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Journal Article

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania.

15 Nov 2010-Iranian biomedical journal (IRANIAN BIOMEDICAL JOURNAL)-Vol. 14, Iss: 3, pp 97-102
TL;DR: Iranian lizard Leishmania ptr1 gene was successfully amplified and cloned into expression vector, and enzymatic assay was performed successfully, indicating PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy.
Abstract: Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6- biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.

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Journal ArticleDOI
TL;DR: The objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously and to describe the mechanisms leading to ZCL in Iran.
Abstract: Objectives Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously. Methods Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites. Results Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies. Conclusion Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans. Objectifs Seul Leishmania major est bien connu comme agent causal de la leishmaniose cutanee zoonotique (LCZ) en Iran. Notre objectif etait de trouver des parasites de Leishmania circulant simultanement chez les hotes reservoirs, les phlebotomes et les humains. Methodes Les phlebotomes, les rongeurs et des frottis prepares a partir d'humains ont ete echantillonnes. L’ADN de Leishmania a ete extrait et deux fragments du gene ITS-ADNr ont ete amplifies par PCR. Le RFLP et le sequencage ont ete utilises pour identifier les parasites Leishmania. Resultats L. major et L. turanica ont ete identifies sans equivoque par le ciblage et le sequencage de l’ITS-ADNr chez les etres humains, les rongeurs et les phlebotomes. Nous avons decouvert une nouvelle espece de Leishmania chez les phlebotomes que nous avons nomme L. iranica (n° d'acces GenBank: EF413076. EF413087). Conclusion Sur base de la detection des parasites par l’ITS-ADNr chez les hotes reservoirs principaux et potentiels et les vecteurs et les humains, nous concluons qu'au moins deux especes de Leishmania sont courantes dans le foyer Turkemen Sahara ZCL. L'analyse phylogenetique a montre que la nouvelle espece Leishmania est etroitement liee a des parasites Leishmania de mammiferes (L. major, L. turanica, L. gerbilli). Son role en tant que principal agent de LCZ est inconnu car elle n'a ete retrouvee que dans les phlebotomes. Nos resultats apportent une nouvelle lumiere sur les cycles de transmission de plusieurs parasites Leishmania dans les phlebotomes, les hotes reservoirs et les humains. Objetivos Solo Leishmania major es un agente causal bien conocido de la Leishmaniasis cutanea zoonotica (LCZ) en Iran. Nuestro objetivo era encontrar parasitos de Leishmania circulando simultaneamente entre hospederos reservorios, moscas de arena y humanos. Metodos Se tomaron muestras de moscas de arena, roedores y frotis preparados de humanos. Se extrajo el ADN de Leishmania y mediante PCR se amplificaron dos fragmentos del gen ITS-ADNr. Se utilizaron el RFLP y la secuenciacion para identificar los parasitos de Leishmania. Resultados Se identifico a L. major y L. turanica de forma inequivoca mediante amplificacion y secuenciacion del ITS-ADNr de muestras de humanos, roedores y moscas de arena. En moscas, hemos descubierto una nueva especie de Leishmania, la cual hemos llamado L. iranica (numeros de acceso de GenBank EF413076; EF413087). Conclusion Basandonos en la deteccion del ITS-ADNr de parasitos en el hospedero principal y otros potenciales, en vectores y en humanos, concluimos que al menos dos especies de Leishmania son comunes en el foco de LCZ de Turkemen Sahara. El analisis filogenetico muestra que la nueva Leishmania esta estrechamente relacionada con especies de Leishmania que parasitan mamiferos (L. major, L. turanica, L. gerbilli). Su papel como el agente principal de LCZ es desconocido puesto que se encontro solamente en moscas de arena. Nuestros hallazgos aportan nuevos datos sobre los ciclos de transmision de varios parasitos de Leishmania en moscas de arena, hospederos reservorios y humanos.

20 citations


Cites background or result from "Molecular cloning, expression and e..."

  • ...Finding variations in L. major and assessing the density of these parasites can be of practical value and experimental evidence for the epidemiological surveillance and control of leishmaniasis in humans, hosts and vectors in Iran (Ready & Rogers 2012; Ready 2013). mania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....

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  • ...It is conceivable that had we put more purposeful efforts into investigating of Sergentomyia sintoni species, and the parasites might have appeared as Sauroleishmania; however, in our previous study, we searched for Leishmania parasite in Sergentomyia sand fly species but found none (Parvizi & Amirkhani 2008; Parvizi & Ready 2008)....

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  • ...Some previous studies were carried out on Sauroleishmania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....

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  • ...…evidence for the epidemiological surveillance and control of leishmaniasis in humans, hosts and vectors in Iran (Ready & Rogers 2012; Ready 2013). mania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....

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Journal Article
TL;DR: The chromosomal and mitochondrial genomes of Leishmania spp.
Abstract: Leishmania is a protozoan parasite belonging to the family Trypanosomatidae, which is found among 88 different countries. The parasite lives as an amastigote in vertebrate macro- phages and as a promastigote in the digestive tract of sand fly. It can be cultured in the laboratory using appropriate culture media. Although the sexual cycle of Leishmania has not been observed during the promastigote and amastigote stages, it has been reported by some researchers. Leishmania has eukaryotic cell organization. Cell culture is convenient and cost effective, and because posttranslational modifications are common processes in the cultured cells, the cells are used as hosts for preparing eukaryotic recombinant proteins for research. Several transcripts of rDNA in the Leishmania genome are suitable regions for conducting gene transfer. Old World Leishmania spp. has 36 chromosomes, while New World Leishmania spp. has 34 or 35 chromo- somes. The genomic organization and parasitic characteristics have been investigated. Leishmania spp. has a unique genomic organization among eukaryotes; the genes do not have introns, and the chromosomes are smaller with larger numbers of genes confined to a smaller space within the nucleus. Leishmania spp. genes are organized on one or both DNA strands and are transcribed as polycistronic (prokaryotic-like) transcripts from undefined promoters. Regulation of gene expres- sion in the members of Trypanosomatidae differs from that in other eukaryotes. The trans-splic- ing phenomenon is a necessary step for mRNA processing in lower eukaryotes and is observed in Leishmania spp. Another particular feature of RNA editing in Leishmania spp. is that mitochon- drial genes encoding respiratory enzymes are edited and transcribed. This review will discuss the chromosomal and mitochondrial (kinetoplast) genomes of Leishmania spp. as well as the phenomenon of RNA editing in the kinetoplast genome.

10 citations


Cites methods from "Molecular cloning, expression and e..."

  • ...It should be noted that most Leishmania genes have no introns (40), and that chromosomal DNA is used as the template for cloning by PCR (41-45)....

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Journal ArticleDOI
TL;DR: Mice immunized with ILL+CpG were protected against the development of the dermal lesion and showed a significant reduction in the parasite load, in comparison to the control groups, indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.
Abstract: Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania major EGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

9 citations


Cites background from "Molecular cloning, expression and e..."

  • ...Leishmania promastigotes that previously were isolated in other countries (28, 29)....

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References
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Book ChapterDOI
TL;DR: An overview of the purine and pyrimidine pathways in Leishmania is presented, comparisons to the equivalent pathways in their mammalian host are made, and how these pathways might be amenable to selective therapeutic targeting is explored.
Abstract: Purines and pyrimidines are indispensable to all life, performing many vital functions for cells: ATP serves as the universal currency of cellular energy, cAMP and cGMP are key second messenger molecules, purine and pyrimidine nucleotides are precursors for activated forms of both carbohydrates and lipids, nucleotide derivatives of vitamins are essential cofactors in metabolic processes, and nucleoside triphosphates are the immediate precursors for DNA and RNA synthesis. Unlike their mammalian and insect hosts, Leishmania lack the metabolic machinery to make purine nucleotides de novo and must rely on their host for preformed purines. The obligatory nature of purine salvage offers, therefore, a plethora of potential targets for drug targeting, and the pathway has consequently been the focus of considerable scientific investigation. In contrast, Leishmania are prototrophic for pyrimidines and also express a small complement of pyrimidine salvage enzymes. Because the pyrimidine nucleotide biosynthetic pathways of Leishmania and humans are similar, pyrimidine metabolism in Leishmania has generally been considered less amenable to therapeutic manipulation than the purine salvage pathway. However, evidence garnered from a variety of parasitic protozoa suggests that the selective inhibition of pyrimidine biosynthetic enzymes offers a rational therapeutic paradigm. In this chapter, we present an overview of the purine and pyrimidine pathways in Leishmania, make comparisons to the equivalent pathways in their mammalian host, and explore how these pathways might be amenable to selective therapeutic targeting.

84 citations


"Molecular cloning, expression and e..." refers background in this paper

  • ..., human, are similar, it is thought that therapeutic manipulation of pyrimidine metabolism in Leishmania would be less effective as compared to manipulation of the purine salvage pathway [6-8]....

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Journal ArticleDOI
TL;DR: It is concluded that this twice a day two-week regimen of aminosidine was inadequate to accelerate the recovery of most cases of cutaneous leishmaniasis, but the ointment did show some clear evidence of parasitologic efficacy and should now be studied in longer or more frequent regimens in an effort to prevent Parasitologic relapse and thus promote clinical improvement.
Abstract: The effect of a two-week regimen of topical aminosidine was investigated in a randomized, double-blind, placebo-controlled trial of 251 selected Iranian patients with zoonotic cutaneous leishmaniasis. Patients underwent clinical and parasitologic assessment before and 15 (end of therapy), 45, and 105 days after starting the treatment. Aminosidine ointment was safe and well-tolerated, and produced significant reductions in the prevalence of parasitologically positive smears on days 15 and 105 (but not day 45) after treatment compared with placebo. However, there was no clear clinical benefit at any stage after treatment. We conclude that this twice a day two-week regimen of aminosidine was inadequate to accelerate the recovery of most cases of cutaneous leishmaniasis. However, the ointment did show some clear evidence of parasitologic efficacy and should now be studied in longer or more frequent regimes in an effort to prevent parasitologic relapse and thus promote clinical improvement.

84 citations

Journal ArticleDOI
TL;DR: TbPTR1 offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis, and is compared with Leishmania major PTR1.
Abstract: The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

61 citations


Additional excerpts

  • ...[29]....

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Journal ArticleDOI
TL;DR: A genomic DNA library was generated with Sau3A cut DNA derived from promastigotes of Leishmania mexicana amazonensis and the lambda vector EMBL3 and the leishmanial beta tubulin coding region was sequenced by the dideoxy method.

52 citations


"Molecular cloning, expression and e..." refers methods in this paper

  • ...Because there is no intron in Leishmania genes [17], PCR was performed using genomic DNA....

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Journal ArticleDOI
TL;DR: Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species, with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircles sequences and in termsof nuclear chromosome patterns on orthogonal gel electrophoresis.

49 citations


Additional excerpts

  • ...[16] reported some differences between lizard Leishmania and mammalian Leishmania with regard to kinetoplast nucleic acid sequences, chromosomes, and membrane lipids, which are not the same as those reported in mammalian Leishmania....

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