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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TL;DR: In this article, the authors evaluated the antileishmanial, antioxidant, and cytotoxic activities of Quercus infectoria Olivier (oak) extract and found that oak significantly inhibited the growth rate of promastigote and amastigotes.
61 citations
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.
55 citations
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.
27 citations
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.
16 citations
TL;DR: Adherence to vaccination is fairly common in this part of the country, however, vaccination delays are still present and should be addressed to improve health care.
Abstract: Objectives: To assess vaccination timeliness, risk factors associated with delays and the reasons for delayed vaccinations among children below the age of 3 years in Jeddah, Kingdom of Saudi Arabia. Methods: This is a cross-sectional study conducted in Jeddah, Saudi Arabia during the period of May 2016 to August 2017. Data were obtained from parents of children under the age of 3 years using a structured questionnaire comprised of questions about sociodemographics, physical well-being of the child and the reasons that are used to justify delayed vaccinations. Vaccinations were considered delayed if they occurred more than 30 days after the time designated on the primary vaccination schedule. Logistic regression was used to assess the risk factors for vaccination delays. Results: The study included 351 children. Delayed vaccinations were observed in 85/351 (24.2%) of the sample. Delays were noted to occur most frequently for Measles, Mumps, Rubella vaccine (MMR), seconddose of meningococcal conjugate quadrivalent vaccine (MCV4), second dose of oral polio vaccine (OPV) and fourth dose of pneumococcal conjugate vaccine (PCV) in 19/125 (15.2%) of the sample. Traveling at the time of vaccination was the most common delay reason and was reported in 31/142 (21.3%) of the sample. Conclusion: Adherence to vaccination is fairly common in this part of the country. However, vaccination delays are still present and should be addressed to improve health care. Saudi Med J 2018; Vol. 39 (4): 347-353 doi: 10.15537/smj.2018.4.21473 How to cite this article: Banjari MA, Alamri AA, Algarni AY, Abualjadayel MH, Alshardi YS, Alahmadi TS. How often do children receive their vaccinations late, and why? Saudi Med J . 2018 Apr;39(4):347-353. doi: 10.15537/smj.2018.4.21473.
14 citations
References
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TL;DR: Using reversed-phase high-performance liquid chromatography, the ability of intact L. donovani to transform [3H]biopterin into tetrahydrofolates was demonstrated and supported the hypothesis that folate transport deficiency in mutant organisms is associated with an inability to transform pterins to reduced folates.
36 citations
TL;DR: The kinetic mechanism and stereochemical course for the catalyzed reaction confirm PTR1's membership within the short chain dehydrogenase/reductase (SDR) family and the existence of a nonproductive binary enzyme-dihydrofolate complex.
Abstract: Pteridine reductase 1 (PTR1) is a novel broad spectrum enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. Overexpression of PTR1 confers methotrexate resistance to these protozoa, arising from the enzyme's ability to reduce dihydrofolate and its relative insensitivity to methotrexate. The kinetic mechanism and stereochemical course for the catalyzed reaction confirm PTR1's membership within the short chain dehydrogenase/reductase (SDR) family. With folate as a substrate, PTR1 catalyzes two rounds of reduction, yielding 5,6,7,8-tetrahydrofolate and oxidizing 2 equiv of NADPH. Dihydrofolate accumulates transiently during folate reduction and is both a substrate and an inhibitor of PTR1. PTR1 transfers the pro-S hydride of NADPH to carbon 6 on the si face of dihydrofolate, producing the same stereoisomer of THF as does dihydrofolate reductase. Product inhibition and isotope partitioning studies support an ordered ternary complex mechanism, with NADPH binding first and NADP + dissociating after the reduced pteridine. Identical kinetic mechanisms and NAD(P)H hydride chirality preferences are seen with other SDRs. An observed tritium effect upon V/K for reduction of dihydrofolate arising from isotopic substitution of the transferred hydride was suppressed at a high concentration of dihydrofolate, consistent with a steady-state ordered kinetic mechanism. Interestingly, half of the binary enzyme - NADPH complex appears to be incapable of rapid turnover. Fluorescence quenching results also indicate the existence of a nonproductive binary enzyme-dihydrofolate complex. The nonproductive complexes observed between PTR1 and its substrates are unique among members of the SDR family and may provide leads for developing antileishmanial therapeutics.
33 citations
TL;DR: Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this important enzyme and support a structure-based approach to discover novel inhibitors of PTR1.
Abstract: The enzyme pteridine reductase (PTR1) has recently been discovered in the protozoan parasite Leishmania and validated as a target for therapeutic intervention. PTR1 is responsible for the salvage of pteridines and also contributes to antifolate drug resistance. Structural analysis, in combination with ongoing biochemical characterization will assist the elucidation of the structure–activity relationships of this important enzyme and support a structure-based approach to discover novel inhibitors. Recombinant L. major PTR1 has been purified from an Escherichia coli expression system and used in crystallization experiments. Orthorhombic crystals have been obtained and data to 2.8 A has been measured. The space group is P21212 or P212121 with unit-cell dimensions of a = 103.9, b = 134.7, c = 96.2 A. One homotetramer, of molecular mass approximately 120 kDa, probably constitutes the asymmetric unit and gives a Matthews coefficient, Vm, of 2.8 A3 Da−1 and 56% solvent volume. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that might represent additional elements of non-crystallographic symmetry. The detail of exactly what constitutes the asymmetric unit will be resolved by structure determination.
19 citations
TL;DR: The cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India will set the basis for inhibition studies as well as for structure-function relationships.
15 citations
"Inhibition of Leishmania major PTR1..." refers methods in this paper
...The membrane was washed three times with TBS-T (TBS-TWEEN 20) and incubated for 1 h at 37 °C with the goat anti-rabbit IgG HRP conjugate solution at a 1:5000 dilution as secondary antibody (13, 16)....
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TL;DR: The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased, suggesting the existence of a potential new target for gene therapy.
Abstract: To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cellsin vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINETM, 2000 (FL2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by westernblot, the growth activity was measured by MTT, and apoptosis was detected by Flow cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.
12 citations
"Inhibition of Leishmania major PTR1..." refers background in this paper
...To inhibit gene expression, different several ways have been reported such as, oligodeoxy nucleotides (ODNs) (28), mRNA antisense (29) and small interfering RNA (siRNA) molecules (30)....
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