initiating progressively larger voltage oscillations. Thus, NE increases the SAN rate by acting primarily on dominant APs in a manner consistent with an increase of I(Ca,L) and I(K) and under conditions where I(f) is either blocked or not activated. NE INITIATES spontaneous discharge by inducing voltage oscillations unrelated to I(f).     

The hyolaryngeal apparatus of three species of Scinax (Scinaxinae: Hylidae: Anura) and comparison with other Hylidae species

The hyolaryngeal apparatus includes the arytenoid, cricoid and hyoid cartilages, and varied sizes and shapes can be distinguished between species. Although the larynx is an important morphological structure for sound production in frogs, few studies describe the structures and processes of its parts in hylids. We studied the hyolaryngeal anatomy of both males and females of Scinax ruber (Laurenti, 1768), using clearing and double staining methods, comparing it with that of Scinax wandae (Pyburn and Fouquette, 1971), Scinax kennedyi (Pyburn, 1973) and other hylid species. We found that S. ruber has the largest arytenoid cartilage of any species of the subfamily Scinaxinae studied to date. We described both the laryngeal differences among three Scinax species and the interspecific variability in the shapes of the projections of the arytenoid in the males of these species. The taxonomic characters (presence/absence of processes of the cartilage of Santorini, oesophageal process or bronchial processes) described here can play a key role in differentiating these species from Scinax species and from other species. In addition, we want to contribute to the natural history and comparison of the larynges in Scinax and within the genus and the Hylidae.     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.