Antimicrobial compounds produced by Lactobacillus sakei subsp sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil

Enterococci have been implicated in severe human infections as a consequence of associated determinants of virulence and antimicrobial resistance. The majority of vancomycin-resistant Enterococcus faecium (VRE(fm)) connected to outbreaks worldwide pertains to the clonal complex 17 (CC17). In Brazil, the majority of VRE(fm) involved in outbreaks reported so far are not related to CC17. VRE(fm) strains responsible for an outbreak and sporadic cases in hospitals located in the city of Campinas, Brazil, were compared to other VRE(fm) strains in the country. Twenty-two out of 23 E. faecium were vancomycin-resistant and harboured the vanA gene. One vancomycin-susceptible E. faecium (VSE(fm)) strain was included in this study because it was isolated from a patient who one week later harboured a VRE(fm). All strains, except VSE, showed the same alteration in the VanA element characterised by deletion of the left extremity of the transposon and insertion of IS1251 between the vanS and vanH genes. Genes codifying virulence factors such as collageneadhesin protein, enterococcal surface protein and hyaluronidase were detected in the VRE(fm) and VSE(fm) studied. Both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed that VRE(fm) and VSE(fm) strains have a clonal relationship. New sequence types (STs) were identified by MLST as ST447, ST448, ST478 and ST412 but all belonged to the CC17. The present study revealed that VRE(fm) outbreaks in Brazil were caused by strains that did not share a common evolutionary history, and that VRE(fm) strains belonging to CC17 could be predominant in Brazil as in other countries. (C) 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Excised Porcine Cornea Integrity Evaluation in an in vitro Model of Iontophoretic Ocular Research

Background/Aims: It is a challenge to adapt traditional in vitro diffusion experiments to ocular tissue. Thus, the aim of this work was to present experimental evidence on the integrity of the porcine cornea, barrier function and maintenance of electrical properties for 6 h of experiment when the tissue is mounted on an inexpensive and easy-to-use in vitro model for ocular iontophoresis. Methods: A modified Franz diffusion cell containing two ports for the insertion of the electrodes and a receiving compartment that does not need gassing with carbogen was used in the studies. Corneal electron transmission microscopy images were obtained, and diffusion experiments with fluorescent markers were performed to examine the integrity of the barrier function. The preservation of the negatively charged corneal epithelium was verified by the determination of the electro-osmotic flow of a hydrophilic and non-ionized molecule. Results: The diffusion cell was able to maintain the temperature, homogenization, porcine epithelial corneal structure integrity, barrier function and electrical characteristics throughout the 6 h of permeation experiment, without requiring CO(2) gassing when the receiving chamber was filled with 25 m M of HEPES buffer solution. Conclusion: The system described here is inexpensive, easy to handle and reliable as an in vitro model for iontophoretic ocular delivery studies. Copyright (C) 2010 S. Karger AG, Basel

In the eye of the storm: exploring the introduction of environmental issues in the production function in Brazilian companies

This article aims to analyse the introduction of environmental issues in the context of the production function, which has been referred to as the organisational area to lead corporate environmental management. With that purpose, the theoretical references for corporate environmental management and the necessary alterations in production function have been organised to include environmental aspects, especially in terms of product and process development, quality management, and logistics. Considering that this research field still lacks empirical evidence for Brazilian companies, four case studies were conducted using companies located in the country. The environmental management maturity level of those companies tends to follow the rate with which the environmental issue is introduced in production sub-areas, especially in the product development process. However, in most cases we found that the companies had difficulties in structuring the insertion of the environmental dimension in logistics. The final notes point out the distance observed between what is recommended by international literature and the reality of Brazilian companies in the challenge of making the production function environmentally friendly.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification.

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, Sao Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 165 rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 165 rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin. (C) 2009 Elsevier Ltd. All rights reserved.

Hyaluronidase Behavior at the Air/Liquid and Air/Lipid Interfaces and Improved Enzymatic Activity by Its Immobilization in a Biomembrane Model

Bovine testicular hyalurphidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid, catalytic hydrolysis, was successfully immobilized on Langmuir- Blodgett films prepared with the sodium salt of dihexadacylphosphoric acid, (DHP-Zn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein, adsorption at the air-liquid interface by means of pendant drop shipe analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information. about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 mu g L(-1) attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at pi -> 0. The surface pressure (pi) area curves obtained for BT-HAase and mixed DPPC- BT-HAase monolayers reveal the presence of the enzyme at the air-lipid interface up to 45 mN m(-1). Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.

Rat osseous plate alkaline phosphatase as Langmuir monolayer - An infrared study at the air-water interface

A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase-OAP) was studied as monolayer (pure and mixed with lipids) at the air-water interface. Surface pressure and surface potential-area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m(-1). Isotherms for mixed dimyristoylphosphatidic acid (DMPA)-OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air-water interface as observed from in situ p-polarized light Fourier transform-infrared reflection-absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed. (C) 2008 Published by Elsevier Inc.

Investigation of the electrical and electrochemical properties of nanocomposites from V2O5, polypyrrole, and polyaniline

In this work, an investigation of the electrical and electrochemical properties responsible for the energy storage capability of nanocomposites has been carried out. We demonstrate that, in the case of the V2O5 xerogel and the nanocomposites polypyrrole (Ppy)/V2O5 and polyaniline (PANI)/V2O5, the quadratic logistic equation (QLE) can be used to fit the inverse of the resistance values as a function of the injected charge in non-steady-state conditions. This contributes to a phenomenological understanding of the lithium ion and electron transport. The departure of the experimental curve from the fitting observed for the V2O5 xerogel can be attributed to the trapping sites formed during the lithium electroinsertion, which was observed by electrochemical impedance spectroscopy. The amount of trapping sites was obtained on the basis of the QLE. Similar values used to fit the inverse of the resistance were also used to fit the absorbance changes, which is also associated with the small polaron hopping from the V(IV) to the V(V) sites. On the other hand, there was good agreement between the experimental and the theoretical data when the profile of the inverse of the resistance as a function of the amount of inserted lithium ions of the nanocomposites Ppy/V2O5 and PANI/ V2O5 was concerned. We suggest that the presence of the conducting polymers is responsible for the different electrical profile of the V2O5 xerogel compared with those of the nanocomposites. In the latter case, interactions between the lithium ions and oxygen atoms from V2O5 are shielded, thus decreasing the trapping effect of lithium ions in the V2O5 sites. The different values of the lithium ion diffusion coefficient into these intercalation materials are in agreement with this hypothesis.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)(3) selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)(3) into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)(3) and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

Study of the antimicrobial peptide indolicidin and mutants in eukaryotic modelled membrane by molecular dynamics simulations

In this work the interaction of the antimicrobial peptide indolicidin (IND) and its mutants CP10A and CP11 with a eukaryotic membrane model was examined by molecular dynamics simulations. The aim was to analyse the behaviour of these antimicrobial peptides when they interact with a eukaryotic modelled membrane, thereby obtaining atomic detailed observations that are not experimentally available. In the simulations, the widely studied dipalmitoylphosphatidylcholine hydrated bilayer was used as a eukaryotic membrane model. In agreement with experimental observations, the peptides IND, CP10A, and CP11 insert into the bilayer differently; the peptides that insert more deeply present the major hemolytic activities. The hydrophobic residues are responsible for the insertion, but some Trp residues of the peptides remain at the bilayer/water interface because they interact with the bilayer choline groups by cation-pi interactions that should be important for recognition of eukaryotic membrane by the three studied peptides.

Differential Toxicity of Disperse Red 1 and Disperse Red 13 in the Ames Test, HepG2 Cytotoxicity Assay, and Daphnia Acute Toxicity Test

Azo dyes are of environmental concern due to their degradation products, widespread use, and low-removal rate during conventional treatment. Their toxic properties are related to the nature and position of the substituents with respect to the aromatic rings and amino nitrogen atom. The dyes Disperse Red 1 and Disperse Red 13 were tested for Salmonella mutagenicity, cell viability by annexin V, and propidium iodide in HepG2 and by aquatic toxicity assays using daphnids. Both dyes tested positive in the Salmonella assay, and the suggestion was made that these compounds induce mainly frame-shift mutations and that the enzymes nitroreductase and O-acetyltransferase play an important role in the observed effect. In addition, it was shown that the presence of the chlorine substituent in Disperse Red 13 decreased the mutagenicity about 14 times when compared with Disperse Red 1, which shows the same structure as Disperse Red 13, but without the chlorine substituent. The presence of this substituent did not cause cytotoxicity in HepG2 cells, but toxicity to the water flea Daphnia similis increased in the presence of the chlorine substituent. These data suggest that the insertion of a chlorine substituent could be an alternative in the design of dyes with low-mutagenic potency, although the ecotoxicity should be carefully evaluated. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol 26: 489-497, 2011.

Porphyrin-phospholipid interaction and ring metallation depending on the phospholipid polar head type

The interaction between a hydrophobically modified 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin and three phospholipids: two negatively charged, DMPA (the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid) and DMPG (the sodium salt of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and a zwitterionic DMPC (dimyristoyl-sn-glycero-phosphatidylcholine), were studied by means of surface pressure isotherms and spectroscopic methods. The interaction results in partial or total metallation of the porphyrin with zinc ions in the presence of negatively charged phospholipids, as attested by UV-vis and luminescence spectroscopy of the transferred films. In the presence of the zwitterionic phospholipid no insertion of zinc ion in the porphyrin ring is detected. These results are relevant for the understanding of photosensitizer-lipid-carrier binding for use in photodynamic therapy. (C) 2010 Elsevier Inc. All rights reserved.