Stachys lavandulifolia Vahl. exhibits promising in vitro and in vivo antileishmanial activity against Leishmania major infection

@#This study aimed to consider the in vitro and in vivo effects of the Stachys lavandulifolia methanolic extract (SLME) (2.5, 5, 10, 25, 50, 100 µg/mL) against Leishmania major infection. The in vitro antileishmanial effects of SLME was studies on promastigote and amastigote forms of L. major. The effect of SLME on the nitric oxide (NO) and apoptosis, secretion of Th1/2 cytokines, and infectivity rate in macrophages cells were also studies. The cytotoxicity of SLME on human (THP-1) and murine (J774-A1 cell) macrophage cells was investigated through the measuring the 50% cytotoxic concentrations (CC50). Moreover, the in vivo effects of SLME for healing the cutaneous leishmaniasis (CL) lesions in infected BALB/c mice studied by assessing the lesions size and the parasite load during four weeks of treatment. The calculated 50% inhibitory concentration (IC50) valuesfor SLME and meglumine antimoniate (MA) against the promastigote stage were 23.4 and 71.1 µg/mL, respectively. For amastigote stage, the IC50 values for SLME and MA were 39.3 µg/mL and 44.3 µg/mL, respectively. Followed by 28 days’ topically therapy with SLME at doses of 50 and 100 mg/kg/day, the CL lesions size as well as parasite load were significantly (p<0.001) reduced; such that the recovery percentage of the infected mice was 80% and 97% after treatment with SLME at the dose of 50 and 100 mg/kg, respectively. SLME also markedly induced the NO production and apoptosis; whereas decreased infection rate in macrophage cells. After incubation of infected macrophages with SLME, the level interferon gamma was meaningfully (p<0.001) elevated as a dose-dependent response; in contrast, release of interleukin 10 (IL-10) and IL-4 markedly (p<0.001) decreased. The CC50 value for SLME against THP-1 and J774-A1 cell was 996.4 µg/mL and 741.3 µg/mL, respectively. The calculated selectivity index of >10 for SLME and MA confirmed their specificity to amastigotes and the low toxicity for macrophages. Our results showed the potent effects of SLME in eliminating and controlling Leishmania parasites in both in vitro and in vivo assays. Based on the current experimental study, SLME can be suggested as an alternative medicine for the isolation and production of a new agent for treating CL caused by L. major. Although, we found some cellular mechanisms of SLME against Leishmania parasites, but, additional surveys are necessary to specify the accurate mechanisms of action, toxicity, and its efficacy mainly in human subjects.

Observation on growth and reproduction of various Leishmania isolates in different culture media / 中国血吸虫病防治杂志

Objective To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. Methods A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. Results The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. Conclusions The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

Observation on growth and reproduction of various Leishmania isolates in different culture media / 中国血吸虫病防治杂志

Objective To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. Methods A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. Results The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. Conclusions The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 µg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 µg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC50) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r) (a furvina-containing antifungal ointment) for the treatment of CL.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves / 中国天然药物

AIM@#The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study.@*METHOD@#In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope.@*RESULTS@#The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 μgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine.@*CONCLUSION@#This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis.

Atividade antiparasitária in vitro e citotóxica de cariofileno e eugenol contra Trypanossoma cruzi e Leishmania brasiliensis / Actividad antiparasitaria in vitro citotóxica de cariofileno y eugenol contra Trypanosoma cruzi y Leishmania brasiliensis / In vitro cytotoxic and antiparasitic activity of caryophyllene and eugenol against Trypanosoma cruzi and Leishmania brasiliensis

Introdução: as doenças negligenciadas persistem por conta de falhas da ciência e acometem principalmente países em desenvolvimento, como exemplos podemos citar a doença de Chagas e a leishmaniose. Objetivo: avaliar o potencial antiparasitário in vitro de um terpenóide componente de óleo essencial, o cariofileno e o eugenol, contra as formas epimastigota e promastigota de T. cruzi e L. brasiliensis, respectivamente, bem como verificar sua citotoxicidade em células de mamíferos. Métodos: para os estudos in vitro de T. cruzi, foi usado o clone B5-CL, estavelmente transfectadas com o gene de Escherichia coli ß-galactosidase (lacZ). Os ensaios de inibição de promastigotas foram realizadas utilizando a estirpe de L. braziliensis, cultivadas a 22 ºC em meio de Schneider de Drosophila suplementado com FBS a 20 %. Para os testes de atividade antiepimastigota, antipromastigota foram utilizados placas de 96 poços com culturas que não tinham atingido a fase estacionária. Os ensaios de citotoxicidade utilizado estirpe de fibroblastos NCTC929 cultivadas em Meio Essencial Mínimo (Sigma). A viabilidade dessas linhagens através da utilização de resazurina como um método colorimétrico. Resultados: as substâncias cariofileno e eugenol foram testadas quanto à atividade antiepimastigota, antipromastigota e quanto à citotoxidade. Foi visto um efeito clinicamente relevante do cariofileno contra os parasitas T. cruzi e L. brasiliensi. Conclusões: os resultados mostram que o cariofileno obteve um melhor resultado quando comparado ao eugenol, sendo capaz de inibir o crescimento dos parasitas testados mostrando uma alternativa contra T. cruzi e L. brasiliensi. Em relação à citotoxidade novos testes deverão ser realizados para futuros testes in vivo.

Introducción: las enfermedades desatendidas persisten debido a los fracasos de la ciencia y afectan principalmente a los países en desarrollo, como ejemplos se pueden citar la enfermedad de Chagas y la leishmaniasis. Objetivo: evaluar el potencial de un componente in vitro terpenoide antiparasitario aceite esencial, el cariofileno y eugenol contra epimastigotes y promastigotes de Trypanosoma cruzi y Leishmania brasiliensis, respectivamente, así como comprobar su citotoxicidad en células de mamífero. Métodos: para los estudios in vitro de Trypanosoma cruzi, se utilizó el clon B5-CL, transfectadas establemente con el gen de la ß galactosidasa de Escherichia coli-(lacZ). Los ensayos de inhibición se realizaron con la cepa promastigotes de Leishmania braziliensis, se cultivó a 22 °C en medio de Schneider de Drosophila suplementado con FBS 20 %. Para la actividad de las pruebas antiepimastigota y antipromastigota, se utilizaron placas de 96 pocillos con cultivos que no habían alcanzado la fase estacionaria. En los ensayos de citotoxicidad se utilizó NCTC929, cepa de fibroblastos cultivados en medio esencial mínimo (Sigma). La viabilidad de estas cepas se evaluó mediante el uso de la resazurina como un método colorimétrico. Resultados: las sustancias cariofileno y eugenol se ensayaron para determinar la actividad antiepimastigota y antipromastigota, respecto a citotoxicidad. Se observó un efecto clínicamente relevante de cariofileno contra los parásitos Trypanosoma cruzi y Leishmania brasiliensis. Conclusiones: los resultados muestran que con el cariofileno se ha obtenido un mejor resultado en comparación con eugenol, siendo capaz de inhibir el crecimiento de parásitos que presentan una alternativa probada contra Trypanosoma cruzi y Leishmania brasiliensis. En cuanto a las pruebas de citotoxicidad, nuevos estudios deben llevarse a cabo para realizar más pruebas in vivo.

Introduction: neglected diseases continue to exist due to failures in science. They mainly affect developing countries. Two examples of neglected diseases are Chagas' disease and leishmaniasis. Objective: evaluate the potential of an in vitro antiparasitic terpenoid essential oil component, caryophyllene and eugenol, against epimastigotes and promastigotes of Trypanosoma cruzi and Leishmania brasiliensis, respectively, and verify their cytotoxicity in mammal cells. Methods: for in vitro studies of Trypanosoma cruzi, the B5-CL clone was used, stably transfected with the beta-galactosidase gene of Escherichia coli-(lacZ). Inhibition assays were performed on the promastigote strain of Leishmania braziliensis, cultured at 22°C in a Schneider's medium of Drosophila supplemented with 20% FBS. For anti-epimastigote and anti-promastigote activity tests, 96-well plates were used with cultures which had not reached their stationary stage. NCTC929 was used in cytotoxicity tests, a strain of fibroblasts cultured in minimum essential medium (Sigma). The viability of these strains was evaluated using resazurine as colorimetric method. Results: the substances caryophyllene and eugenol were assayed to determine their anti-epimastigote and anti-promastigote activity with respect to cytotoxicity. it was found that caryophyllene has a clinically relevant effect against the parasites Trypanosoma cruzi and Leishmania brasiliensis. Conclusions: results show that caryophyllene was more effective than eugenol, inhibiting the growth of parasites and thus constituting a proven alternative against Trypanosoma cruzi and Leishmania brasiliensis. As to cytotoxicity testing, new studies should be conducted in which more in vivo tests are performed.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

Atividade antileishmania do extrato hidroalcoólico e de frações obtidas de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck / Antileishmanial activity of hydroalcoholic extract and fractions obtained from leaves of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.)

A atividade biológica do extrato bruto e as várias frações obtidas de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck foi avaliada em Leishmania amazonensis. Este estudo incluiu o processo de extração e o fracionamento biomonitorado pelo método de cromatografia de adsorção. Um aumento progressivo no efeito antileishmania foi observado durante o processo de purificação. O extrato hidroalcoólico solúvel em água (EBA) apresentou concentração inibitória 50 por cento (IC50) igual a 167 µg/mL enquanto o extrato hidroalcoólico solúvel em acetato de etila (EBAcOEt) mostrou um IC50 de 30 mg/mL sobre o crescimento de formas promastigotas após 48 h de cultivo. A fração hexano (FHex) apresentou uma atividade antileishmania maior que o EBAcOEt com IC50 de 21,5 µg/mL. Análises de citotoxicidade indicaram que as concentrações tóxicas do EBA, EBAcOEt e das frações foram maiores para macrófagos J774G8 do que para os protozoários.

Biological activity of the crude extract and several fractions obtained from Piper regnellii var. pallescens was assessed on Leishmania amazonensis. This study included the extraction process and bioassay-guided fractionation by the adsorption chromatography method. A progressive increase in the antileishmanial effect was observed in the course of the purification process. The hydroalcoholic extract water soluble (EBA) had a 50 percent inhibitory concentration (IC50) at 167 µg/mL whereas the hydroalcoholic extract acetate soluble (EBAcOEt) showed an IC50 of 30 µg/mL against the growth of promastigote forms after 48 h of culturing. The hexan fraction (FHex) showed an antileishmanial activity greater than EBAcOEt with IC50 at 21.5 µg/mL. Analysis of cytotoxicity indicated that the toxic concentrations of the EBA, EBAcOEt, and fractions were higher for J774G8 macrophages than for the protozoans.

Detection of P-4 and GP-46 Expression in Leishmania amazonensis Amastigotes and Promastigotes by RT-PCR / 中国寄生虫学与寄生虫病杂志

Objective To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. Methods Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions,infected J774.G8 macrophages,and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction(PCR)was used to amplify the specific fragments of amastigote-specific nuclease(P-4)and promastigote-specific membrane glycoprotein(GP-46). PCR products were analyzed in 1.5% agarose gel. Results A P-4-specific band(273 bp)was observed in all three types of amastigotes with similar density,but it was almost undetectable in promastigotes. In contract,a GP-46-specific band(325 bp)was expressed at a higher level in promastigotes than in all three types of amastigotes. Conclusion Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.

Comparative Proteomic Analysis of the Promastigotes and Amastigotes of Leishmania donovani / 中国寄生虫学与寄生虫病杂志

Objective To explore the protein profile and identify developmentally regulated proteins of the promastigotes and axenic amastigotes with comparative proteomics technique.Methods The total proteins of promas-tigotes and axenic amastigotes of Leishmania donovani SC6 strain were separated by two-dimensional electrophoresis(2-DE) in a broad pH range(3-10) , and the gel was stained with Coomassie blue.The images were analyzed by PDQuest 1.0 software, and the major developmentally regulated proteins were identified by electrospray mass spectrometry.Results Approximately 700 protein spots were revealed in equivalent proteins of the promastigotes and axenic amastigotes separated by 2-DE, among which more than 90% protein spots showed equivalent quantity and distribution, with 6 proteins up-regulated and 3 proteins down-regulated in axenic amastigotes compared with promastigotes.Five of the 6 up-regulated proteins were with known function, respectively ascribed as Reiske iron-sulfur protein precursor, ?-tubulin, peroxidoxin 1, dihydrolipoamide acetyltransferase precursor, and mannose-1-phosphate guanyltransferase.Two of the 3 down-regulated proteins were identified as heat shock protein 70 and ?-tubulin.The functions of the developmentally regulated proteins were related to the carbohydrate/energy metabolism, stress response, or formation of cell membrane/cytoskeleton.Conclusion The findings demonstrate the differences in protein expression profiles between promastigotes and amastigotes.