20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

Numerical-experimental method for the validation of a controlled stiffness femoral prosthesis

The aim of this paper is to describe a new numerical-experimental method to determine the stiffness of a conceptual proximal femoral prototype. The methodology consists of the comparison of the numerical and experimental displacement distributions of the prosthesis loaded as a cantilever beam to validate a design concept: controlled stiffness prosthesis. The manufactured prototype used to test the applicability of the numerical-experimental procedure integrates a stiff metal core bonded to a composite material made of an epoxy resin reinforced with carbon-glass braided pre-forms. The prosthesis with an embedded controlled stiffness concept was obtained by varying the geometry of the core with the composite layer thickness.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.     

Evaluation of a Western blot test,Helico blot 2.1, in the diagnosis of Helicobacter pylori infection in a pediatric population

Background. Noninvasive diagnostic tests are useful as screening tools for Helicobacter pylori infection in pediatric populations. The aim of this study was to evaluate performance of the immunoblot assay, Helico Blot 2.1, for the diagnosis of H. pylori infection in symptomatic children. Materials and Methods. Immunoblot assay was used for detection of IgG antibodies to specific H. pylori proteins and to a recombinant H. pylori antigen, CIM marker. The study was performed on sera collected from 134 symptomatic, untreated children (mean age, 9.1 ± 3.2 years; range, 1–14 years). H. pylori infection status was determined by culture, histology and rapid urease test. Results. Immunoblot assay yielded a positive result in 71 of the 72 infected patients (sensitivity 98.6%) and in eight of the 62 noninfected ones (specificity 87.1%). The predictive values for a positive and a negative result were 89.9% and 98.2%, respectively. The performance of the CIM band alone, as a marker for H. pylori infection status, was also evaluated. This band was present on the blot of 71 infected patients and on four of the 62 H. pylori‐negative patients. The sensitivity, specificity, PPV and NPV of the CIM antigen were 98.6%, 93.5%, 94.7% and 98.3%, respectively. Conclusions. The immunoblot assay Helico Blot 2.1 is a suitable noninvasive test for the serodiagnosis of H. pylori infection in children. The good level of performance demonstrated by the novel recombinant antigen CIM suggests it may be a useful contribution to the qualitative and quantitative performance of the Helico Blot 2.1 in pediatric populations.     

Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in the State of Rio de Janeiro,Brazil

Experimental releases of female Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus were performed in August and September 1999, in an urban area of Nova Igua u, State of Rio de Janeiro, Brazil, to estimate their flight range in a circular area of 1,600 m where 1,472 ovitraps were set. Releases of 3,055 Ae. aegypti and 2,225 Ae. albopictus females, fed with rubidium (Rb)-marked blood and surgically prevented from subsequent blood-feeding, were separated by 11 days. Rb was detected in ovitrap-collected eggs by atomic emission spectrophotometry. Rb-marked eggs of both species were detected up to 800 m from the release point. Eggs of Ae. albopictus were more numerous and more heterogeneously distributed in the area than those of Ae. aegypti. Eggs positively marked for Rb were found at all borders of the study area, suggesting that egg laying also occurred beyond these limits. Results from this study suggest that females can fly at least 800 m in 6 days and, if infected, potentially spread virus rapidly.     

Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.     

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf=0.60, as a result of dye degradation.