The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

A Multicenter,Randomized, Controlled Trial of Osteopathic Manipulative Treatment on Preterms

BackgroundDespite some preliminary evidence, it is still largely unknown whether osteopathic manipulative treatment improves preterm clinical outcomes.ResultsA total of 695 newborns were randomly assigned to either the study group (n= 352) or the control group (n=343). A statistical significant difference was observed between the two groups for the primary outcome (13.8 and 17.5 days for the study and control group respectively, p<0.001, effect size: 0.31). Multivariate analysis showed a reduction of the length of stay of 3.9 days (95% CI -5.5 to -2.3, p<0.001). Furthermore, there were significant reductions with treatment as compared to usual care in cost (difference between study and control group: 1,586.01€; 95% CI 1,087.18 to 6,277.28; p<0.001) but not in daily weight gain. There were no complications associated to the intervention.ConclusionsOsteopathic treatment reduced significantly the number of days of hospitalization and is cost-effective on a large cohort of preterm infants.     

The Saccharomyces cerevisiae PDS1 and RAD9 checkpoint genes control different DNA double-strand break repair pathways

In response to DNA damage, the Saccharomyces cerevisiae securin Pds1 blocks anaphase promotion by inhibiting ESP1-dependent degradation of cohesins. PDS1 is positioned downstream of the MEC1- and RAD9-mediated DNA damage-induced signal transduction pathways. Because cohesins participate in postreplicative repair and the pds1 mutant is radiation sensitive, we identified DNA repair pathways that are PDS1-dependent. We compared the radiation sensitivities and recombination phenotypes of pds1, rad9, rad51 single and double mutants, and found that whereas pds1 rad9 double mutants were synergistically more radiation sensitive than single mutants, pds1 rad51 mutants were not. To determine the role of PDS1 in recombinational repair pathways, we measured spontaneous and DNA damage-associated sister chromatid exchanges (SCEs) after exposure to X rays, UV and methyl methanesulfonate (MMS) and after the initiation of an HO endonuclease-generated double-strand break (DSB). The rates of spontaneous SCE and frequencies of DNA damage-associated SCE were similar in wild type and pds1 strains, but the latter exhibited reduced viability after exposure to DNA damaging agents. To determine whether pds1 mutants were defective in other pathways for DSB repair, we measured both single-strand annealing (SSA) and non-homologous end joining (NHEJ) in pds1 mutants. We found that the pds1 mutant was defective in SSA but efficient at ligating cohesive ends present on a linear plasmid. We therefore suggest that checkpoint genes control different pathways for DSB repair, and PDS1 and RAD9 have different roles in recombinational repair.     

Oxidative stress,erythrocyte ageing and plasma non-protein-bound iron in diabetic patients

Increased oxidative stress and decreased life span of erythrocytes (RBCs) are repeatedly reported in diabetes. In the aim to elucidate the mechanism of the latter, i.e. the events leading to erythrocyte ageing, this study determined in RBCs from diabetic patients iron release in a free desferrioxamine-chelatable form (DCI), methemoglobin (MetHb) formation, binding of autologous IgG to membrane proteins and in plasma non-protein-bound iron (NPBI), F₂-Isoprostanes (F₂-IsoPs) and advanced oxidation protein products (AOPP). DCI and MetHb were higher in diabetic RBCs than in controls and autologous IgG binding occurred in a much higher percentage of diabetic patients than controls. A significant correlation between DCI and IgG binding was found in diabetic RBCs. Plasma NPBI, esterified F₂-IsoPs and AOPP were higher in diabetic patients and a significant correlation was found between plasma NPBI and intra-erythrocyte DCI. The increased DCI and autologous IgG binding appear to be important factors in the accelerated removal of RBCs from the blood stream in diabetes and the increase in plasma NPBI could play an important role in the increased oxidative stress.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67^phox, one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67^phox membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.     

Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.     

The golden beauty: brain response to classical and renaissance sculptures

Is there an objective, biological basis for the experience of beauty in art? Or is aesthetic experience entirely subjective? Using fMRI technique, we addressed this question by presenting viewers, naïve to art criticism, with images of masterpieces of Classical and Renaissance sculpture. Employing proportion as the independent variable, we produced two sets of stimuli: one composed of images of original sculptures; the other of a modified version of the same images. The stimuli were presented in three conditions: observation, aesthetic judgment, and proportion judgment. In the observation condition, the viewers were required to observe the images with the same mind-set as if they were in a museum. In the other two conditions they were required to give an aesthetic or proportion judgment on the same images. Two types of analyses were carried out: one which contrasted brain response to the canonical and the modified sculptures, and one which contrasted beautiful vs. ugly sculptures as judged by each volunteer. The most striking result was that the observation of original sculptures, relative to the modified ones, produced activation of the right insula as well as of some lateral and medial cortical areas (lateral occipital gyrus, precuneus and prefrontal areas). The activation of the insula was particularly strong during the observation condition. Most interestingly, when volunteers were required to give an overt aesthetic judgment, the images judged as beautiful selectively activated the right amygdala, relative to those judged as ugly. We conclude that, in observers naïve to art criticism, the sense of beauty is mediated by two non-mutually exclusive processes: one based on a joint activation of sets of cortical neurons, triggered by parameters intrinsic to the stimuli, and the insula (objective beauty); the other based on the activation of the amygdala, driven by one''s own emotional experiences (subjective beauty).     

Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.