N-Alpha-Acetylation of α-Synuclein Increases Its Helical Folding Propensity,GM1 Binding Specificity and Resistance to Aggregation

A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson’s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.     

Isolation and Identification of 1-Ribosyl Pyridone Nucleosides from Human Urine

Abstract

1-Ribosylpyridin-4-one-3-carboxamide with lesser amounts of 1-ribosylpyridin-2-one-5-carboxamide have been isolated quantitatively from human urine using ion exchange column chromatography. Absorption spectra and mass spectrometry were used in the identification.     

Footwear and Foam Surface Alter Gait Initiation of Typical Subjects

Gait initiation is the task commonly used to investigate the anticipatory postural adjustments necessary to begin a new gait cycle from the standing position. In this study, we analyzed whether and how foot-floor interface characteristics influence the gait initiation process. For this purpose, 25 undergraduate students were evaluated while performing a gait initiation task in three experimental conditions: barefoot on a hard surface (barefoot condition), barefoot on a soft surface (foam condition), and shod on a hard surface (shod condition). Two force plates were used to acquire ground reaction forces and moments for each foot separately. A statistical parametric mapping (SPM) analysis was performed in COP time series. We compared the anterior-posterior (AP) and medial-lateral (ML) resultant center of pressure (COP) paths and average velocities, the force peaks under the right and left foot, and the COP integral x force impulse for three different phases: the anticipatory postural adjustment (APA) phase (Phase 1), the swing-foot unloading phase (Phase 2), and the support-foot unloading phase (Phase 3). In Phase 1, significantly smaller ML COP paths and velocities were found for the shod condition compared to the barefoot and foam conditions. Significantly smaller ML COP paths were also found in Phase 2 for the shod condition compared to the barefoot and foam conditions. In Phase 3, increased AP COP velocities were found for the shod condition compared to the barefoot and foam conditions. SPM analysis revealed significant differences for vector COP time series in the shod condition compared to the barefoot and foam conditions. The foam condition limited the impulse-generating capacity of COP shift and produced smaller ML force peaks, resulting in limitations to body-weight transfer from the swing to the support foot. The results suggest that footwear and a soft surface affect COP and impose certain features of gait initiation, especially in the ML direction of Phase 1.     

Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans

The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on their resting or germinating conidia.

Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the diploid strain as well as mitotic recombination frequencies.

We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.     

No evidence for biased co-transmission of speciation islands in Anopheles gambiae

Genome-scale scans have revealed highly heterogeneous levels of divergence between closely related taxa in many systems. Generally, a small number of regions show high differentiation, with the rest of the genome showing no or only low levels of divergence. These patterns have been interpreted as evidence for ongoing speciation-with-gene-flow, with introgression homogenizing the whole genome except loci involved in reproductive isolation. However, as the number of selected loci increases, the probability of introgression at unselected loci decreases unless there is a transmission ratio distortion causing an over-representation of specific combinations of alleles. Here we examine the transmission of three 'speciation islands' that contain fixed differences between the M and S forms of the mosquito, Anopheles gambiae. We made reciprocal crosses between M and S parents and genotyped over 2000 F(2) individuals, developing a hierarchical likelihood model to identify specific genotypes that are under- or over-represented among the recombinant offspring. Though our overall results did not match the expected number of F(2) genotypes, we found no biased co-transmission among M or S alleles in the three islands. Our likelihood model did identify transmission ratio distortion at two of the three islands, but this distortion was small (approx. 3%) and in opposite directions for the two islands. We discuss how our results impinge on hypotheses of current gene flow between M and S and ongoing speciation-with-gene-flow in this system.     

TGFβ and BMP-2 regulate epicardial cell invasion via TGFβR3 activation of the Par6/Smurf1/RhoA pathway

Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial-mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3^−/− mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFβR3 are not well described. TGFβR3-dependent endocardial EMT stimulated by either TGFβ2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFβ to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFβR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3^+/+ cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3^−/− epicardial cells which have a decreased ability to invade collagen gels in response to TGFβ2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFβR3 in Tgfbr3^−/− cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFβR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFβR3 in epicardial cells.