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.
Abstract: Background: 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.
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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.
8 citations
TL;DR: It could be concluded that since SNWCLSOs could silence Cpb gene with no remarkable toxicity, they are good choice for treat cutaneous leishmaniasis in future.
4 citations
TL;DR: In this article , the authors summarized the updated progress in gene therapy by taking an edge on the unique properties of the gold nanoparticles, which will be an important outlook for future research.
Abstract: Simple Summary Successful gene therapy mainly depends on the fabrication of efficient and nontoxic carriers that can compact genetic materials and adequately deliver them to target cells. Nanoscale gold-mediated gene delivery systems have been considered a powerful tool for gene therapy because of their inherent potential of nontoxicity, high specificity, and therapeutic efficacy. Here, we summarize the updated progress in gene therapy by taking an edge on the unique properties of the gold nanoparticles. Distinct employed by these nano-carriers for gene-silencing and gene-editing are a great deal of optimism for the treatment of human fatal genetic disorders. From other published reviews on gold theranostics, this article discusses the recent advances of gold nanostructures in gene therapeutics for diseases caused by a single gene in humans. The promising advancements employed by these nano-carriers for gene-silencing and gene-editing are a great deal of optimism for the treatment of human fatal genetic disorders. Abstract Gold nanoparticles (AuNPs) have gained increasing attention as novel drug-delivery nanostructures for the treatment of cancers, infections, inflammations, and other diseases and disorders. They are versatile in design, synthesis, modification, and functionalization. This has many advantages in terms of gene editing and gene silencing, and their application in genetic illnesses. The development of several techniques such as CRISPR/Cas9, TALEN, and ZFNs has raised hopes for the treatment of genetic abnormalities, although more focused experimentation is still needed. AuNPs, however, have been much more effective in trending research on this subject. In this review, we highlight recently well-developed advancements that are relevant to cutting-edge gene therapies, namely gene editing and gene silencing in diseases caused by a single gene in humans by taking an edge of the unique properties of the AuNPs, which will be an important outlook for future research.
2 citations
10 Jun 2013
TL;DR: Results were supported by the pharmacophore modeling, indicating that kauren-19-oic acids possess significant structural features for inhibition of PTR1, which is an excellent starting point for future studies of structural optimization of this kind of compounds.
Abstract: Pteridine reductase-1 (PTR1) is one of enzymes discovered as being responsible for the reduced susceptibility to antifolates (eg, metotrexato) of trypanosomic parasites, which has become a chemotherapeutic target. Therefore, thirteen known kaurane-related diterpenes were evaluated in silico against PTR1 by molecular docking and pharmacophore modeling. The molecular docking exhibited clear polar interactions with some active site residues of PTR1, which was primarily dependent on the presence of a carboxyl group at C19. These results were supported by the pharmacophore modeling, indicating that kauren-19-oic acids possess significant structural features for inhibition of PTR1, which is an excellent starting point for future studies of structural optimization of this kind of compounds.
2 citations
TL;DR: The results showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20, which indicated that the use of antisense RNA reducedNT4 gene expression in L. major.
Abstract: Background & objectives: Leishmania parasites cause various clinical symptoms in humans such as cutaneous ulcers and fatal visceral diseases. These parasites cannot synthesize purine rings de novo and must uptake purines from their hosts via salvage. Salvage is regulated by permeases in the cell membrane. There are hundreds of membrane transporter proteins to receive nutrients in Leishmania. Nucleoside transporter 4 (NT4) is one of the purine transporters that is involved in enhancing the uptake of adenine in Leishmania major. They are important new drug targets for the treatment of leishmaniasis because they can be used to transport toxic purine analogs to kill parasitic cells, thus preventing the progression of the infection. The present study was conducted to silence the NT4 nucleobase involved in the salvage pathway to interrupt purine nucleotide membrane transport in the cells of L. major. Methods: In this study, a 502 bp segment of NT4 gene sequence was selected and designed as antisense transcripts after insertion in the parasite. The NT4 construct was transfected into L. major promastigotes for in vitro study of gene expression. Then, BALB/c mice infected with transgenic Leishmania and wild-type strain along with the number and size of lesions were studied in vivo. Results: The study showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20. The percentages and the number of amastigotes in infected macrophages with wild-type strain L. major were more than infected macrophages with mutant parasites. Infected BALB/c mice with transgenic Leish- mania showed a lower number and size of lesions than the BALB/c mice infected with wild-type strain. Interpretation & conclusion: The results of the study indicated that the use of antisense RNA reduced NT4 gene expression in L. major. Further, studies are needed to ascertain that the use of antisense can be considered as a new treatment for leishmaniasis.
1 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
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
215,169 citations
TL;DR: Improvements in ODN chemistry and cellular delivery techniques now allow for more potent and specific gene inhibition.
Abstract: Antisense oligodeoxynucleotides (ODNs) have great promise as agents for the specific manipulation of gene expression. Until recently, nonspecific effects of ODNs often confounded the interpretation of antisense studies. Improvements in ODN chemistry and cellular delivery techniques now allow for more potent and specific gene inhibition. This review critically evaluates recent progress in the development of antisense ODNs.
825 citations
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.
564 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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543 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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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.
252 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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