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Luciferase leishmania

Adriano C Coelho, Jordana C Oliveira, Caroline R Espada, Juliana Q Reimão, Cristiana T Trinconi, Silvia R B Uliana
BACKGROUND: Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine...
May 2016: PLoS Neglected Tropical Diseases
Tahereh Taheri, Negar Seyed, Sima Rafati
Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail...
2016: Methods in Molecular Biology
Cristiana T Trinconi, Juliana Q Reimão, Adriano C Coelho, Silvia R B Uliana
OBJECTIVES: The objective of this study was to characterize in vitro interactions and evaluate the antileishmanial activity of tamoxifen and miltefosine combinations. METHODS: Interactions between drugs were evaluated in vitro against Leishmania amazonensis promastigotes and intracellular amastigotes by a modified isobologram method. Four different drug ratios were used to calculate the FIC index (FICI) and the mean sum of FICI. Treatment of L. amazonensis-infected BALB/c mice was initiated 4 weeks post-infection...
May 2016: Journal of Antimicrobial Chemotherapy
Samira Seif, Fereshteh Kazemi, Elham Gholami, Negar Seyed, Yasaman Taslimi, Sima Habibzadeh, Bahareh Azarian, Shahram Jamshidi, Mehrdad Hashemi, Sima Rafati, Tahereh Taheri
Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel...
May 2016: Applied Microbiology and Biotechnology
Mozna Khraiwesh, Susan Leed, Norma Roncal, Jacob Johnson, Richard Sciotti, Philip Smith, Lisa Read, Robert Paris, Thomas Hudson, Mark Hickman, Max Grogl
Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries...
February 2016: American Journal of Tropical Medicine and Hygiene
Somayeh Sadeghi, Negar Seyed, Mohammad-Hossein Etemadzadeh, Saeid Abediankenari, Sima Rafati, Tahereh Taheri
Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain...
August 2015: Korean Journal of Parasitology
Raquel García-Hernández, Verónica Gómez-Pérez, Santiago Castanys, Francisco Gamarro
Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio...
April 2015: PLoS Neglected Tropical Diseases
Juliana Q Reimão, Jordana C Oliveira, Cristiana T Trinconi, Paulo C Cotrim, Adriano C Coelho, Silvia R B Uliana
BACKGROUND: The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease...
February 2015: PLoS Neglected Tropical Diseases
Filipa Teixeira, Helena Castro, Tânia Cruz, Eric Tse, Philipp Koldewey, Daniel R Southworth, Ana M Tomás, Ursula Jakob
Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown...
February 17, 2015: Proceedings of the National Academy of Sciences of the United States of America
Tahereh Taheri, Hana Saberi Nik, Negar Seyed, Fatemeh Doustdari, Mohammad-Hossein Etemadzadeh, Fatemeh Torkashvand, Sima Rafati
Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice...
March 2015: Experimental Parasitology
Sushmita Das, Ayan Kumar Ghosh, Shikha Singh, Bhaskar Saha, Ashish Ganguly, Pradeep Das
Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species-specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88...
February 2015: Journal of Leukocyte Biology
Suman Gupta, Vanessa Yardley, Preeti Vishwakarma, Rahul Shivahare, Bhawna Sharma, Delphine Launay, Denis Martin, Sunil K Puri
OBJECTIVES: The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). METHODS: A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining...
February 2015: Journal of Antimicrobial Chemotherapy
Fernanda A H Batista, Glessler S Almeida, Thiago V Seraphim, Kelly P Silva, Silvane M F Murta, Leandro R S Barbosa, Júlio C Borges
The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L...
January 2015: FEBS Journal
Selene Maia de Morais, Nadja Soares Vila-Nova, Claudia Maria Leal Bevilaqua, Fernanda Cristina Rondon, Carlos Henrique Lobo, Arlindo de Alencar Araripe Noronha Moura, Antônia Débora Sales, Ana Paula Ribeiro Rodrigues, José Ricardo de Figuereido, Claudio Cabral Campello, Mary E Wilson, Heitor Franco de Andrade
In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi...
November 1, 2014: Bioorganic & Medicinal Chemistry
Anny Fortin, Diana P Caridha, Susan Leed, Franklyn Ngundam, Jenell Sena, Tom Bosschaerts, Sandi Parriott, Mark R Hickman, Thomas H Hudson, Max Grogl
BACKGROUND: Cutaneous leishmaniasis (CL) represents a range of skin diseases caused by infection with Leishmania parasites and associated with tissue inflammation and skin ulceration. CL is clinically widespread in both the Old and New World but lacks treatments that are well tolerated, effective and inexpensive. Oleylphosphocholine (OlPC) is a new orally bioavailable drug of the alkylphosphocholine family with potent antileishmanial activity against a broad range of Leishmania species/strains...
September 2014: PLoS Neglected Tropical Diseases
Job D F Inacio, Luiza Gervazoni, Marilene M Canto-Cavalheiro, Elmo E Almeida-Amaral
BACKGROUND: Leishmaniasis is a parasitic disease associated with extensive mortality and morbidity. The treatment for leishmaniasis is currently based on pentavalent antimonials and amphotericin B; however, these drugs result in numerous adverse side effects. Natural compounds have been used as novel treatments for parasitic diseases. In this paper, we evaluated the effect of (-)-epigallocatechin 3-O-gallate (EGCG) on Leishmania braziliensis in vitro and in vivo and described the mechanism of EGCG action against L...
August 2014: PLoS Neglected Tropical Diseases
Radika Soysa, Nicola S Carter, Phillip A Yates
Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals...
June 2014: Molecular and Biochemical Parasitology
Jessica L Martin, Phillip A Yates, Radika Soysa, Joshua F Alfaro, Feng Yang, Kristin E Burnum-Johnson, Vladislav A Petyuk, Karl K Weitz, David G Camp, Richard D Smith, Phillip A Wilmarth, Larry L David, Gowthaman Ramasamy, Peter J Myler, Nicola S Carter
The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity...
February 2014: PLoS Pathogens
Mary Dan-Goor, Abedelmajeed Nasereddin, Hanan Jaber, Charles L Jaffe
Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780) is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E...
2013: PloS One
Andrew A Voak, Karin Seifert, Nuala A Helsby, Shane R Wilkinson
Many of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes and Leishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity against Leishmania major...
2014: Antimicrobial Agents and Chemotherapy
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