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Acinetobacter baumannii is considered an important pathogen of clinical significance that is responsible for a wide range of nosocomial infections. Carbapenem resistance among A. baumannii isolates has increased dramatically in the Past years because of the emergence and dissemination of specific epidemic clones. We aimed to characterize the population structure of A. baumannii isolates from Porto Alegre city, Southern Brazil, in two distinct periods: during the first carbapenem-resistant A. baumannii (CRAB) outbreak (2007–2008) and 5 years later when the CRAB reached endemic levels (2013–2014). The study included 49 CRAB isolates collected in two periods: 2007–2008 (31 isolates) and 2013–2014 (18 isolates). Multilocus sequence typing (MLST) was performed according to Institute Pasteur, followed by eBURST analysis. PCR was used to detect integrase gene, blaNDM, and oxacillinase genes, and also to detect the ISAba1 element upstream blaOXA-23. The eBURST analysis identified the clonal complexes (CCs) CC15, CC32, CC79, CC216, CC221, and CC464 in the first period (2007–2008) and CC1, CC2, CC15, CC79, and CC162 during the endemic period (2013–2014). Molecular analysis by MLST identified 13 new sequence types. As we found A. baumannii with the blaOXA-23 gene of several CCs, it can be concluded that the increase of CRAB infections are not related to a specific clone. Furthermore, the high-risk CC15 and CC79 were related to the first CRAB outbreak and these CCs persisted up to 2014 in Porto Alegre city. The international clones CC1 and CC2 were observed for the first time in only the second period (2013–2014), alerting to the emergence of these clones in Southern Brazil. [ABSTRACT FROM AUTHOR]

Abstract: During plant–pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24 h of wounding or infection, in tubers from two potato cultivars with different resistance to P. infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion. [Copyright &y& Elsevier]

Abstract: In the search of new therapeutic tools for the treatment of American Trypanosomiasis, the largest parasitic disease burden in the American continent, three series of novel ruthenium complexes of the formula [RuCl2(HL)2], [RuCl3(dmso)(HL)] and [RuCl(PPh3)(L)2] with bioactive 5-nitrofuryl containing thiosemicarbazones as ligands (HL neutral, L monoanionic) were synthesized and characterized. Their in vitro growth inhibition activity against Trypanosoma cruzi and the effect of co-ligands in related physicochemical properties i.e. nitro moiety redox potential, lipophilicity and free radical scavenger capacity were evaluated. Results show that although a loss of activity was observed as a consequence of ruthenium complexation, lipophilicity and free radical scavenger capacity of the obtained complexes could be correlated to their trypanocidal effect. [Copyright &y& Elsevier]

Abstract: Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s). [Copyright &y& Elsevier]

The article focuses on the ability of Solanum tuberosum aspartic proteases (StAPs) to produce a direct antimicrobial effect. StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. The proteins interact directly with the surface of spores and hypae of F. Solania and P. infestans.

Abstract: Myofibrils from pre- and post-spawned female hake were depleted of thin and thick filaments by KI-treatment and the cytoskeletal ultrastructure of the preparations was investigated by electron microscopy. Irrespective of the gonadal stage of the fish, transmission (TEM) and scanning (SEM) electron micrographs showed an extensive network of filaments connecting Z-structures, which appeared as two circular rings held together. Z-structures of KI-treated myofibrils from post-spawned hake were more compact than those from pre-spawned fish. This ultrastructural difference was absent when KI-treated myofibrils from hake in both gonadal conditions were prepared in the presence of a proteinase inhibitor cocktail (1 mM EDTA+1 mM PMSF+1 mM iodoacetic acid). The total lipid (TL) content of the I-Z-I fraction from fish in pre-spawned condition was about 3.7 mg/g I-Z-I and non-polar lipids (NPL) represent 86.5% of TL. TL and NPL contents of the I-Z-I fraction from post-spawned hake were about 50% and 78.2% lower than those obtained from this fraction in pre-spawning fish. No significant changes (p>0.05) were observed in phospholipids (PL) and acidic phospholipids (AP). No significant differences (p<0.01) were observed among the corresponding lipid fractions of I-Z-I purified from pre- and post-spawning fish in the presence of a protease inhibitor cocktail. [Copyright &y& Elsevier]

The ATPase activities and proteolysis of myofibrils from pre- and postspawned flounder stored on ice were studied. At zero time of storage, no significant differences (P > 0.01) were observed in the Mg 2+ Ca 2+ and Mg 2+ (EGTA) ATPase activities of pre- and postspawned flounder myofibrils. No significant changes (P > 0.01) were observed thereafter in both enzymatic activities of postspawned myofibrils. Conversely, a significant decrease (P < 0.01) in the Mg 2+ Ca 2+ and Mg 2+ (EGTA) ATPase activities occurs in myofibrils from prespawned fish during storage. No significant changes (P > 0.01) in the Ca 2+ sensitivity and no evidence of proteolysis were detected in myofibrils from pre- and postspawned fish stored on ice. [ABSTRACT FROM AUTHOR]

Abstract: In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [MIICl2(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [MIICl2(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds. [Copyright &y& Elsevier]

Abstract: Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity. [Copyright &y& Elsevier]

Objective: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. Design: Controlled laboratory study. Setting: Three research laboratories at a university of biologic science. Animal(s) and Donor(s): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. Intervention(s): The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. Main Outcome Measure(s): Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. Result(s): The StAPs reduced sperm motility in a dose-dependent manner, and 25 μM of StAP1 and 35 μM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. Conclusion(s): The spermicidal effect of StAPs involves plasma membrane permeabilization. [Copyright &y& Elsevier]

Objectives To evaluate the emergence of New Delhi metallo-β-lactamase 1 (NDM-1)-producing Enterobacteriaceae isolates in Brazil. Methods From April to October 2013, following the detection of the first NDM-1-producing isolate, a surveillance study was performed for the detection of blaNDM-1 among Enterobacteriaceae isolates with reduced susceptibility to carbapenems in 17 hospitals of Porto Alegre, Brazil. Real-time PCR was used to determine the presence of carbapenemase genes, which were further sequenced. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE). Results A total of 1134 isolates were evaluated. blaNDM-1 was detected in 11 (0.97%) isolates: nine Enterobacter cloacae complex (eight belonging to a single clone recovered from two distinct hospitals and the other strain from a third hospital) and two Morganella morganii (belonging to a single clone recovered from one hospital). Most isolates presented high-level resistance to carbapenems. Conclusions NDM-1-producing Enterobacteriaceae have emerged rapidly in the hospitals of the Brazilian city where they were first detected. The emergence of NDM-1 in Brazil is of great concern, since it is a severe threat to antimicrobial therapy against Enterobacteriaceae in this country. [ABSTRACT FROM AUTHOR]