Ca-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca²⁺-calmodulin (Ca²⁺CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca²⁺ sparks and waves in cardiac cells. In contrast, it is well known that Ca²⁺CaM inhibits [³H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [³H]ryanodine binding technique does not reflect the effects of Ca²⁺CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca²⁺CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca²⁺CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M₁ and M₂, in a voltage-dependent manner. Ca²⁺CaM significantly alters the equilibrium between these two states. The results suggest that Ca²⁺CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca²⁺CaM at the single-channel level are not mirrored by [³H]ryanodine binding studies.     

Major histocompatibility complex class I restricted T-cell autoreactivity in human peripheral blood mononuclear cells

During selection in the thymus or any subsequent response, T-cells recognize peptides bound to major histocompatibility complex (MHC) molecules. Peptides produced by lysosomes or by proteasome/immunoproteasome stimulate CD4+ or CD8+ T-cell, respectively. Inflammation alters components of both antigen-processing pathways resulting in the production of different peptides. The role of such changes in self/non-self discrimination was examined in autologous mixed peripheral blood mononuclear cell cultures. Stimulator cells were incubated in the presence or absence of INF-gamma, with or without lysosome inhibitors (ammonium chloride/chloroquine), cathepsin inhibitor (E-64), or proteasome/immunoproteasome inhibitor (epoxomicin). Responder cells were added and zeta-chain phosphorylated forms were used as read out. INF-gamma did not affect zeta-chain phosphorylated forms, which means that the expected INF-gamma induced alterations in antigen processing machinery do not influence self/non-self discrimination. Surprisingly, the completely phosphorylated 23-kDa zeta-chain was always present except in the case of epoxomicin, indicating the presence of MHC class I restricted autoreactive CD8+ T-cells but not of MHC class II restricted autoreactive CD4+ T-cells, possibly due to more efficient negative selection in the thymus of the latter. Autoimmunity is prevented due to absence of help by CD4+ T-cells. This conclusion was confirmed by the lack of differences in IL-2 levels in cell culture supernatants, as well as, by the absence of differences in cell proliferation under the various conditions described above.     

Domestication does not narrow MHC diversity in Sus scrofa

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.     

Side reactions in the SPPS of Cys-containing peptides

Alkylation of sensitive amino acids during synthesis of biologically important peptides is a common and well-documented problem in Fmoc-based strategy. Herein, we probed for the first time an unexpected S-alkylation of Cys-containing peptides that occur during the final TFA cleavage of peptides from the Wang solid support. Through a battery of approaches (NMR, UV and LC–MS) the formed by-product was assigned as the alkylation of the cysteine sulfydryl group by the p-hydroxyl benzyl group derived from the acidic Wang linker decomposition. Factors affecting this side reaction were monitored and a protocol that minimizes the presence of the by-product is reported.     

Molecular Evolution of a Pervasive Natural Amino-Acid Substitution in Drosophila cryptochrome

Genetic variations in circadian clock genes may serve as molecular adaptations, allowing populations to adapt to local environments. Here, we carried out a survey of genetic variation in Drosophila cryptochrome (cry), the fly’s dedicated circadian photoreceptor. An initial screen of 10 European cry alleles revealed substantial variation, including seven non-synonymous changes. The SNP frequency spectra and the excessive linkage disequilibrium in this locus suggested that this variation is maintained by natural selection. We focused on a non-conservative SNP involving a leucine - histidine replacement (L232H) and found that this polymorphism is common, with both alleles at intermediate frequencies across 27 populations surveyed in Europe, irrespective of latitude. Remarkably, we were able to reproduce this natural observation in the laboratory using replicate population cages where the minor allele frequency was initially set to 10%. Within 20 generations, the two allelic variants converged to approximately equal frequencies. Further experiments using congenic strains, showed that this SNP has a phenotypic impact, with variants showing significantly different eclosion profiles. At the long term, these phase differences in eclosion may contribute to genetic differentiation among individuals, and shape the evolution of wild populations.     

Angiopoietin-2 Primes Infection-Induced Preterm Delivery

Current knowledge on the participation of angiopoietin-2 (Ang-2) in the inflammatory process and on the importance of bacterial endotoxins (LPS) in the induction of preterm delivery (PTD) led us to investigate the role of Ang-2/LPS interplay in the pathogenesis of PTD. At a first stage, Ang-2 was measured at the end of the first trimester of pregnancy in the serum of 50 women who delivered prematurely; of 88 women well-matched for age and parity who delivered full-term; and of 20 non-pregnant healthy women. Ang-2 was greater in pregnant than in non-pregnant women. The time until delivery was shorter among those with Ang-2 greater than 4 ng/ml (odds ratio for delivery until week 34; p: 0.040). To further investigate the role of Ang-2 for PTD, an experimental model of PTD induced by the intraperitoneal injection of LPS in mice was used. Ang-2 was administered intraperitoneally before LPS on day 14 of pregnancy. When Ang-2 was administered before the LPS diluent, all mice delivered full-term. However, administration of Ang-2 prior LPS accelerated further the time until delivery. Sacrifice experiments showed that the effect of Ang-2 was accompanied by decrease of the penetration of Evans Blue in the embryos and by increase of its penetration in maternal tissues. In parallel, the concentration of tumour necrosis factor-alpha in the maternal circulation, in fetal tissues and in the placentas was significantly decreased. Results indicate that Ang-2 accelerated the phenomena of PTD induced by LPS. This is related with deprivation of fetal perfusion.     

Use of Wild Bird Surveillance,Human Case Data and GIS Spatial Analysis for Predicting Spatial Distributions of West Nile Virus in Greece

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723–1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841–6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence.     

The Temporal Structure of Vertical Arm Movements

The present study investigates how the CNS deals with the omnipresent force of gravity during arm motor planning. Previous studies have reported direction-dependent kinematic differences in the vertical plane; notably, acceleration duration was greater during a downward than an upward arm movement. Although the analysis of acceleration and deceleration phases has permitted to explore the integration of gravity force, further investigation is necessary to conclude whether feedforward or feedback control processes are at the origin of this incorporation. We considered that a more detailed analysis of the temporal features of vertical arm movements could provide additional information about gravity force integration into the motor planning. Eight subjects performed single joint vertical arm movements (45° rotation around the shoulder joint) in two opposite directions (upwards and downwards) and at three different speeds (slow, natural and fast). We calculated different parameters of hand acceleration profiles: movement duration (MD), duration to peak acceleration (D PA), duration from peak acceleration to peak velocity (D PA-PV), duration from peak velocity to peak deceleration (D PV-PD), duration from peak deceleration to the movement end (D PD-End), acceleration duration (AD), deceleration duration (DD), peak acceleration (PA), peak velocity (PV), and peak deceleration (PD). While movement durations and amplitudes were similar for upward and downward movements, the temporal structure of acceleration profiles differed between the two directions. More specifically, subjects performed upward movements faster than downward movements; these direction-dependent asymmetries appeared early in the movement (i.e., before PA) and lasted until the moment of PD. Additionally, PA and PV were greater for upward than downward movements. Movement speed also changed the temporal structure of acceleration profiles. The effect of speed and direction on the form of acceleration profiles is consistent with the premise that the CNS optimises motor commands with respect to both gravitational and inertial constraints.