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Journal Article

Inhibition of Leishmania major PTR1 Gene Expression by Antisense in Escherichia coli.

30 Jun 2012-Iranian Journal of Public Health (Tehran University of Medical Sciences)-Vol. 41, Iss: 6, pp 65-71

TL;DR: In inhibition of Iranian L. coli strain M15, major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more.

AbstractBackground: Protozoa related to Trypanosome family including Leishmania , synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase (DHFR). Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. Methods: PTR1 gene  was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Results: Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Conclusion: Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

Topics: Sense (molecular biology) (59%), Antisense RNA (57%), Dihydrofolate reductase (56%), Plasmid (54%), Gene expression (52%)

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Citations
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Journal ArticleDOI
Abstract: Currently, there is no effective vaccine available, and chemotherapy is the main approach for treatment of cutaneous leishmaniasis (CL) During recent decades, studies have demonstrated that a number of plant-derived compounds may act as new therapeutic tools against leishmaniasis This study was evaluated the antileishmanial, antioxidant, and cytotoxic activities of Quercus infectoria Olivier (oak) extract The total amount of phenolic and flavonoid compounds was measured in oak extract High performance liquid chromatography (HPLC) analysis was also performed to determine the amount of quercetin and gallic acid in this plant This extract (0-80g/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of Leishmania major (MRHO/IR/75/ER) using MTT assay and in a macro-phage model, respectively Then oak extract was tested on CL in infected male BALB/c mice with L major in order to evaluate the antileishmanial activity topically Moreover, cytotoxicity effects of oak in murine macrophage cells were tested by MTT assay Antioxidative activity of oak was also determined by the 2,2-diphenyl-1,1-picrylhydrazyl (DPPH) scavenging test The amount of phenolic and flavonoid compounds in the oak extract was 5750 and 186%, respectively The amount of quercetin and gallic acid in the oak extract was 00064 and 022%, respectively The findings revealed that oak significantly (P<005) inhibited the growth rate of promastigote of (IC50 1265μg/mL) and amastigotes (IC50 1031μg/mL) as a dose-dependent response In the in vivo assay, after 4 weeks of treatment, 916, 6666, and 50% recovery was observed in the infected mice treated with 20, 10, and 5mg/kg of oak extract, respectively After treatment of the infected mice with the concentration of 10 and 20mg/kg of oak, the mean diameter of lesions, parasite load and mean number of parasites was significantly (P<005) reduced Selectivity index of greater than 10 for oak revealed that oak extract had no cytotoxic effects on macrophage cells Moreover, DPPH test demonstrated that radical inhibition occurred at greater power with increasing the concentration of oak To conclude, the present study showed potent antileishmanial and antioxidant activity of oak extract; whereas this plant had no toxic effect on mammalian cells

43 citations


Journal ArticleDOI
TL;DR: This review focuses on recent findings in drugs used for the treatment of leishmaniasis including; chemical and natural antileishmanial moieties, different potential targets, as well as various trials of vaccination development.
Abstract: Leishmaniasis affects over 150 million people all over the world, especially in subtropical regions. Currently used antileishmanial synthesized drugs are associated with some drawbacks such as resistance and cytotoxicity, which hamper the chances of treatment. Furthermore, effective leishmanial vaccines are not well developed. Promising chemotherapy, either from natural or synthetic compounds, was or still is the most promising treatment. This review focuses on recent findings in drugs used for the treatment of leishmaniasis including; chemical and natural antileishmanial moieties, different potential targets, as well as various trials of vaccination development. Special emphasis has been paid to the mechanisms of the drugs, their safety and where possible, the structure-activity relationship to enable guided future drug discovery.

34 citations


Journal ArticleDOI
TL;DR: Through molecular docking it was found that two sesquiterpene coumarins are promising leads for the P TR1 and TryS inhibition purposes, and some xanthanolides also exhibited better affinity towards PTR1 and CS binding.
Abstract: A great number of sesquiterpenes are reported in the available literature as good antileishmanial leads. However, their mode of action at the molecular level has not been elucidated. The lack of molecular studies could be considered an impediment for studies seeking to improve sesquiterpene-based drug design. The present in silico study allows us to make important observations about the molecular details of the binding modes of a set of antileishmanial sesquiterpenes against four drug-enzyme targets [pteridine reductase-1 (PTR1), N-myristoyl transferase (NMT), cysteine synthase (CS), trypanothione synthetase (TryS)]. Through molecular docking it was found that two sesquiterpene coumarins are promising leads for the PTR1 and TryS inhibition purposes, and some xanthanolides also exhibited better affinity towards PTR1 and CS binding. In addition, the affinity values were clustered by Principal Component Analysis and drug-like properties were analyzed for the strongest-docking sesquiterpenes. The results are an excellent starting point for future studies of structural optimization of this kind of compounds.

23 citations


Journal ArticleDOI
TL;DR: It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major and hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L.major.
Abstract: The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.

14 citations


DOI
05 Dec 2016
TL;DR: The present research showed potent antileishmanial activity of S. khuzestanica essential oil; additionally this plant had no toxic effect on mammalian cells.
Abstract: Background and Aim: Leishmaniasis is endemic in 98 countries including Iran. Pentavalent antimony compounds resistance as first-line therapy is increasing in some local areas. Also side effects of these drugs are limited at the beginning of treatment, but the toxicity increases with time. The aim of this study was to assess the effect of Satureja khuzestanica essential oil (SKEO) on promastigote and amastigote Leishmania major forms. Materials and Methods: The components of S. khuzestanica oil were identified by gas chromatography/mass spectroscopy (GC/MS) analysis. To evaluate antipromastigote activity the different concentrations of extract and glucantime were added to the wells that contained L. major . The plates were incubated at 26±1°C for a week. On days 1, 3 and 5, the number of live promastigotes in each well was counted. For assessment of SKEO effect on intracellular amastigotes, mouse peritoneal macrophages were isolated and infected with promastigotes. Different concentrations of the extract and glucantime were added to the cultures. The cultures were incubated at 37°C and CO 2 5%. The number of infected macrophages and amastigotes within each macrophage were counted. Toxicity assessment of SKEO on macrophages was done by MTT method. Results and Conclusions: The mean number of promastigotes, infected macrophages and amastigotes in a macrophage in the control and treated groups had significantly difference. So that their number in the treated groups was less as a dose-dependent response. The present research showed potent antileishmanial activity of, SKEO; additionally this plant had no toxic effect on mammalian cells.

7 citations


References
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Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

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TL;DR: Improvements in ODN chemistry and cellular delivery techniques now allow for more potent and specific gene inhibition.
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TL;DR: Despite several disadvantages, amphotericin B is the only drug available for use in these areas and should be used as first‐line drug instead of Sbv, and the new oral antileishmanial drug miltefosine is likely to be the first-line drug in future.
Abstract: Throughout the world, pentavalent antimonial compounds (Sb(v)) have been the mainstay of antileishmanial therapy for more than 50 years. Sb(v) has been highly effective in the treatment of Indian visceral leishmaniasis (VL: kala-azar) at a low dose (10 mg/kg) for short durations (6-10 days). But in the early 1980s reports of its ineffectiveness emerged, and the dose of Sb(v) was eventually raised to 20 mg/kg for 30-40 days. This regimen cures most patients with VL except in India, where the proportion of patients unresponsive to Sb(v) has steadily increased. In hyperendemic districts of north Bihar, 50-65% patients fail treatment with Sb(v). Important reasons are rampant use of subtherapeutic doses, incomplete duration of treatment and substandard drugs. In vitro experiments have established emergence of Sb(v) resistant strains of Leishmania donovani, as isolates from unresponsive patients require 3-5 times more Sb(v) to reach similarly effectiveness against the parasite as in Sb(v) responders. Anthroponotic transmission in India has been an important factor in rapid increase in the Sb(v) refractoriness. Pentamidine was the first drug to be used and cured 99% of these refractory patients, but over time even with double the amount of initial doses, it cures only 69-78% patients now and its use has largely been abandoned in India. Despite several disadvantages, amphotericin B is the only drug available for use in these areas and should be used as first-line drug instead of Sb(v). The new oral antileishmanial drug miltefosine is likely to be the first-line drug in future. Unfortunately, development of newer antileishmanial drugs is rare; two promising drugs, aminosidine and sitamaquine, may be developed for use in the treatment of VL. Lipid associated amphotericin B has an excellent safety and efficacy profile, but remains out of reach for most patients because of its high cost.

548 citations


"Inhibition of Leishmania major PTR1..." refers background in this paper

  • ...In addition to reports in the world about Leishmania which is resistant to drugs (19, 20) in Iran 10 to 15 percent of people infected with cutaneous leishmaniasis have been treated with Glucantime; they have no response to treatment that one of the reasons is drug-resistant parasites (21)....

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Journal ArticleDOI

540 citations


"Inhibition of Leishmania major PTR1..." refers background in this paper

  • ...On the other hand, PTR1 causes synthesis and reduction of pteridine that increases the resistance of parasite against oxidative stress-related drug or reactions of the host (1, 2, 12)....

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Journal ArticleDOI
TL;DR: It is evident from in vitro test data that the SAMT is an extremely powerful and highly accurate technique for the prediction and determination of drug sensitivity of leishmanial isolates, as well as a means to screen for anti-leishmania agents.
Abstract: We report that in vitro sensitivity to pentavalent antimony (Sb5) of 35 Leishmania isolates as determined by the semiautomated microdilution technique (SAMT) showed an 89% and 86% correlation with clinical outcome after Pentostam and Glucantime treatment, respectively. These results suggest that in over 85% of the cases, the clinical outcome of treatment (cure or failure) could have been predicted by using the SAMT technique. Furthermore, the results clearly indicate that drug resistance is a problem, and that at least in some instances, failure to respond to treatment is due to the parasite as well as patient factors. Strains from Sb5-treated patients with American cutaneous and mucocutaneous disease who fail at least one complete course of Pentostam are as highly nonresponsive to this drug as laboratory-proven drug-resistant Leishmania strains. It was determined that some Leishmania isolates are innately less susceptible to Sb5 than others, and that moderate resistance to Sb5 exists in nature. A 10- and 17-fold increase was detected in the 50% inhibitory concentration (IC50) of Sb5 for L. mexicana and L. braziliensis isolates after subcurative treatment of the patients, when compared with the mean IC50 of seven and six isolates from the same endemic areas in Guatemala and Peru, respectively. Thus, we have correlated subcurative treatment to a decrease in drug sensitivity in at least these two cases. Collectively, these results indicate that under Sb5 pressure from undermedication, the parasites inherently most drug resistant are favored. The degree of resistance of a strain to antimony in association with host-specific factors will determine whether the clinical response to treatment with this drug is a total cure or a partial response followed by relapse(s), and possibly secondary unresponsiveness resulting in total resistance to antimony. It is evident from our in vitro test data that the SAMT is an extremely powerful and highly accurate technique for the prediction and determination of drug sensitivity of leishmanial isolates, as well as a means to screen for anti-leishmanial agents.

251 citations


"Inhibition of Leishmania major PTR1..." refers background or methods in this paper

  • ...As was mentioned DHFR in parasitic DNA synthesis has an important role and inhibit this enzyme is causing the death....

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  • ...Methotrexate, one of anti-metabolite drugs, is an antagonist for folic acid, which competitively will inhibit DHFR, and its result is the death of parasite (24-25)....

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  • ...The enzymatic activity of PTR1 is similar to dihydrofolate reductase (DHFR) (10, 11)....

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  • ...Genesis drugs on metabolism in mediating cell proliferation are important affect; one *Corresponding Author: Tel: +9821 22439956, E-mail address: kazemi@sbmu.ac.ir Available at: http://ijph.tums.ac.ir 66 Available at: http://ijph.tums.ac.ir of the mechanisms of drug is inhibition of DHFR-TS (1)....

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  • ...Since PTR1 activity is similar to DHFR causes the decrease of inhibition effect of drug (10, 11)....

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