Rosangela Maria de Araújo Soares

Antileishmanial Activity of a Linalool-Rich Essential Oil from Croton cajucara

ABSTRACT The in vitro leishmanicidal effects of a linalool-rich essential oil from the leaves of Croton cajucara against Leishmania amazonensis were investigated. Morphological changes in L. amazonensis promastigotes treated with 15 ng of essential oil per ml were observed by transmission electron microscopy; leishmanial nuclear and kinetoplast chromatin destruction, followed by cell lysis, was observed within 1 h. Pretreatment of mouse peritoneal macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these macrophages and L. amazonensis, with a concomitant increase by 220% in the level of nitric oxide production by the infected macrophages. Treatment of preinfected macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these cells and the parasites, which led to a 60% increase in the amount of nitric oxide produced by the preinfected macrophages. These results provide new perspectives on the development of drugs with activities against Leishmania, as linalool-rich essential oil is a strikingly potent leishmanicidal plant extract (50% lethal doses, 8.3 ng/ml for promastigotes and 8.7 ng/ml for amastigotes) which inhibited the growth of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and which presented no cytotoxic effects against mammalian cells.

Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol

R.C. GOMES, L.T.A.S. SEME(r)DO, R.M.A. SOARES, C.S. ALVIANO, L.F. LINHARES and R.R.R. COELHO.2000.The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.

Supplementing the Antimicrobial Effects of Chemomechanical Debridement with Either Passive Ultrasonic Irrigation or a Final Rinse with Chlorhexidine: A Clinical Study

INTRODUCTION The ability of 2 different approaches to supplement the antimicrobial effects of chemomechanical debridement in infected root canals was compared in vivo. METHODS Samples from necrotic root canals of teeth with apical periodontitis were taken at the baseline (S1), after preparation with rotary nickel-titanium BioRaCe instruments and 2.5% NaOCl irrigation (S2), and then after either passive ultrasonic irrigation (PUI) for activation of NaOCl (n = 13) or a final rinse with 2% chlorhexidine (CHX) (n = 14) (S3). The incidence of positive culture for bacteria and fungi as well as positive broad-range polymerase chain reaction (PCR) results for bacteria, fungi, and archaea was determined. RESULTS All S1 samples were positive for bacteria in all methods. Fungi were not detected, and archaea occurred in only one S1 sample. Treatment procedures were significantly effective in reducing the incidence of positive culture and PCR results. Although both supplementary approaches reduced the incidence of positive bacteriologic results when compared with postinstrumentation samples, reduction was not statistically significant (P > .05). There was no significant difference for intergroup comparisons either (P > .05). CONCLUSIONS Although supplementary disinfection with either PUI or a final rinse with CHX can reduce the number of cases with positive culture and PCR results for bacteria, many cases still remain with detectable bacteria in the main root canal. Research on alternative or supplementary antimicrobial methods or substances should be encouraged.

Identification of sialic acids on the cell surface of Candida albicans

The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1. 6x10(6) to 2.8x10(6). The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated beta-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are alpha2,6- and alpha2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The alpha2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.

Heterogeneity of metallo and serine extracellular proteinases in oral clinical isolates of Candida albicans in HIV-positive and healthy children from Rio de Janeiro, Brazil

Candida yeasts frequently cause life-threatening systemic infections in immunocompromised hosts. In the present study, gelatin-SDS-PAGE analysis was used to characterize extracellular proteinases in 44 oral clinical isolates of Candida albicans from HIV-positive (29/50) and healthy children (15/50). Our survey indicates that these oral clinical isolates of C. albicans have complex extracellular proteolytic activity profiles, which illustrates the heterogeneity of this species. We showed four distinct proteolytic patterns composed of distinct serine (30-58 kDa) and metalloproteinase (64-95 kDa) activities, based on the inhibition profile with phenylmethylsulfonyl fluoride and 1,10-phenanthroline, respectively. This is the first report on secreted serine and metalloproteinases present in the culture supernatant fluids of C. albicans; however, we did not observe a significant correlation between proteolytic profile expressed by the C. albicans isolates from HIV-positive children and CD4(+) T cell count and plasma viral load.

Use of proteolytic enzymes as an additional tool for trypanosomatid identification.

The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri, H. samuelpessoai, H. mariadeanei, H. roitmani, H. muscarum ingenoplastis, H. muscarum muscarum, H. megaseliae, H. dendoderi, Herpetomoas sp., Crithidia oncopelti, C. deanei, C. acanthocephali, C. harmosa, C. fasciculata, C. guilhermei, C. luciliae, Blastocrithidia culicis, Leptomonas samueli and Lept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens, P. mcgheei, Trypanosoma cruzi and Leishmania amazonensis) by in situ detection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2 Herpetomonas (H. anglusteri and H. samuelpessoai) and 3 Crithidia (C. fasciculata, C. guilhermei and C. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification.

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.

Oral Candida colonization and its relation with predisposing factors in HIV-infected children and their uninfected siblings in Brazil: the era of highly active antiretroviral therapy.

OBJECTIVES To evaluate predisposing factors such as orofacial manifestations, immunosuppression status and antiretroviral therapy in relation to oral colonization by Candida spp. in Brazilian HIV-infected children and their uninfected siblings in the era of highly active antiretroviral therapy (HAART). METHODS Whole stimulated saliva was collected from 65 HIV-infected children (HIV+) and 40 uninfected siblings (HIV-), followed by assessment of orofacial manifestation, caries indexes and the number of cavitated dentinal carious teeth (CDT). The salivary samples were cultured and the colonies were counted. After which they were identified by sugar assimilation and fermentation (API 20C). Data was analyzed using chi-square, Mann-Whitney, Spearman tests and logistic regression. RESULTS Regarding positive growth, HIV+ presented 80% (52/65) and HIV- 57.5% (23/40) (P = 0.013). Absence of antiretroviral therapy and HAART increased the probability of Candida isolation (P < 0.05). Mean CD4%, immune-status and history of recurrent oral candidiasis (OC) had no influence on Candida isolation. Mixed Candida spp. cultures were observed in HIV+ (40%) and HIV- (52%): C. albicans was more frequently found in both groups, with a higher prevalence in HIV+ (P = 0.05); other non-albicans species were isolated in HIV+ and HIV-. Low prevalence of orofacial manifestations was observed in HIV+ (10.7% of OC). There was an association between means of CDT and Candida growth (P < 0.05) and a positive correlation between number of CDT and Candida cfu-counts in HIV+ and HIV-. Mean CD4% and immune-status had no influence on Candida isolation. Absence of antiretroviral therapy and HAART increased the probability of Candida isolation (P < 0.05). CONCLUSIONS The HIV infected children had a significantly higher prevalence of oral Candida spp. compared to their uninfected siblings. Absence of HAART and presence of dentinal carious teeth increased significantly Candida spp. colonization in these children.

Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released by Herpetomonas samuelpessoai

In previous studies, we showed that Herpetomonas samuelpessoai produced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 from Leishmania spp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by living H. samuelpessoai cells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS-PAGE to determine the secretory protein profile and on gelatin-SDS-PAGE to identify the proteolytic activity. The results demonstrated that H. samuelpessoai secreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6.0 at 37 degrees C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin of L. amazonensis demonstrated the presence of similar molecules on the cell-surface of H. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid of H. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide from H. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from the H. samuelpessoai surface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed in Leishmania.

Proteolytic expression in Blastocrithidia culicis : influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.