Aristolochia trilobata: Identification of the Anti-Inflammatory and Antinociceptive Effects.

Aristolochia trilobata, popularly known as "mil-homens," is widely used for treatment of stomach aches, colic, asthma, pulmonary diseases, diabetes, and skin affection. We evaluated the antinociceptive and anti-inflammatory activities of the essential oil (EO) and the main constituent, 6-methyl-5-hepten-2-yl acetate (sulcatyl acetate, SA). EO and SA (1, 10, and 100 mg/kg, p.o.) were evaluated using chemical (formalin-induced licking) and thermal (hot-plate) models of nociception or inflammation (carrageenan-induced cell migration into the subcutaneous air pouch, SAP). The mechanism of antinociceptive activity was evaluated using opioid, cholinergic receptor antagonists (naloxone and atropine), or nitric oxide synthase inhibitor (L-NAME). EO and SA presented a central antinociceptive effect (the hot-plate model). In formalin-induced licking response, higher doses of EO and SA also reduced 1st and 2nd phases. None of the antagonists and enzyme inhibitor reversed antinociceptive effects. EO and SA reduced the leukocyte migration into the SAP, and the cytokines tumor necrosis factor and interleukin-1 (TNF-α and IL-1ß, respectively) produced in the exudate. Our results are indicative that EO and SA present peripheral and central antinociceptive and anti-inflammatory effects.

Melanin particles isolated from the fungus Fonsecaea pedrosoi activates the human complement system

BACKGROUND Melanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America. OBJECTIVE The aim of this study was to evaluate the effects of acid-basic extracted F. pedrosoi melanin particles and fungal cell ghosts obtained by Novozym 234 treatment on their ability to activate the human complement system. METHODS The ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). FINDINGS Unsensitised melanin particles and melanin ghosts presented complement consumption of 82.67 ± 2.08% and 96.04 ± 1.13%, respectively. Immunofluorescence assays revealed intense deposition of the C3 and C4 fragments on the surface of melanin particles and ghosts extracted from F. pedrosoi. Deposition of the C3, C4, and C5 fragments onto melanin samples and zymosan was confirmed by ELISA. Deposition of small amounts of C1q and C9 onto melanin samples and zymosan was detected by ELISA. CONCLUSION Fonsecaea pedrosoi melanin particles and fungal cell ghosts activated the complement system mainly through an alternative pathway.

Apoptosis-inducing effects of Melissa officinalis L. essential oil in glioblastoma multiforme cells.

Current therapies for glioblastoma multiforme (GBM) are not effective. This study investigated the activity of the M. officinalis essential oil (EO) and its major component (citral) in GBM cell lines. Both EO and citral decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation and caspase-3 activation. Antioxidant prevented citral-induced death, indicating its dependence on the production of reactive oxygen species. Citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). These results show that EO, through its major component, citral, may be of potential interest for the treatment of GBM.

Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes.

Abstract Lippia alba (Miller) N.E. Brown is an aromatic plant known locally as "Erva-cidreira-do-campo" that has great importance in Brazilian folk medicine. The aim of our study was to evaluate the antidermatophytic potential of linalool-rich essential oil (EO) from L. alba and analyze the ability of this EO to inhibit peptidase and keratinase activities, which are important virulence factors in dermatophytes. The minimum inhibitory concentrations (MICs) of L. alba EO were 39, 156 and 312 µg/mL against Trichophyton rubrum, Epidermophyton floccosum and Microsporum gypseum, respectively. To evaluate the influence of L. alba EO on the proteolytic and keratinolytic activities of these dermatophytes, specific inhibitory assays were performed. The results indicated that linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, and this inhibition could be a possible mechanism of action against dermatophytes. Due to the effective antidermatophytic activity of L. alba EO, further experiments should be performed to explore the potential of this linalool-rich EO as an alternative antifungal therapy.

Prevalence of Group B Streptococcus serotypes III and V in pregnant women of Rio de Janeiro, Brazil

GBS serotypes III and V were the most prevalent in pregnant women and exhibited resistance to tetracycline, clindamycin and sulfamethoxazole/trimethoprim. Serotype III showed high sialic acid content and PFGE analysis discerned 33 heterogeneous profiles. Phenotypic and genotypic characterization could be relevant to control GBS infections unaffected by intra-partum chemoprophylaxis.

Sialoglycoproteins in morphological distinct stages of Mucor polymorphosporus and their influence on phagocytosis by human blood phagocytes.

The possible role of sialic acids in host cells-fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.

Antimicrobial action and anti-corrosion effect against sulfate reducing bacteria by lemongrass (Cymbopogon citratus) essential oil and its major component, the citral.

The anti-corrosion effect and the antimicrobial activity of lemongrass essential oil (LEO) against the planktonic and sessile growth of a sulfate reducing bacterium (SRB) were evaluated. Minimum inhibitory concentration (MIC) of LEO and its major component, the citral, was 0.17 mg ml-1. In addition, both LEO and citral showed an immediate killing effect against SRB in liquid medium, suggesting that citral is responsible for the antimicrobial activity of LEO against SRB. Transmission electron microscopy revealed that the MIC of LEO caused discernible cell membrane alterations and formed electron-dense inclusions. Neither biofilm formation nor corrosion was observed on carbon steel coupons after LEO treatment. LEO was effective for the control of the planktonic and sessile SRB growth and for the protection of carbon steel coupons against biocorrosion. The application of LEO as a potential biocide for SRB growth control in petroleum reservoirs and, consequently, for souring prevention, and/or as a coating protection against biocorrosion is of great interest for the petroleum industries.

Does the essential oil of Lippia sidoides Cham. (pepper-rosmarin) affect its endophytic microbial community?

BACKGROUND: Lippia sidoides Cham., also known as pepper-rosmarin, produces an essential oil in its leaves that is currently used by the pharmaceutical, perfumery and cosmetic industries for its antimicrobial and aromatic properties. Because of the antimicrobial compounds (mainly thymol and carvacrol) found in the essential oil, we believe that the endophytic microorganisms found in L. sidoides are selected to live in different parts of the plant. RESULTS: In this study, the endophytic microbial communities from the stems and leaves of four L. sidoides genotypes were determined using cultivation-dependent and cultivation-independent approaches. In total, 145 endophytic bacterial strains were isolated and further grouped using either ERIC-PCR or BOX-PCR, resulting in 76 groups composed of different genera predominantly belonging to the Gammaproteobacteria. The endophytic microbial diversity was also analyzed by PCR-DGGE using 16S rRNA-based universal and group-specific primers for total bacteria, Alphaproteobacteria, Betaproteobacteria and Actinobacteria and 18S rRNA-based primers for fungi. PCR-DGGE profile analysis and principal component analysis showed that the total bacteria, Alphaproteobacteria, Betaproteobacteria and fungi were influenced not only by the location within the plant (leaf vs. stem) but also by the presence of the main components of the L. sidoides essential oil (thymol and/or carvacrol) in the leaves. However, the same could not be observed within the Actinobacteria. CONCLUSION: The data presented here are the first step to begin shedding light on the impact of the essential oil in the endophytic microorganisms in pepper-rosmarin.

Biological activities of α-pinene and ß-pinene enantiomers.

The antimicrobial activities of the isomers and enantiomers of pinene were evaluated against bacterial and fungal cells. The agar diffusion test showed that only the positive enantiomers of the α- and ß-isomers of pinene were active. The minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of these monoterpenes were also determined, confirming that the positive enantiomers exhibited microbicidal activity against all fungi and bacteria tested with MICs ranging from 117 to 4,150 µg/mL. However, no antimicrobial activity was detected with the negative enantiomers up to 20 mg/mL. Time-kill curves showed that (+)-α-pinene and (+)-ß-pinene were highly toxic to Candida albicans, killing 100% of inoculum within 60 min. By contrast, the bactericidal effect occurred after 6 h in methicillin-resistant Staphylococcus aureus (MRSA). In combination with commercial antimicrobials, ciprofloxacin plus (+)-α-pinene or (+)-ß-pinene presented synergistic activity against MRSA whereas an indifferent effect against all fungi was detected when amphotericin B was combined with the positive enantiomers of pinene. The potential of (+)-α-pinene and (+)-ß-pinene to inhibit phospholipase and esterase activities was also evaluated, and the best inhibition results were obtained with Cryptococcus neoformans. C. albicans biofilm formation was prevented with the MIC concentration of (+)-α-pinene and twice the MIC value of (+)-ß-pinene. Finally, the cytotoxicity of the positive enantiomers of pinene to murine macrophages was evaluated, and 250 µg/mL of (+)-α-pinene and (+)-ß-pinene reduced the cell viability to 66.8% and 57.7%, respectively.

Microbial colonization in orthodontic mini-implants

Peri-implant inflammation contributes for loss of secondary stability of orthodontic mini-implants. The investigation of microbial colonization in this area would benefit its control, and consequently favor the long-term success of mini-implants. Therefore, the aim of this study was to determine the establishment and the evolution of microbial colonization process in orthodontic mini-implants for 3 months, since the time of their installation. One-hundred and fifty samples collected from 15 mini-implants were investigated from baseline up to 3 months. The biological material was obtained from peri-implant area using paper points. Nonspecific, Streptococcus spp, Lactobacillus casei and Candida spp colonizations were analyzed by cell growth methods. Porphyromonas gingivalis colonization was observed by 16S rDNA-directed polymerase chain reaction. Data from cell growth were submitted to the Wilcoxon sign rank test and results from molecular analysis were presented in a descriptive way. There was no significant difference in the microbial colonization among the examined time intervals, except for Streptococcus spp, between baseline and 24 h, which characterized the initial colonization in this time interval. Lactobacillus casei and Candida spp colonizations were insignificant. No Porphyromonas gingivalis was detected among the analyzed samples. The microbial colonization of mini-implants did not significantly change during the study. However, it should be monitored by orthodontists, since it is an important factor for mini-implants success.

A inflamação peri-implantar contribui para a perda da estabilidade secundária dos mini-implantes ortodônticos. A investigação da colonização microbiana desta área beneficiaria o seu controle e, consequentemente, favoreceria o sucesso dos mini-implantes a longo prazo. Portanto, o objetivo dos autores foi determinar o estabelecimento e evolução do processo de colonização microbiana em mini-implantes ortodônticos por três meses desde a instalação. Cento e cinquenta amostras coletadas de 15 mini-implantes foram investigadas desde o tempo inicial até 3 meses. O material biológico foi obtido da área peri-implantar com auxílio de cones de papel absorvente. As colonizações inespecíficas de Streptococcus spp, Lactobacillus casei e Candida spp foram analisadas por métodos de crescimento celular. A colonização por Porphyromonas gingivalis foi observada por meio da reação em cadeia da polimerase 16S rDNA. Os dados do crescimento celular foram submetidos ao teste de Wilcoxon sign rank e os resultados da biologia molecular foram apresentados de modo descritivo. Não houve diferença estatisticamente significante da colonização microbiana entre os intervalos de tempo avaliados, exceto para Streptococcus spp entre os tempos inicial e 24 h, o que caracterizou o início da colonização neste intervalo de tempo. As colonizações por Lactobacillus casei e Candida spp foram insignificantes. Não foi detectada a presença de Porphyromonas gingivalis nas amostras analisadas. A colonização microbiana nos mini-implantes não se alterou significativamente durante o estudo. No entanto, deve ser monitorada por ortodontistas, uma vez que é um fator importante para o sucesso dos mini-implantes.

Phospholipase and esterase production by clinical strains of Fonsecaea pedrosoi and their interactions with epithelial cells.

Fonsecaea pedrosoi is the major etiologic agent of chromoblastomycosis. The virulence of F. pedrosoi is a meagerly explored phenomenon. The ability to interact with host cells and the production of hydrolytic enzymes are thought to be important virulence mechanisms of fungal pathogens. Here, we measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by three clinical strains of F. pedrosoi isolated from chromoblastomycosis lesions, as well as their capabilities to interact with epithelial cells. All the strains were excellent esterase producers, generating elevated hydrolytic halos after 5 days of growth. Conversely, phospholipase activity was detected only after 10 days, except for the most recent strain of F. pedrosoi (Magé) in which measurable phospholipase activity was detected on day 5. The ability to interact with epithelial cells was also investigated. Regarding the adhesion capability, an indirect connection was observed in relation to the adaptation time of each strain in axenic culture, in which Magé strain showed the best adhesion ability followed by LDI 11428 and 5VPL strains. Both 5VPL and Magé strains were also detected inside the epithelial cells, while the LDI 11428 strain was rarely detected in cytoplasmatic vacuolar compartments. Moreover, these F. pedrosoi strains were able to cause injury in epithelial cells.

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae.

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.

Antigiardial activity of Ocimum basilicum essential oil.

In this study, we investigated the effects of Ocimum basilicum essential oil on Giardia lamblia and on the modulation of the interaction of these parasites by peritoneal mouse macrophage. The essential oil (2 mg/ml) and its purified substances demonstrated antigiardial activity. Linalool (300 microg/ml), however, was able to kill 100% parasites after 1 h of incubation, which demonstrates its high antigiardial potential. Pretreatment of peritoneal mouse macrophages with 2 mg/ml essential oil dilution reduced in 79% the association index between these macrophages and G. lamblia, with a concomitant increase by 153% on nitric oxide production by the G. lamblia-ingested macrophages. The protein profiles and proteolitic activity of these parasite trophozoites, previously treated or not with 2 mg/ml essential oil or with the purified fractions, were also determined. After 1 and 2 h of incubation, proteins of lysates and culture supernatants revealed significant differences in bands patterns when compared to controls. Besides, the proteolitic activity, mainly of cysteine proteases, was clearly inhibited by the essential oil (2 mg/ml) and the purified linalool (300 microg/ml). These results suggest that, with G. lamblia, the essential oil from O. basilicum and its purified compounds, specially linalool, have a potent antimicrobial activity.

Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors.

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.

The major chromoblastomycosis fungal pathogen, Fonsecaea pedrosoi, extracellularly releases proteolytic enzymes whose expression is modulated by culture medium composition: implications on the fungal development and cleavage of key's host structures.

We investigated the possible secretion of peptidases by F. pedrosoi, when conidial cells were cultured in two distinct media. Aspartyl proteolytic activity was detected on the Czapeck-Dox-derived supernatant, which was blocked by pepstatin, and only active in extremely acidic conditions. The supernatant obtained after conidia growth in Kauffman medium presented metallopeptidase activity, which was active over a broad pH range and sensitive to 1,10-phenanthroline and EGTA. Additionally, both culture supernatants were able to cleave a wide range of proteinaceous substrates, including important human serum proteins (e.g. albumin and immunoglobulin G) and extracellular matrix components (e.g. fibronectin and laminin). As peptidases participate in different cellular metabolic pathways, we also tested the influence of proteolytic inhibitors on the F. pedrosoi conidia development in vitro. The metallopeptidase inhibitors, 1,10-phenanthroline, EGTA and EDTA, strongly abrogated the growth of conidial forms by approximately 95%, 85% and 60%, respectively. Moreover, 1,10-phenanthroline blocked the differentiation process from conidia to mycelia, an essential step during the F. pedrosoi life cycle. Phenylmethanesulfonyl fluoride, a serine peptidase inhibitor, slightly reduced the conidial growth, whereas proteolytic inhibitors of cysteine (E-64) and aspartic (pepstatin) type peptidases did not alter conidial developmental behavior. In summary, our results showed for the first time the expression of extracellular proteolytic activity by F. pedrosoi conidial cells.

Antileishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum.

Leishmaniasis is a group of diseases with a large spectrum of clinical manifestations caused by protozoans of the genus Leishmania. Here we demonstrate the leishmanicidal activity of the essential oil of Ocimum gratissimum as well as its main constituent, eugenol. The eugenol-rich essential oil of O. gratissimum progressively inhibited Leishmania amazonensis growth at concentrations ranging from 100 to 1000 microg/ml. The IC50 (sub-inhibitory concentration) of the essential oil for promastigotes and amastigotes were respectively 135 and 100 microg/ml and the IC50 of eugenol was 80 microg/ml for promastigote forms. L. amazonensis exposed to essential oil at concentrations corresponding to IC50 for promastigotes and for amastigotes underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Two or more nuclei or flagella were observed in 31% and 23.3% of treated amastigote and promastigote forms, respectively, suggesting interference in cell division. Considerable mitochondrial swelling was observed in essential oil-treated promastigotes and amastigotes, which had the inner mitochondrial membrane altered, with a significant increase in the number of cristae; in some amastigotes the mitochondrial matrix became less electron-dense. The minimum inhibitory concentration for both promastigotes and amastigotes was 150 microg/ml. Pretreatment of mouse peritoneal macrophages with 100 and 150 microg/ml essential oil reduced the indices of association between promastigotes and the macrophages, followed by increased in nitric oxide production by the infected macrophages. The essential oil showed no cytototoxic effects against mammalian cells. This set of results suggests that O. gratissimum essential oil and its compounds could be used as sources for new antileishmanial drugs.

Melissa officinalis L. essential oil: antitumoral and antioxidant activities.

Melissa officinalis L (lemon balm) is a traditional herbal medicine used widely as a mild sedative, spasmolytic and antibacterial agent. This paper focuses on the analysis of the chemical composition and the biological activities of M. officinalis essential oil obtained under controlled harvesting and drying conditions. An in-vitro cytotoxicity assay using MTT indicated that this oil was very effective against a series of human cancer cell lines (A549, MCF-7, Caco-2, HL-60, K562) and a mouse cell line (B16F10). This oil possessed antioxidant activity, as evidenced by reduction of 1,1-diphenyl-2-picryl-hydrazyl (DPPH). These results pointed to the potential use of M. officinalis essential oil as an antitumoral agent.

The effect of a catechin-rich extract of Cocos nucifera on lymphocytes proliferation.

Catechins are polyphenols with antioxidant activity. The fruit Cocos nucifera (Palmae) has a fi ber husk rich in catechins and the local population of northeast Brazil uses it as a medicine against various diseases. An anti-bacterial and anti-viral activity has been already observed using this substance. Plant extracts, from other sources, rich in catechins are inhibitory to tumour cells, suppressing their proliferation. The aim of the present work was to verify if catechins isolated from Cocos nucifera were capable of inhibiting cell proliferation. An extract obtained from Cocos nucifera was purified through adsorption chromatography using the resin XAD-2. The purified material was used in cultures of an erythroleukaemia cell line (K562) and on normal human peripheral blood lymphocytes. Cell viability was assessed using MTT. Cellular proliferation was measured by [(3)H]-thymidine incorporation and cell cycle analysis in a fl ow cytometer. A dose-dependent inhibitory effect was observed on tumour cells and on lymphocytes activated by phytohemaglutinin (PHA) or phorbol ester. For PHA this effect was irreversible being already established on the fi rst four hours of culture.

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.

Antileishmanial activity of a linalool-rich essential oil from Croton cajucara.

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.