A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Effect of photodynamic therapy on the virulence factors of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria who integrate the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation process (PDI) is based in the combined use of a light source, an oxidizing agent like oxygen and an intermediary agent (a photosensitizer). These three components interact to form cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. In fact, PDI is being shown to be a promising alternative to the antibiotic approach in the inactivation of pathogenic microorganisms. However, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus. For this, as photosensitizer the 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus (one reference strain, one strain with 1 enterotoxin, two strains with 3 enterotoxins and two strains resistant to methicillin, MRSA – one with 5 enterotoxins and the other without enterotoxins) were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production and resistance to methicillin was assessed. The results indicate that the expression of some virulence factors in the cells subjected to this therapy is affected. Additionally the susceptibility of the strains to PDI did not decrease upon successive treatments.

MECHANISM OF ACTION OF COBALT IONS ON RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by cobalt ions in a similar way as by magnesium ions. For concentrations up to 1 mu M, the Chelex-treated enzyme was stimulated by cobalt ions, showing K-d = 6.0 mu M, V = 977.5 U/mg, and site-site interactions (n = 2.5). Cobalt-enzyme was highly unstable at 37 degrees C, following a biphasic inactivation process with inactivation constants of about 0.0625 and 0.0015 min(-1). Cobalt ions stimulated the enzyme synergistically in the presence of magnesium ions (K-d = 5.0 mu M; V = 883.0 U/mg) or in the presence of zinc ions (K-d = 75.0 mu M; V = 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.

Autism spectrum disorder in a girl with a de novo x;19 balanced translocation

Balanced X-autosome translocations are rare, and female carriers are a clinically heterogeneous group of patients, with phenotypically normal women, history of recurrent miscarriage, gonadal dysfunction, X-linked disorders or congenital abnormalities, and/or developmental delay. We investigated a patient with a de novo X;19 translocation. The six-year-old girl has been evaluated due to hyperactivity, social interaction impairment, stereotypic and repetitive use of language with echolalia, failure to follow parents/caretakers orders, inconsolable outbursts, and persistent preoccupation with parts of objects. The girl has normal cognitive function. Her measurements are within normal range, and no other abnormalities were found during physical, neurological, or dysmorphological examinations. Conventional cytogenetic analysis showed a de novo balanced translocation, with the karyotype 46,X,t(X;19)(p21.2;q13.4). Replication banding showed a clear preference for inactivation of the normal X chromosome. The translocation was confirmed by FISH and Spectral Karyotyping (SKY). Although abnormal phenotypes associated with de novo balanced chromosomal rearrangements may be the result of disruption of a gene at one of the breakpoints, submicroscopic deletion or duplication, or a position effect, X; autosomal translocations are associated with additional unique risk factors including X-linked disorders, functional autosomal monosomy, or functional X chromosome disomy resulting from the complex X-inactivation process.

Characterization of selectivity and pharmacophores of type 1 sea anemone toxins by screening seven Na-v sodium channel isoforms

During their evolution, animals have developed a set of cysteine-rich peptides capable of binding various extracellular sites of voltage-gated sodium channels (VGSC). Sea anemone toxins that target VGSCs delay their inactivation process, but little is known about their selectivities. Here we report the investigation of three native type 1 toxins (CGTX-II, delta-AITX-Bcg1a and delta-AITX-Bcg1b) purified from the venom of Bunodosoma cangicum. Both delta-AITX-Bcg1a and delta-AITX-Bcg1b toxins were fully sequenced. The three peptides were evaluated by patch-clamp technique among Nav1.1-1.7 isoforms expressed in mammalian cell lines, and their preferential targets are Na(v)1.5 > 1.6 > 1.1. We also evaluated the role of some supposedly critical residues in the toxins which would interact with the channels, and observed that some substitutions are not critical as expected. In addition, CGTX-II and delta-AITX-Bcg1a evoke different shifts in activation/inactivation Boltzmann curves in Nav1.1 and 1.6. Moreover, our results suggest that the interaction region between toxins and VGSCs is not restricted to the supposed site 3 (S3-54 linker of domain IV), and this may be a consequence of distinct surface of contact of each peptide vs. targeted channel. Our data suggest that the contact surfaces of each peptide may be related to their surface charges, as CGTX-II is more positive than delta-AITX-Bcg1a and delta-AITX-Bcg1b. (C) 2011 Elsevier Inc. All rights reserved.

Complex genetic background in a large family with Brugada syndrome.

The Brugada syndrome (BrS) is an inherited arrhythmia characterized by ST-segment elevation in V1-V3 leads and negative T wave on standard ECG. BrS patients are at risk of sudden cardiac death (SCD) due to ventricular tachyarrhythmia. At least 17 genes have been proposed to be linked to BrS, although recent findings suggested a polygenic background. Mutations in SCN5A, the gene coding for the cardiac sodium channel Nav1.5, have been found in 15-30% of index cases. Here, we present the results of clinical, genetic, and expression studies of a large Iranian family with BrS carrying a novel genetic variant (p.P1506S) in SCN5A. By performing whole-cell patch-clamp experiments using HEK293 cells expressing wild-type (WT) or p.P1506S Nav1.5 channels, hyperpolarizing shift of the availability curve, depolarizing shift of the activation curve, and hastening of the fast inactivation process were observed. These mutant-induced alterations lead to a loss of function of Nav1.5 and thus suggest that the p.P1506S variant is pathogenic. In addition, cascade familial screening found a family member with BrS who did not carry the p.P1506S mutation. Additional next generation sequencing analyses revealed the p.R25W mutation in KCNH2 gene in SCN5A-negative BrS patients. These findings illustrate the complex genetic background of BrS found in this family and the possible pathogenic role of a new SCN5A genetic variant.

MacroH2A1 Regulation During the Cell Cycle of Mouse Embryonic Stem Cells

MacroH2A is a core histone variant that plays an important role in the X-inactivation process during differentiation of embryonic stem cells. It has been shown that macroH2A changes in localization during the cell cycle of somatic cells. This study aims to determine how macroH2A changes during the cell cycle of embryonic stem cells. Male and female mouse embryonic stem cells were transfected with a GFP::macroH2A construct and the relationship between macroH2A and the cell cycle was determined using FACS. This study shows that macroH2A is altered during the cell cycle of embryonic stem cells as it is in somatic cells and that in randomly cycling cells, there is a correlation between macroH2A expression and the phases of the cell cycle. High GFP expressing cells are mostly in the G2/M phase and low GFP expressing cells are mostly in the G1 phase. This correlation indicated that macroH2A is replicated with cellular DNA during the S phase resulting in higher expression in the G2/M phase. Future research, such as RT-PCR and differentiation experiments, is needed to further study this relationship and determine whether this change is at the protein or RNA level and how it changes during differentiation.

Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist.

During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated.

Predicting the thermal inactivation of bacteria in a solid matrix: simulation studies on the relative effects of microbial thermal resistance parameters and process conditions

A combined mathematical model for predicting heat penetration and microbial inactivation in a solid body heated by conduction was tested experimentally by inoculating agar cylinders with Salmonella typhimurium or Enterococcus faecium and heating in a water bath. Regions of growth where bacteria had survived after heating were measured by image analysis and compared with model predictions. Visualisation of the regions of growth was improved by incorporating chromogenic metabolic indicators into the agar. Preliminary tests established that the model performed satisfactorily with both test organisms and with cylinders of different diameter. The model was then used in simulation studies in which the parameters D, z, inoculum size, cylinder diameter and heating temperature were systematically varied. These simulations showed that the biological variables D, z and inoculum size had a relatively small effect on the time needed to eliminate bacteria at the cylinder axis in comparison with the physical variables heating temperature and cylinder diameter, which had a much greater relative effect. (c) 2005 Elsevier B.V All rights reserved.

Development of a log-quadratic model to describe microbial inactivation, illustrated by thermal inactivation of Clostridium botulinum

In the commercial food industry, demonstration of microbiological safety and thermal process equivalence often involves a mathematical framework that assumes log-linear inactivation kinetics and invokes concepts of decimal reduction time (DT), z values, and accumulated lethality. However, many microbes, particularly spores, exhibit inactivation kinetics that are not log linear. This has led to alternative modeling approaches, such as the biphasic and Weibull models, that relax strong log-linear assumptions. Using a statistical framework, we developed a novel log-quadratic model, which approximates the biphasic and Weibull models and provides additional physiological interpretability. As a statistical linear model, the log-quadratic model is relatively simple to fit and straightforwardly provides confidence intervals for its fitted values. It allows a DT-like value to be derived, even from data that exhibit obvious "tailing." We also showed how existing models of non-log-linear microbial inactivation, such as the Weibull model, can fit into a statistical linear model framework that dramatically simplifies their solution. We applied the log-quadratic model to thermal inactivation data for the spore-forming bacterium Clostridium botulinum and evaluated its merits compared with those of popular previously described approaches. The log-quadratic model was used as the basis of a secondary model that can capture the dependence of microbial inactivation kinetics on temperature. This model, in turn, was linked to models of spore inactivation of Sapru et al. and Rodriguez et al. that posit different physiological states for spores within a population. We believe that the log-quadratic model provides a useful framework in which to test vitalistic and mechanistic hypotheses of inactivation by thermal and other processes. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Photocatalytic Inactivation of Escherichia coli with LbL Fabricated Immobilized TiO2 Thin Films

Photocatalysis using semiconductor catalyst such as TiO2, in presence of UV light, is a promising technique for the inactivation of various microorganisms present in water. In the current study, the photocatalytic inactivation of Escherichia coli bacteria was studied with commercial Degussa Aeroxide TiO2 P25 (Aeroxide) and combustion synthesized TiO2 (CS TiO2) catalysts immobilized on glass slides in presence of UV irradiation. Thin films of the catalyst and polyelectrolytes, poly(allyl amine hydrochloride) and poly(styrene sulfonate sodium salt), were deposited on glass slides by layer by layer (LbL) deposition method and characterized by SEM and AFM imaging. The effect of various parameters, namely, catalyst concentration, surface area and number of bilayers, on inactivation was studied. Maximum inactivation of 8-log reduction in the viable count was observed with 1.227 mg/cm(2) of catalyst loaded slides. With this loading, complete inactivation was observed within 90 min and 75 min of irradiation, for Aeroxide and CS TiO2, respectively. Further increase in the catalyst concentration or increasing number of bilayers had no significant effect on inactivation. The effect of surface area on the inactivation was studied by increasing the number of slides and the inactivation was observed to increase with increasing surface area. It was also observed that the immobilized catalyst slides can be used for several cycles leading to an economic process. The study shows potential application of TiO2, for the inactivation of bacteria, in its fixed form by a simple immobilization technique.

Inactivation of macrophage Rab7 by Burkholderia cenocepacia

Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the transmissible ET12 clone, recruited Rab5 and synthesized phosphatidylinositol-3-phosphate, indicating progression to the early phagosomal stage. Despite the fact that the B. cenocepacia-containing vacuoles rarely fused with lysosomes, they could nevertheless acquire the late phagosomal markers CD63 and Rab7. Fluorescence recovery after photobleaching and use of a probe that detects Rab7-guanosine triphosphate indicated that activation of Rab7 was impaired by B. cenocepacia, accounting at least in part for the inability of the vacuole to merge with lysosomes. The Rab7 defect was not due to excessive cholesterol accumulation and was confined to the infected vacuoles. Jointly, these experiments indicate that B. cenocepacia express virulence factors capable of interfering with Rab7 function and thereby with membrane traffic.

Photodynamic inactivation of bacteria by cationic porphyrins : their cellular targets and potential environmental applications

Photodynamic inactivation (PDI) is defined as the process of cell destruction by oxidative stress resulting from the interaction between light and a photosensitizer (PS), in the presence of molecular oxygen. PDI of bacteria has been extensively studied in recent years, proving to be a promising alternative to conventional antimicrobial agents for the treatment of superficial and localized infections. Moreover, the applicability of PDI goes far beyond the clinical field, as its potential use in water disinfection, using PS immobilized on solid supports, is currently under study. The aim of the first part of this work was to study the oxidative modifications in phospholipids, nucleic acids and proteins of Escherichia coli and Staphylococcus warneri, subjected to photodynamic treatment with cationic porphyrins. The aims of the second part of the work were to study the efficiency of PDI in aquaculture water and the influence of different physicalchemical parameters in this process, using the Gram-negative bioluminescent bacterium Vibrio fischeri, and to evaluate the possibility of recycling cationic PS immobilized on magnetic nanoparticles. To study the oxidative changes in membrane phospholipids, a lipidomic approach has been used, combining chromatographic techniques and mass spectrometry. The FOX2 assay was used to determine the concentration of lipid hydroperoxides generated after treatment. The oxidative modifications in the proteins were analyzed by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Changes in the intracellular nucleic acids were analyzed by agarose gel electrophoresis and the concentration of doublestranded DNA was determined by fluorimetry. The oxidative changes of bacterial PDI at the molecular level were analyzed by infrared spectroscopy. In laboratory tests, bacteria (108 CFU mL-1) were irradiated with white light (4.0 mW cm-2) after incubation with the PS (Tri-Py+-Me-PF or Tetra-Py+-Me) at concentrations of 0.5 and 5.0 μM for S. warneri and E. coli, respectively. Bacteria were irradiated with different light doses (up to 9.6 J cm-2 for S. warneri and up to 64.8 J cm-2 for E. coli) and the changes were evaluated throughout the irradiation time. In the study of phospholipids, only the porphyrin Tri-Py+-Me-PF and a light dose of 64.8 J cm-2 were tested. The efficiency of PDI in aquaculture has been evaluated in two different conditions: in buffer solution, varying temperature, pH, salinity and oxygen concentration, and in aquaculture water samples, to reproduce the conditions of PDI in situ. The kinetics of the process was determined in realtime during the experiments by measuring the bioluminescence of V. fischeri (107 CFU mL-1, corresponding to a level of bioluminescence of 105 relative light units). A concentration of 5.0 μM of Tri-Py+-Me-PF was used in the experiments with buffer solution, and 10 to 50 μM in the experiments with aquaculture water. Artificial white light (4.0 mW cm-2) and solar irradiation (40 mW cm-2) were used as light sources.

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK.

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.