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1.
Mem Inst Oswaldo Cruz ; 117: e220065, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35920504

RESUMO

BACKGROUND: Leishmania (Mundinia) enriettii is a species commonly found in the guinea pig, Cavia porcellus. Although it is a dermotropic species, there is still an uncertainty regarding its ability to visceralise during Leishmania life cycle. OBJECTIVE: Here, we investigated the ability of L. enriettii (strain L88) to visceralise in lungs, trachea, spleen, and liver of C. porcellus, its natural vertebrate host. METHODS: Animals were infected sub-cutaneously in the nose and followed for 12 weeks using histological (hematoxilin-eosin) and molecular tools (polymerase chain reaction-restriction fragment length polymorphism - PCR-RFLP). To isolate parasite from C. porcellus, animals were experimentally infected for viscera removal and PCR typing targeting hsp70 gene. FINDINGS: Histological analysis revealed intense and diffuse inflammation with the presence of amastigotes in the trachea, lung, and spleen up to 12 weeks post-infection (PI). Molecular analysis of paraffin-embedded tissues detected parasite DNA in the trachea and spleen between the 4th and 8th weeks PI. At the 12th PI, no parasite DNA was detected in any of the organs. To confirm that the spleen could serve as a temporary site for L. enriettii, we performed additional in vivo experiments. During 6th week PI, the parasite was isolated from the spleen confirming previous histopathological and PCR observations. MAIN CONCLUSION: Leishmania enriettii (strain L88) was able to visceralise in the trachea, lung, and spleen of C. porcellus.


Assuntos
Leishmania enriettii , Leishmania , Animais , Cobaias , Leishmania/genética , Pulmão , Baço , Traqueia
2.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 744-748, May-June, 2020. ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1128947

RESUMO

Leishmaniasis is a parasitic disease of worldwide spread. It is caused by protozoa of the genus Leishmania and is transmitted to animals and humans through the bite of sand flies. In Brazil, leishmaniasis is one of the zoonoses of major importance and expansion. The objective of this work is to describe the clinical, pathological, immunohistochemical and molecular findings of cutaneous leishmaniasis by Leishmania enriettii in guinea pig (Cavia porcellus). Three animals had nodular and alopecia lesions on the muzzle, ears and ulcerated lesions on the distal extremities of the pelvic limbs. The males (2) also had diffuse thickening of the scrotal skin. Samples of the ulcerated cutaneous lesions were evaluated by cytology which were observed as amastigote forms of Leishmania. One of the animals was euthanized and necropsied. Histopathology showed abundant dermal infiltrate of macrophages, plasma cells, lymphocytes and multinucleated giant cells. Numerous macrophages contained parasitoid vacuoles with amastigote forms, evidenced by immunohistochemical examination. The molecular characterization based on the SSUrDNA gene identified the species as L. enrietti. The diagnosis of cutaneous leishmaniasis in these cases was based on pathological findings and confirmed by immunohistochemistry, PCR and sequencing.(AU)


A leishmaniose é uma doença parasitária de distribuição mundial. É causada por protozoários do gênero Leishmania e é transmitida para animais e seres humanos por meio da picada de flebotomíneos. No Brasil, a leishmaniose é uma das zoonoses de maior importância e expansão. O objetivo deste trabalho é descrever os achados clínicos, patológicos, imuno-histoquímicos e moleculares de leishmaniose cutânea por Leishmania enriettii em cobaia (Cavia porcellus). Três animais apresentavam lesões nodulares e alopécicas no focinho e orelhas, além de lesões ulceradas nas extremidades distais dos membros pélvicos. Nos machos (2), foi observado espessamento difuso da pele escrotal. Amostras das lesões cutâneas ulceradas foram avaliadas por citologia, nas quais foram observadas formas amastigotas de Leishmania. Um dos animais foi submetido à eutanásia e necropsiado. Na histopatologia, foi observado infiltrado dérmico abundante de macrófagos, plasmócitos, linfócitos e com células gigantes multinucleadas. Numerosos macrófagos continham vacúolos parasitóforos com formas amastigotas, evidenciados por meio do exame de imuno-histoquímica. A caracterização molecular baseada no gene de SSUrDNA identificou a espécie como L. enrietti. O diagnóstico de leishmaniose cutânea nesses casos foi baseado nos achados patológicos e confirmado pelas técnicas de imuno-histoquímica, PCR e sequenciamento.(AU)


Assuntos
Animais , Cobaias , Leishmaniose Cutânea/veterinária , Leishmania enriettii/isolamento & purificação , Cobaias/microbiologia , Imuno-Histoquímica/veterinária , Zoonoses , Reação em Cadeia da Polimerase/veterinária , Alopecia
4.
PLoS Negl Trop Dis ; 9(9): e0004060, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26367424

RESUMO

BACKGROUND: Despite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date. METHODOLOGY/PRINCIPAL FINDINGS: Females from colonies of the midges Culicoides nubeculosus Meigen and C. sonorensis Wirth & Jones and the sand fly Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae) were experimentally infected with two different species of Leishmania, originating from Australia (Leishmania sp. AM-2004) and Brazil (Leishmania enriettii). In addition, the infectivity of L. enriettii infections generated in guinea pigs and golden hamsters for Lu. longipalpis and C. sonorensis was tested by xenodiagnosis. Development of L. enriettii in Lu. longipalpis was relatively poor compared to other Leishmania species in this permissive vector. Culicoides nubeculosus was not susceptible to infection by parasites from the L. enriettii complex. In contrast, C. sonorensis developed late stage infections with colonization of the thoracic midgut and the stomodeal valve. In hamsters, experimental infection with L. enriettii led only to mild symptoms, while in guinea pigs L. enriettii grew aggressively, producing large, ulcerated, tumour-like lesions. A high proportion of C. sonorensis (up to 80%) feeding on the ears and nose of these guinea pigs became infected. CONCLUSIONS/SIGNIFICANCE: We demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".


Assuntos
Ceratopogonidae/parasitologia , Insetos Vetores , Leishmania enriettii/isolamento & purificação , Animais , Feminino , Trato Gastrointestinal/parasitologia , Cobaias , Leishmaniose/parasitologia , Leishmaniose/patologia , Leishmaniose/transmissão , Mesocricetus , Psychodidae/parasitologia
5.
Int J Parasitol ; 45(11): 679-84, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26099650

RESUMO

An active case detection approach with PCR diagnosis was used in the Ho District of the Volta Region, Ghana that identified individuals with active cutaneous leishmaniasis. Three isolates were successfully cultured and DNA sequences from these were analysed (ribosomal RNA internal transcribed spacer 1; ribosomal protein L23a intergenic spacer; RNA polymerase II large subunit), showing them to be Leishmania, identical to each other but different from all other known Leishmania spp. Phylogenetic analysis showed the parasites to be new members of the Leishmania enriettii complex, which is emerging as a possible new subgenus of Leishmania parasites containing human pathogens.


Assuntos
Leishmania enriettii/classificação , Leishmania enriettii/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Feminino , Gana , Humanos , Leishmania enriettii/genética , Leishmaniose Cutânea/diagnóstico , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , RNA Polimerase II/genética , Proteínas Ribossômicas/genética , Análise de Sequência de DNA , Adulto Jovem
6.
Parasit Vectors ; 8: 31, 2015 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-25595203

RESUMO

BACKGROUND: Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. METHODS: Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. RESULTS: LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(ß1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF-α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). CONCLUSION: The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.


Assuntos
Glicolipídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Leishmania enriettii/fisiologia , Leishmaniose/parasitologia , Fosfolipídeos/metabolismo , Animais , Células CHO , Cricetulus , Reservatórios de Doenças , Cobaias , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Óxido Nítrico , Psychodidae/parasitologia
7.
PLoS Negl Trop Dis ; 8(12): e3339, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474647

RESUMO

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis.


Assuntos
Leishmania enriettii/isolamento & purificação , Leishmaniose Visceral/parasitologia , Filogenia , Humanos , Leishmania enriettii/classificação , Leishmania enriettii/genética , Leishmaniose Visceral/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Tailândia
8.
J Med Entomol ; 49(5): 967-70, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23025175

RESUMO

Biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae) have recently been implicated as vectors of kinetoplastid parasites in the Leishmania enrietti complex. This study assesses susceptibility of one of the few successfully colonized Ceratopogonidae, Culicoides nubeculosus Meigen, to infection with Leishmania parasites infecting humans. While Leishmania infantum initially developed in the midgut of C. nubeculosus until 2 d postfeeding, parasite populations on day 3 were considerably reduced. Despite this, a polymerase chain reaction-based assay continued to indicate presence of L. infantum for up to 7 d after the bloodmeal. These findings are discussed within the wider context of implicating arthropods as vectors of Leishmania and it is suggested that conventional polymerase chain reaction use in vector-competence studies should be accompanied by direct microscopical observations.


Assuntos
Ceratopogonidae/parasitologia , Insetos Vetores/fisiologia , Leishmania enriettii/fisiologia , Animais , Feminino , Interações Hospedeiro-Parasita , Leishmaniose/transmissão
9.
J Antimicrob Chemother ; 63(6): 1179-90, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19377065

RESUMO

OBJECTIVES: The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites. METHODS: Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effects of different combinations of quinacrine and an apigenin dimer on modulating the pentamidine resistance levels of LePentR50 and LdAG83PentR50 using an MTS proliferation assay. We then measured the accumulation level of pentamidine using HPLC. The fractional inhibitory concentration index (FICI) method was used to evaluate the interaction between quinacrine and the apigenin dimer on reversing pentamidine resistance in Leishmania. RESULTS: LePentR50 and LdAG83PentR50 promastigotes were approximately 8.6- and 4.6-fold more resistant to pentamidine than their wild-type parents. Amastigotes derived from LePentR50 and LdAG83PentR50 were also pentamidine-resistant. We found that quinacrine can increase the susceptibility of Leishmania to pentamidine. Quinacrine, when used at 6 microM, can increase the IC(50) of pentamidine by 3.8-, 3.4-, 3.5- and 6.3-fold in wild-type Leishmania enriettii Le, LePentR50, wild-type Leishmania donovani LdAG83 and LdAG83PentR50, respectively. Quinine, quinidine and verapamil did not show any sensitizing effect. The sensitizing effect of quinacrine was: (i) dose-dependent; (ii) not associated with an increase in pentamidine accumulation; and (iii) only observed in pentamidine-resistant but not sodium stibogluconate-resistant or vinblastine-resistant parasites. Other than quinacrine, we also found that an apigenin dimer (compound 9d), previously shown to be able to inhibit ABCB1-mediated cancer drug resistance in mammalian cells, can also increase the pentamidine susceptibility of Leishmania. 9d, when used at 6 microM, can increase the IC(50) of pentamidine by 2.5-, 4.2-, 1.6- and 1.9-fold in Le, LePentR50, LdAG83 and LdAG83PentR50, respectively. Unlike quinacrine, sensitization by 9d was accompanied by an increase in pentamidine accumulation, presumably due to the inhibition of an ABC transporter. Using the FICI method, we found that quinacrine and 9d can act synergistically. When they are used in a 1:1 ratio, they sensitize LePentR50 to pentamidine by 19-fold, with an FICI of 0.48 (P < 0.005), indicating that they might act synergistically. CONCLUSIONS: Our findings support the notion that the pentamidine susceptibility of Leishmania is mediated by multiple targets. Quinacrine and apigenin dimer 9d, each inhibiting its own target, can have a synergistic effect when used together to sensitize Leishmania to pentamidine.


Assuntos
Antiprotozoários/farmacologia , Apigenina/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmania enriettii/efeitos dos fármacos , Pentamidina/farmacologia , Quinacrina/farmacologia , Animais , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Concentração Inibidora 50
10.
Antimicrob Agents Chemother ; 51(3): 930-40, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17194831

RESUMO

Drug resistance by overexpression of ATP-binding cassette (ABC) transporters is an impediment in the treatment of leishmaniasis. Flavonoids are known to reverse multidrug resistance (MDR) in Leishmania and mammalian cancers by inhibiting ABC transporters. Here, we found that synthetic flavonoid dimers with three (compound 9c) or four (compound 9d) ethylene glycol units exhibited a significantly higher reversing activity than other shorter or longer ethylene glycol-ligated dimers, with approximately 3-fold sensitization of pentamidine and sodium stibogluconate (SSG) resistance in Leishmania, respectively. This modulatory effect was dosage dependent and not observed in apigenin monomers with the linker, suggesting that the modulatory effect is due to its bivalent nature. The mechanism of reversal activity was due to increased intracellular accumulation of pentamidine and total antimony in Leishmania. Compared to other MDR modulators such as verapamil, reserpine, quinine, quinacrine, and quinidine, compounds 9c and 9d were the only agents that can reverse SSG resistance. In terms of reversing pentamidine resistance, 9c and 9d have activities comparable to those of reserpine and quinacrine. Modulators 9c and 9d exhibited reversal activity on pentamidine resistance among LeMDR1(-/-), LeMDR1(+/+), and LeMDR1-overexpressed mutants, suggesting that these modulators are specific to a non-LeMDR1 pentamidine transporter. The LeMDR1 copy number is inversely related to pentamidine resistance, suggesting that it might be involved in importing pentamidine into the mitochondria. In summary, bivalency could be a useful strategy for the development of more potent ABC transporter modulators and flavonoid dimers represent a promising reversal agent for overcoming pentamidine and SSG resistance in parasite Leishmania.


Assuntos
Antifúngicos/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Flavonoides/farmacologia , Leishmania enriettii/efeitos dos fármacos , Pentamidina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Apigenina/farmacologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Flavonoides/química , Leishmania enriettii/genética , Espectrometria de Massas , Relação Estrutura-Atividade
11.
Mol Biochem Parasitol ; 150(2): 278-87, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17018238

RESUMO

In parasitic protozoan Leishmania enriettii, the role of a multidrug resistance (mdr) gene LeMDR1 (L. enriettii multidrug resistance 1) in mediating vinblastine resistance has been previously demonstrated by association, transfection and "gene knockout" studies. LeMDR1 has been shown to be located intracellularly and it was proposed to mediate drug resistance by sequestering drugs into intracellular organelles rather than by active efflux. Here we compared LeMDR1 overexpressed cell lines (Vint3 and V160), wild type (Le) and LeMDR1 "double knockout" mutant (LeMDR1-/-) and demonstrated that LeMDR1 gene copy number was associated with (1) higher level of intracellular iron, (2) increased sensitivity to an iron-dependent antibiotic, streptonigrin and (3) increased enzyme activity of an iron-sulfur-containing mitochondrial enzyme, aconitase. This result suggests that the normal function of LeMDR1 is related to mitochondrial iron homeostasis. To test such hypothesis, we have used the LeMDR1-overexpressing mutant V160 and LeMDR1-/- mutant to determine how iron level can affect its resistance level to drugs targeting either cytosol (vinblastine) or mitochondria (rhodamine 123 and pentamidine). It was found that the resistance level of V160 to vinblastine can be increased by iron whereas resistance to both rhodamine 123 and pentamidine can be increased by iron depletion and vice versa. Iron treatment can potentiate rhodamine 123 and pentamidine accumulation whereas iron deprivation can cause the reduction of rhodamine 123 accumulation. Our result highly suggests that LeMDR1's function in mediating drug resistance is iron-dependent.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antiprotozoários/farmacologia , Resistência a Múltiplos Medicamentos , Ferro/metabolismo , Leishmania enriettii/efeitos dos fármacos , Proteínas de Protozoários/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Aconitato Hidratase/metabolismo , Animais , Dosagem de Genes , Homeostase/fisiologia , Concentração Inibidora 50 , Proteínas Ferro-Enxofre/metabolismo , Leishmania enriettii/genética , Leishmania enriettii/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Pentamidina/farmacologia , Proteínas de Protozoários/genética , Rodamina 123/farmacologia , Vimblastina/farmacologia
12.
Mol Biochem Parasitol ; 135(1): 89-100, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15287590

RESUMO

Previous studies have established that the ISO1 glucose transporter of Leishmania enriettii resides primarily in the flagellar membrane, whereas the ISO2 glucose transporter is located in the pellicular plasma membrane surrounding the cell body. This pronounced difference in subcellular targeting is conferred by the NH2-terminal domain of the transporters, since this is the only region of the two permeases that differs in sequence. Analysis of the 130 residue NH2-terminal domain of ISO1 using multiple terminal deletion mutants and various internal deletion mutants established that a sequence located between amino acids 84 and 100 of this domain is required for flagellar trafficking. In addition, chimeras between ISO1 and ISO2 indicated that the region between residues 110 and 118 of ISO1 is also required for flagellar targeting. These results imply that flagellar targeting information for this integral membrane protein does not constitute a simple linear sequence of amino acids but is at least bipartite in structure.


Assuntos
Flagelos/metabolismo , Leishmania enriettii/genética , Leishmania enriettii/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Genes Reporter , Proteínas de Membrana/química , Microscopia de Fluorescência , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Deleção de Sequência
13.
Mol Microbiol ; 51(6): 1563-75, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15009885

RESUMO

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Leishmania enriettii/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose/tratamento farmacológico , Proteínas de Protozoários/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antiprotozoários/farmacologia , Transporte Biológico , Linhagem Celular , Resistência a Múltiplos Medicamentos/genética , Retículo Endoplasmático/metabolismo , Fluoresceínas/farmacologia , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Leishmania enriettii/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Proteínas de Protozoários/análise , Proteínas Recombinantes de Fusão/metabolismo , Rodamina 123/farmacologia , Vimblastina/farmacologia
14.
Acta Trop ; 76(2): 131-8, 2000 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-10936572

RESUMO

A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, Leishmania infanturn, Leishmania enriettii, and Leishmania major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.


Assuntos
Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Naftoquinonas/farmacologia , Animais , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania enriettii/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/parasitologia
15.
J Biol Chem ; 274(41): 29543-8, 1999 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-10506220

RESUMO

The surface membranes of eukaryotic flagella and cilia are contiguous with the plasma membrane. Despite the absence of obvious physical structures that could form a barrier between the two membrane domains, the lipid and protein compositions of flagella and cilia are distinct from the rest of the cell surface membrane. We have exploited a flagellar glucose transporter from the parasitic protozoan Leishmania enriettii as a model system to characterize the first targeting motif for a flagellar membrane protein in any eukaryotic organism. In this study, we demonstrate that the flagellar membrane-targeting motif is recognized by several species of Leishmania. Previously, we demonstrated that the 130 amino acid NH(2)-terminal cytoplasmic domain of isoform 1 glucose transporter was sufficient to target a nonflagellar integral membrane protein into the flagellar membrane. We have now determined that an essential flagellar targeting signal is located between amino acids 20 and 35 of the NH(2)-terminal domain. We have further analyzed the role of specific amino acids in this region by alanine replacement mutagenesis and determined that single amino acid substitutions did not abrogate targeting to the flagellar membrane. However, individual mutations located within a cluster of five contiguous amino acids, RTGTT, conferred differences in the degree of targeting to the flagellar membrane and the flagellar pocket, implying a role for these residues in the mechanism of flagellar trafficking.


Assuntos
Flagelos/química , Leishmania enriettii/metabolismo , Proteínas de Membrana/química , Proteínas de Protozoários , Animais , Imunofluorescência , Proteínas de Membrana/genética , Microscopia Confocal , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Mutagênese , Proteínas Recombinantes de Fusão , Transfecção
16.
Parasitol Res ; 84(7): 559-64, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9694372

RESUMO

Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.


Assuntos
Bisbenzimidazol , Corantes Fluorescentes , Leishmania enriettii/isolamento & purificação , Pneumocystis/isolamento & purificação , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Núcleo Celular/ultraestrutura , Feminino , Leishmania enriettii/ultraestrutura , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Pulmão/microbiologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Monócitos/parasitologia , Pneumocystis/ultraestrutura , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/microbiologia , Ratos , Ratos Wistar
17.
Exp Parasitol ; 87(2): 98-111, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9326885

RESUMO

Intracellular killing of Leishmania parasites within activated murine macrophages is thought to result from the toxic activities of nitrogen oxidation products (referred to as NO) released by the activated cells. In order to determine possible mechanisms of NO toxicity for these microorganisms, promastigotes of Leishmania major and Leishmania enriettii were exposed to NO generated chemically from acidified nitrite, S-nitrosocysteine, diethylamine NONOate, or nitroprusside. Treatment with these agents led to loss of viability (as determined from decreased motility and inhibition of [3H]TdR uptake upon reincubation in NO-free medium) with kinetics characteristic for each compound L. major was less sensitive to these effects than L. enriettii, and amastigotes displayed the same sensitivity as promastigotes of the same species. The early effects of NO toxicity could be detected within minutes of exposure to the NO donors; they included decreased respiration rate and inhibition of glucose, proline, and adenine incorporation. Inhibition of the activities of glyceraldehyde 3-phosphate dehydrogenase and of aconitase were also evidenced. In order to determine whether these phenomena reflected the mechanisms of toxicity of bona fide NO generated by macrophages, promastigotes were exposed to IFN-gamma + LPS-activated macrophages across permeable membranes. This resulted in marked inhibition of proline and adenine uptake in the parasites, which was restored, however, to control levels when macrophages were activated in the presence of the nitric oxide synthase inhibitor NGMMA. These results indicate that several cellular targets may be subject to NO toxicity in Leishmania parasites, including enzymes of glycolysis and respiratory metabolism as well as trans-membrane transport systems.


Assuntos
Leishmania/efeitos dos fármacos , Nitrogênio/metabolismo , S-Nitrosotióis , Aconitato Hidratase/metabolismo , Adenina/metabolismo , Animais , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/toxicidade , Relação Dose-Resposta a Droga , Feminino , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Cobaias , Hidrazinas/metabolismo , Hidrazinas/toxicidade , Concentração de Íons de Hidrogênio , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Leishmania enriettii/efeitos dos fármacos , Leishmania enriettii/crescimento & desenvolvimento , Leishmania enriettii/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Leishmania major/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Nitrogênio/toxicidade , Óxidos de Nitrogênio , Oxirredução , Consumo de Oxigênio , Prolina/metabolismo , Nitrito de Sódio/metabolismo , Nitrito de Sódio/toxicidade
18.
Mem. Inst. Oswaldo Cruz ; 92(3): 377-87, May-Jun. 1997. ilus
Artigo em Inglês | LILACS | ID: lil-189311

RESUMO

There are 20 named species of the genus Leishmania at present recognized in the New York, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have till now been found to cause human leismaniasis; namely, Leishmania (Leishmania) enrietti, L. (L.) deanei, L. (L.) aristidesi, L. (L.) forattinii and L. (Viannia) equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s) and sandfly vector of L. (L.) enrietti. Doubt is cast on the validity of the species L. herreri, described in Costa Rica sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors.


Assuntos
Animais , Leishmania enriettii/microbiologia , Leishmania/classificação , Artrópodes/parasitologia , Brasil , América Latina
19.
Mem Inst Oswaldo Cruz ; 92(3): 377-87, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9332605

RESUMO

There are 20 named species of the genus Leishmania at present recognized in the New World, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have not till now been found to cause human leishmaniasis; namely, Leishmania (Leishmania) enriettii, L. (L.) hertigi, L. (L.) deanei, L. (L.) aristidesi, L. (L.) forattinii and L. (Viannia) equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s) and sandfly vector of L. (L.) enriettii. Doubt is cast on the validity of the species L. herreri, described in Costa Rican sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors.


Assuntos
Leishmania/fisiologia , Animais , Leishmania/classificação , Leishmania enriettii/fisiologia
20.
J Cell Biol ; 139(7): 1775-83, 1997 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-9412471

RESUMO

The major glucose transporter of the parasitic protozoan Leishmania enriettii exists in two isoforms, one of which (iso-1) localizes to the flagellar membrane, while the other (iso-2) localizes to the plasma membrane of the cell body, the pellicular membrane. These two isoforms differ only in their cytosolic NH2-terminal domains. Using immunoblots and immunofluorescence microscopy of detergent-extracted cytoskeletons, we have demonstrated that iso-2 associates with the microtubular cytoskeleton that underlies the cell body membrane, whereas the flagellar membrane isoform iso-1 does not associate with the cytoskeleton. Deletion mutants that remove the first 25 or more amino acids from iso-1 are retargeted from the flagellum to the pellicular membrane, suggesting that these deletions remove a signal required for flagellar targeting. Unlike the full-length iso-1 protein, these deletion mutants associate with the cytoskeleton. Our results suggest that cytoskeletal binding serves as an anchor to localize the iso-2 transporter within the pellicular membrane, and that the flagellar targeting signal of iso-1 diverts this transporter into the flagellar membrane and away from the pellicular microtubules.


Assuntos
Citoesqueleto/metabolismo , Leishmania enriettii/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Sítios de Ligação , Fracionamento Celular , Detergentes/farmacologia , Epitopos de Linfócito B , Flagelos/metabolismo , Transportador de Glucose Tipo 2 , Leishmania enriettii/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Octoxinol/farmacologia , Sinais Direcionadores de Proteínas/metabolismo , Ratos , Solubilidade
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