Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A.flavus solution containing 1 x 10(8) spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 X 10(8) spores, ears covered; T3, inoculated with E verticillioides plus A. flavus solution containing 1 x 10(8) spores of each, ears covered; T4, sprayed with sterile phosphate-buffered saline, ears covered; TS, non-sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B-1 and B-2 and the frequency of F. verticillioides was statistically verified in all treatments. (C) 2007 Society of Chemical Industry.

Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Background and Objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT. Material and Methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed. Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits. Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2 Delta AB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2 Delta AB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2 Delta AB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in Sao Paulo, Brazil

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in Sao Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels. (C) 2008 Elsevier Inc. All rights reserved.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

New mechanical samples positioning system for irradiations on a radial channel at nuclear research reactor in a full-power continuous operation

This paper describes a new mechanical samples positioning system that allows the safe placement and removal of biological samples for prolonged irradiation, in a nuclear reactor during full-power continuous operation. Also presented herein the materials of construction and operating principles. Additionally, this sample positioning system is compared with an existing pneumatic and automated transfer system, already available at the research reactors. The system consists of a mechanical arm with a claw, which can deliver the samples for irradiations without reactor shutdown. It was installed in the lEA-R1 research reactor at Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, Brazil, and for the past 5 years, the system has successfully operated and allowed the conducting of important experiments. As a result of its introduction, the facility has been in a position to positively respond to the increased demand in studies of biology, medicine, physics, engineering, detector/dosimeter calibrations, etc. It is one example of the appropriated technologies that save energy and resources. (C) 2010 Elsevier Ltd. All rights reserved.

Effect on the heavy-ion fusion and elastic scattering cross sections of common approximations assumed in coupled-channel calculations

We study the effects of several approximations commonly used in coupled-channel analyses of fusion and elastic scattering cross sections. Our calculations are performed considering couplings to inelastic states in the context of the frozen approximation, which is equivalent to the coupled-channel formalism when dealing with small excitation energies. Our findings indicate that, in some cases, the effect of the approximations on the theoretical cross sections can be larger than the precision of the experimental data.

Absolute determination of soluble potassium in tea infusion by gamma-ray spectroscopy

Potassium content in tea brew was determined by gamma-ray spectroscopy, Using the 1461 keV gamma-ray fro M (40)K, the naturally occurring radioactive isotope of potassium. We measured radiation with a shielded HPGe detector from individual test samples of tea leaves, before and after infusion preparation, and from commercial instant tea powder. The correction factor for the gamma-ray self-absorption in the extended source was determined with the help of Monte-Carlo simulations. This gamma-ray spectroscopy technique enabled the absolute determination of potassium content with a relative uncertainty smaller than 4%, at the one standard deviation confidence level, showing the feasibility of this method. An experiment to evaluate a possible systematic Uncertainty due to K distribution heterogeneity in the sample was performed, with file result that the corresponding relative standard deviation is smaller than 2% at 95% confidence level. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a novel ligand binding motif in the transthyretin channel

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR: 1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15` and a water-mediated hydrogen bond with Thr119`. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern. (C) 2009 Published by Elsevier Ltd.