Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes.

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Heterogeneity of the induction of HLA-DR expression by human immune interferon on glioma cell lines and their clones.

The modulation of HLA-DR and HLA-A, -B, and -C by human recombinant immune interferon (IFN-gamma) was studied on 10 malignant glioma cell lines established in our laboratory, on 8 clones or subclones derived from these lines, and on a fetal astrocyte cell line. Comparative studies were performed with recombinant leukocyte interferon (IFN-alpha). The results not only confirmed the selective activity of IFN-gamma on the modulation of HLA-DR expression, as opposed to that of IFN-alpha, but also demonstrated a marked heterogeneity in the response of glioma cell lines and their clones to the two types of IFN tested. For example, all 3 clones of an inducible cell line could be modulated to express HLA-DR, whereas only 2 of 5 clones derived from a noninducible line were modulated. This heterogeneity did not seem to be due to the absence of the receptor for IFN-gamma on the surface of these cells, since almost all of the cell lines or clones tested (17 of 19) responded to IFN-gamma by the induction or enhancement of the expression for either HLA-DR or HLA-A, -B, and -C (or both). The heterogeneity of induction was also demonstrated between clones derived from a glioma line that did not express HLA-DR after IFN-gamma treatment. The production of HLA-DR by one of the clones was abundant enough to be confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Identification of opsA, a gene involved in solute stress mitigation and survival in soil, in the polycyclic aromatic hydrocarbon-degrading bacterium Novosphingobium sp. strain LH128.

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.

Cyst-based ecotoxicological tests using Anostracans: comparison of two species of Streptocephalus

Cyst-based ecotoxicological tests are simple and low-cost methods for assessing acute toxicity. Nevertheless, only a few comparative studies on their sensitivity are known. In the present study, the suitability of the use of two freshwater Anostracan species, Streptocephalus rubricaudatus and S. texanus, was assessed. The impact of 16 priority pollutants (4 heavy metals, 11 organic, and 1 organometallic compounds) on these two species, as well as on Artemia salina (Artoxkit M), Daphnia magna (International Organization for Standardization 6341), and S. proboscideus (Streptoxkit F) was assessed. For indicative comparison, bioassays using Brachionus calyciflorus (Rotoxkit F) and Photobacterium phosphoreum (Microtox) were also performed. For heavy metals (K2Cr2O7, Cd2+, Zn2+, Cu2+), the sensitivity of the two studied Streptocephalus species was slightly higher than that of D. magna. It was significantly more elevated than for the marine A. salina. For organic and organometallic micropollutants [phenol, 3,5-dichlorophenol, pentachlorophenol (PCP), hydroquinone, linear alkylbenzene sulfonate, sodium dodecyl sulfate, tributylphosphate, dimethylphthalate, atrazine, lindane, malathion, tributyltin chloride (TBT-Cl)], the sensitivity of the 4 anostracan species was of the same order of magnitude as that of D. magna. Artemia salina was slightly less sensitive to some organic compounds (PCP, hydroquinone, TBT-Cl). The sensitivity of S. rubricaudatus to organic solvents was low. On the other hand, this anostracan was quite sensitive to NaCl. Thus, its use is restricted to freshwater samples. The evaluation of global practicability of these two tests confirms that cyst-based freshwater anostracans may be used to perform low-cost tests at a sensitivity comparable to that of D. magna (24 h immobilization test).

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Identification of promising antigenic components in latent fingermark residues

An analysis of latent fingermark residues by Sodium-Dodecyl-Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) followed by silver staining allowed the detection of different proteins, from which two major bands, corresponding to proteins of 56 and 64 kDa molecular weight, could be identified. Two other bands, corresponding to proteins of 52 and 48 kDa were also visualizable along with some other weaker bands of lower molecular weights. In order to identify these proteins, three antibodies directed against human proteins were tested on western blots of fingermarks residues: anti-keratin 1 and 10 (K1/10), anti-cathepsin-D (Cat.D) and anti-dermcidin (Derm.). The corresponding antigens are known to be present in the stratum corneum of desquamating stratified epithelium (K1/10, Cat.D) and/or in eccrine sweat (Cat.D, Derm.). The two major bands were identified as consistent with keratin 1 and 10. The pro-form and the active form of the cathepsin-D have also been identified from two other bands. Dermcidin could not be detected in the western blot. In addition, these antibodies have been tested on latent fingermarks left on polyvinylidene fluoride (PVDF) membrane, as well as on whitened and non-whitened paper. The detection of fingermarks was successful with all three antibodies.

Grape epicatechin conjugates prevent erythrocyte membrane protein oxidation

Epicatechin conjugates obtained from grape have shown antioxidant activity in various systems. However, how these conjugates exert their antioxidant benefits has not been widely studied. We assessed the activity of epicatechin and epicatechin conjugates on the erythrocyte membrane in the presence and absence of a peroxyl radical initiator, to increase our understanding of their mechanisms. Thus, we studied cell membrane fluidity by fluorescence anisotropy measurements, morphology of erythrocytes by scanning electron microscopy, and finally, red cell membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data showed that incubation of red cells in the presence of epicatechin derivatives altered membrane fluidity and erythrocyte morphology but not the membrane protein pattern. The presence in the medium of the peroxyl radical initiator 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) resulted in membrane disruptions at all levels analyzed, causing changes in membrane fluidity, cell morphology, and protein degradation. The presence of antioxidants avoided protein oxidation, indicating that the interaction of epicatechin conjugates with the lipid bilayer might reduce the accessibility of AAPH to membranes, which could explain in part the inhibitory ability of these compounds against hemolysis induced by peroxidative insult.

Determinação da concentração micelar crítica de ácidos húmicos por medidas de condutividade e espectroscopia

The aim of this study was the determination of the critical micelle concentration (CMC) of the sodium dodecyl sulfate (SDS) surfactant using spectroscopic and conductimetric determinations and to compare these methodologies in the determination of the CMC of different humic acids (HA). The CMC obtained by conductimetric determination was satisfactory. By spectroscopic determination two values of the CMC were obtained for HA. These values can be to due the intra and intermolecular interactions in the HA structure.

O pKa de indicadores ácido-base e os efeitos coloidais

An experiment is proposed that can be included in undergraduate courses of chemistry. The subject is the acidity of organic compounds, which are employed as pH indicators, particularly in acid-base titrations. The indicators used are methyl orange, bromophenol blue and bromocresol green in aqueous medium. The influence of colloidal systems on the equilibrium is evaluated by the pKa. The colloids employed are surfactants like sodium dodecyl sulfate, cetyl-trimethylammonium bromide and a polymeric non-ionic F127 (pluronics). The effect of stabilization promoted by the system on the acidic or basic structureof the indicator establishes the action mechanism of the colloid on the pKa values.

Determinação potenciométrica da concentração micelar crítica de surfactantes: uma nova aplicação metodológica no ensino de Química

The participation of the students in the planning, execution and discussion of experimental results is important for the valuation of the role of them in the construction of the chemical knowledge implicited in the approach between education and research. This work relates either the construction of selective membrane electrodes for surfactants and its application in the potenciometric determination of the critical micellar concentration of the sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) surfactants. In adition it´s possible to discuss the potenciometric results in matching with the data valued for the tensiometric and condutimetric traditionally used in the physical-chemistry lessons.

Auto-associação do dodecilsulfato de sódio (SDS) com o polímero hidrofobicamente modificado etil(hidroxietil) celulose (EHEC)

Mixtures of ethyl(hydroxyethyl)cellulose (EHEC) and Sodium Dodecyl Sulfate (SDS) were investigated using surface tension, conductivity and viscosity measurements in aqueous solutions. The parameters of the surfactant to polymer association processes such as the critical aggregation concentration (cac) and saturation of the polymer by SDS (psp) were determined from the plots of surface tension and specific conductivity versus surfactant concentration. Through the final results we see that there was no specific link of polymer with the surfactant, implying therefore a phenomenon of only cooperative association.

Foam-forming properties of Ilex paraguariensis (mate) saponin: foamability and foam lifetime analysis by Weibull equation

Saponins are natural soaplike foam-forming compounds widely used in foods, cosmetic and pharmaceutical preparations. In this work foamability and foam lifetime of foams obtained from Ilex paraguariensis unripe fruits were analyzed. Polysorbate 80 and sodium dodecyl sulfate were used as reference surfactants. Aiming a better data understanding a linearized 4-parameters Weibull function was proposed. The mate hydroethanolic extract (ME) and a mate saponin enriched fraction (MSF) afforded foamability and foam lifetime comparable to the synthetic surfactants. The linearization of the Weibull equation allowed the statistical comparison of foam decay curves, improving former mathematical approaches.

Flotação da mistura argila-quartzo com surfactantes e estudos de pilarização

Clay is often employed as a catalyst, but quartz impurities can decrease the catalytic efficiency. Fine particles of clay can be purified by flotation. We examined the cationic surfactant hexadecyltrimethylammonium bromide (HTAB), the anionic sodium dodecyl sulfate (SDS) and the non-ionic TRITON X-100 for separating the quartz impurities from clay. Using X-ray diffraction, the separation was monitored for changes in the peaks corresponding to clay and quartz. Cationic surfactant HTAB was most effective in separating the quartz-clay mixture and the selectivity can be explained by internal adsorption of the surfactant onto the clay and external adsorption onto the quartz.