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Dihydrofolate reductase (DHFR), a housekeeping enzyme in primary metabolism, has been extensively studied as a model of acid-base catalysis and a clinic drug target. Herein, we investigated the enzymology of a DHFR-like protein SacH in safracin (SAC) biosynthesis, which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance. Furthermore, based on the crystal structure of SacH-NADPH-SAC-A ternary complexes and mutagenesis, we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore. These findings expand the functions of DHFR family proteins, reveal that the common reaction can be catalyzed by distinct family of enzymes, and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.
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@#Introduction: Disease-modifying anti rheumatic drugs (DMARDs) provide the mainstay for the treatment of rheumatoid arthritis (RA). Adverse effects (AEs) in DMARDs among RA patients are usually related with methotrexate (MTX) use, the common conventional DMARDs. Genetic variant such as single nucleotide polymorphism (SNP) in gene transcribing dihydrofolate reductase (DHFR) (i.e, 829C>T, rs12517451) has been correlated with drug AEs in MTX-treated RA. The prevalence of the DHFR rs12517451 SNP has been reported in other populations, but not in Malaysian. The aim of this study was to determine the prevalence of the DHFR rs12517451 SNP and its association with drug AEs among MTX-treated RA patients from Kelantan, Malaysia. Methods: A total of 78 RA patients receiving MTX (alone or in combination) were included in this study. Based on evidence of clinically perceived drug AEs in MTX-treated RA patients, 33 and 45 samples were assigned as cases and controls, respectively. The genotype of the patients was determined using the polymerase chain reaction-restriction fragment length polymorphism method and validated by sequencing analysis. Results: Minor allele frequency (MAF) for DHFR rs12517451 in cases and controls were 28.8% and 32.2% but there was no significant difference (p=0.727) for the possession of the minor allele T between the two groups. The most reported AEs among cases were haematological effects, gastrointestinal toxicity, and skin problems resulting in 21% withdrawal of MTX. Conclusion: We did not find significant association of the DHFR rs12517451 with drug AEs in MTX-treated RA patients. Our findings warrant replication in a larger patient cohort.
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To explore the immobilization of target proteins for screening libraries of ligand mixtures, magnetic submicron particles (MSP) functionalized with Ni²⁺-NTA and carboxyl were compared for the immobilization of Mycobacterium tuberculosis dihydrofolate reductase (MtDHFR). MtDHFR fused with 6×His was expressed, purified and characterized for kinetics. MtDHFR was immobilized on Ni²⁺-NTA-functionalized MSP directly and carboxyl-functionalized MSP upon activation. The immobilization capacity, residual activity, thermostability and affinities for putative inhibitors were characterized. MtDHFR immobilized on Ni²⁺-NTA-functionalized MSP retained about 32% activity of the free one with the immobilization capacity of (93±12) mg/g of MSP (n=3). Ni²⁺ and EDTA synergistically inhibited MtDHFR activity, while Fe³⁺ had no obvious interference. MtDHFR immobilized on carboxyl-functionalized MSP retained (87±4)% activity of the free one with the immobilization capacity of (8.6±0.6) mg/g MSP (n=3). In 100 mmol/L HEPES (pH 7.0) containing 50 mmol/L KCl, there was no significant loss of the activities of the free and immobilized MtDHFR after storage at 0 °C for 16 h, but nearly 60% and 35% loss of their activities after storage at 25 °C for 16 h, respectively. The inhibition effects of methotrexate on the immobilized and free MtDHFR were consistent (P>0.05). The immobilization of MtDHFR on carboxyl-functionalized MSP was thus favorable for higher retained activity and better thermostability, with promise for rapid screening of its ligand mixtures.
Subject(s)
Enzyme Stability , Enzymes, Immobilized , Hydrogen-Ion Concentration , Kinetics , Ligands , Magnetite Nanoparticles , Mycobacterium tuberculosis , Temperature , Tetrahydrofolate DehydrogenaseABSTRACT
Malaria is a parasitic disease which threatens human life and health seriously. Sulfadoxine-pyrimethamine (SP) has been recommended for intermittent preventive treatment of malaria in children and pregnant women, and also used as a compound component of artemisinin based therapy. The mechanisms of SP resistance in P. falciparum involve point mutations in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), and the drug pressure can also lead to the mutations in the two genes of P. vivax. To provide the information for the formulation of anti-malarial strategies, this article reviews the discovery, application, effect of SP, and the resistance mechanism and research progress of the related genes in P. vivax.
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Malaria is a parasitic disease which threatens human life and health seriously. Sulfadoxine-pyrimethamine (SP) has been recommended for intermittent preventive treatment of malaria in children and pregnant women, and also used as a compound component of artemisinin based therapy. The mechanisms of SP resistance in P. falciparum involve point mutations in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), and the drug pressure can also lead to the mutations in the two genes of P. vivax. To provide the information for the formulation of anti-malarial strategies, this article reviews the discovery, application, effect of SP, and the resistance mechanism and research progress of the related genes in P. vivax.
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Knowing that dihydrofolate reductase (DHFR) is the primary target enzyme for antifolate drugs and 1,3,5-triazine derivatives containing various amino groups at position 2, 4 or 6 have been known as potent anticancer drugs, two series of tri-amino-substituted 1,3,5-triazine derivatives were designed, synthesized and evaluated as cytotoxic agents against non-small cell lung cancer (A549). The first series are N2-(4-phenylthiazol-2-yl)-1,3,5-triazine-2,4,6-triamine analogs and the second series are4-((4,6-Diamino-1,3,5-triazin-2-yl)amino)-4H-1,2,4-triazole-3-thiol analogs. Out of twenty two synthesized compounds there were thirteen compounds showed a higher cytotoxic activity against A549 cell line than methotrexate and four compounds were equipotent to methotrexate. Compounds 8e, 9a, 10e and 11e showed the highest cytotoxic activity with IC50 values of 50,42, 62 and 28 nM respectively. Molecular docking study was performed to interpret the comparative differences in the binding interactions of the synthesized novel compounds at molecular level as inhibitors of human dihydrofolate reductase (hDHFR)and to understand the structure activity relationships. The excellent anticancer activity of synthesized analogs presented in this study needs further investigation as highly promising cytotoxic lead agents against lung cancer.
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Objective K93T point mutation exists in the quinoid dihydropteridine reductase ( QDPR) of OLEFT rats which catalyzes QDPR into tetrahydrobinopterin(BH4), while dihydrofolate reductase(DHFR) can reduce QDPR to BH4, which implies crosstalk between hydrobiopterin and folate metabolism.By investigating the influence of QDPR expression on DHFR expression of NRK-52E cells, the article aimed to find out the possible underlying mechanism of QDPR gene in diabetic nephropathy ( DN). Methods Western blot was performed to identify the expression level in NRK-52E cell under high glucose ambience and DHFR pro-tein expression of OLETF rats.NRK-52E cells were transfected by the lentivirus to establish no-load overexpression, overexpressed QDPR and knockdown QDPR models.Each group was given 5.4 mmol/L normal sugar medium and 30mmol/L in high glucose ambi-ence for 72 hours'cell cultivation to simulate DN model.Observation was made on the influence of QDPR gene expression levels on DHFR in high glucose ambience. Results The western blot analysis revealed that DHFR protein decreased in NHG group( [0.33 ± 0.16] vs [0.64 ±0.5], P<0.05) and OLETF rats cortex ([0.56 ±0.16] vs [1.03 ±0.12], P<0.01).In high glucose ambi-ence, compared with LV-OCON-HG group, the protein expression of DHFR was significantly decreased in LV-QDPR-HG group ([0.12 ±0.09] vs [0.63 ±0.08], P<0.01).No difference was found in the comparison of DHFR expression levels between LV-SHQDPR-HG and LV-SHCON-HG group. Conclusion DHFR protein expression decreases in NRK-52E cells of high glucose and LOLETF rat model, which suggests that DHFR protein plays an important role in the development of DN.QDPR overexpression leads to the decreased expression of DHFR, which implies that overexpressed QDPR influences the occurrence and process of DN by down-regulating DHFR expression level.
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Background and purpose:Dihydrofolate reductase (DHFR) is expressed highly in platinum-resis-tant ovarian cancer. This study aimed to explore the relationship between the silence ofDHFR gene and platinum drug resistance in ovarian cancer, and lay the foundation for the treatment of platinum-resistant ovarian cancer.Methods:To design targeting hairpin siRNA ofDHFR gene, the optimal siRNA silent sequence was selected, and lentiviral vector carryingDHFR gene was constructed successfully, named DHFR-pGCSIL-SKOV3 cell. Flow cytometry was used to detect the cell apoptosis of DHFR-pGCSIL-SKOV3 cells, pGCSIL-SKOV3 cells and SKOV3 cells incubated in various concentrations of cisplatin (2.5, 5.0, 10.0 and 20.0 μg/mL) at different time points (24, 48 and 72 h), and cell cycle changes of these cells at IC50 cisplatin concentration (4.4 μg/mL). High performance liquid chromatography was used to test intracellular concentration of cisplatin at different induction concentration of cisplatin (2.5, 5.0 and 7.5 μg/mL) and various time points (24 and 48 h). Ultrastructural changes of these cells at concentration of cisplatin IC50 (4.4 μg/mL) were observed by transmission electron microscope.Results:After annealing double-strand nucleotide was connected to pGCSIL/GFP vector, sequencing result was correct. SKOV3 cell were transfected with virus particles followed by Western blot detection of interference effect. Flow cytometry was used to detect apoptosis in three groups of cells, and increased apoptosis rate was found at the raised cisplatin concentration (2.5, 5.0, 10.0 and 20.0 μg/mL) at 24, 48 and 72 h in DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3 and SKOV3 cells. The apoptosis rate in DHFR-pGCSIL-SKOV3 was signiifcantly higher than that in pGCSIL-SKOV3 and SKOV3 cells at 24 and 48 h (P<0.05). Flow cytometry was adopted to test cells cycle of 3 groups at different time period under IC50 cisplatin concentration (4.4 μg/mL), the results indicated that G0/G1 phase cell rate of DHFR-pGCSIL-SKOV3 was much more than the others, of which G2/M and S phase cell rates were on the contrary. While at 72 h, 3 groups were mainly G2/M and S phase cell rates, DHFR-pGC-SIL-SKOV3 was lower than the others. High performance liquid chromatography method was used to detect intracellu-lar cisplatin concentration at 24 and 48 h after the cells were incubated at various concentrations of cisplatin (2.5 and 5.0μg/mL). The results showed the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was signiifcantly higher than that of pGCSIL-SKOV3 and SKOV3 cells. However, after incubation at cisplatin concentration of 7.5 μg/mL, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was signiifcantly lower than that of pGCSIL-SKOV3 and SKOV3 cells at 24 h, while higher than pGCSIL-SKOV3 and SKOV3 cells at 48 h (P=0.034,P=0.014). We observed ultrastructural changes of three different cell lines induced by IC50 cisplatin concentration(4.4 μg/mL) at different time points by the electron microscope. We found that the microiflaments were increased and gathered together and mitochondrial structure was also changed obviously without the drug. However, there was rare microiflament in three groups of cells at 24 and 48 h, while at 72 h, obviously increased inordinate microiflaments were observed.Conclusion:We successfully constructed pGCSIL lentivirus interference carrier carryingDHFR gene. The research indicates that down-regulation ofDHFR gene is related to cisplatin drug resistance in ovarian cancer. The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.
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Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.
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Objective Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) are among the most common congenital birth defects worldwide. Several lines of evidence point to the involvement of folate, as well as folate metabolizing enzymes in risk reduction of orofacial clefts. Dihydrofolate reductase (DHFR) enzyme participates in the metabolic cycle of folate and has a crucial role in DNA synthesis, a fundamental feature of gestation and development. A functional polymorphic 19-bp deletion within intron-1 of DHFR has been associated with the risk of common congenital malformations. The present study aimed to evaluate the possible association between DHFR 19-bp deletion polymorphism and susceptibility to NS-CL/P in an Iranian population. Material and Methods The current study recruited 100 NS-CL/P patients and 100 healthy controls. DHFR 19-bp deletion was determined using an allele specific-PCR method. Results We observed the DHFR 19-bp homozygous deletion genotype (D/D) vs. homozygous wild genotype (WW) was more frequent in controls than in NS-CL/P patients (25% vs. 13%), being associated with a reduced risk of NS-CL/P in both codominant (OR=0.33, P=0.027) and recessive (OR=0.45, P=0.046) tested inheritance models. We also stratified the cleft patients and reanalyzed the data. The association trend for CL+CL/P group compared to the controls revealed that the DD genotype in both codominant (OR=0.30, P=0.032) and recessive models (OR=0.35, P=0.031) was associated with a reduced risk of CL+CL/P. Conclusions Our results for the first time suggested the DHFR 19-bp D/D genotype may confer a reduced risk of NS-CL/P and might act as a protective factor against NS-CL/P in the Iranian subjects. .
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Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Young Adult , Brain/abnormalities , Cleft Lip/genetics , Cleft Palate/genetics , Gene Deletion , Polymorphism, Genetic/genetics , Tetrahydrofolate Dehydrogenase/genetics , Case-Control Studies , Gene Frequency , Genetic Association Studies , Logistic Models , Polymerase Chain Reaction , Reference Values , Risk AssessmentABSTRACT
Objective To investigate the effects of dihydrofolate reductase (DHFR) gene expression up-regulating on cisplatin resistance in epithelial ovarian cancer cell lines.Methods The cDNA length of DHFR gene was amplified by PCR and was connected to lentiviral vector pWPI, the recombinant retroviral vector DHFR-pWPI was infected SKOV3 cells by lipofectamine 2000.The groups included DHFR-pWPI-SKOV3 cell, pWPI-SKOV3 cell and SKOV3 cell group.Western blot was used to detect the expression of DHFR.Flow cytometry was applied to measure the cell apoptosis rate of 3 groups cells in different cisplatin concentrations (2.5, 5.0, 10.0, 20.0 μg/ml) and at different time period (24, 48 and 72 hours), and half maximal inhibitory concentration (IC50) treated with cisplatin concentration (6.0, 4.0, 4.9 μg/ml).High performance liquid chromatography (HPLC) was applied to test intracellular cisplatin concentration in different cisplatin concentration (4.0, 6.0, 8.0 μg/ml) at 24 and 48 hours.Transmission electron microscope was used to observe ultrastructural changes cells under IC50 cisplatin concentration.Results The recombinant plasmid DHFR-pWPI was constructed and then infected into SKOV3 cell successfully.(1) The expression of DHFR detected by western blot in transfection group was higher than those in the negative control group and blank control group (10.280±0.009 vs 2.050±0.003 vs 3.480±0.003;P＜0.01).(2) Treated with cisplatin concentration (2.5, 5.0, 10.0, 20.0 μg/ml) at 24, 48 hours, the apoptosis rate detected by flow cytometry results shown that they were lower than those in the negative control group and blank control group (P＜0.05), while treated at the concentration of 5.0 and 10.0 μg/ml for 72 hours, whose apoptosis rate in transfection group was higher than those in the negative control group and blank control group (P＜0.05).When treated cells under IC50 cisplatin concentration (6.0, 4.0, 4.9 μg/ml) at for 24 and 48 hours, the results indicated thatthere were mainly G0/G1 stage cell cycle rate in 3 groups, it was obviously higher in transfection group than those in two control groups (P＜0.05).However, mainly G2/M, S stage cell cycle rate for 72 hours, and S stage cell cycle rate in transfection group obviously higher than those in two control groups, but G2/M stage cell cycle rate were lower (P＜0.01).(3) After treated with cisplatin concentration (4.0 μg/ml) for 24, 48 hours and cisplatin concentration (6.0 μg/ml) for 24 hours, the intracellular cisplatin content tested by HPLC method in the transfection group were significantly lower than those in two control groups (P＜0.01).While, at 6.0 μg/ml of cisplatin concentration for 48 hours and 8.0 μg/ml of cisplatin concentration for 24 and 48 hours, the intracellular cisplatin content of transfection group were obviously higher than those two control groups (P＜0.05).(4) Treated with IC50 (6.0, 4.0, 4.9 μg/ml) cisplatin concentration at different time to obeserve ultrastructural changes by transmission electron microscopy.The results shown that the microwire gathered together at 24 and 48 hours, and the number and structure of mitochondria had obvious change in transfection group, while there was rare microfilament, the number of mitochondria decreased but structure change was not apparent in two control groups.There were appeared expansion of endoplasmic reticulum and had rare normal organelles among three groups.After treated with cisplatin for 72 hours, there were inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in two control groups, and there were rare microfilament in transfection group, nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells on the verge of death.Conclusion The lentiviral expressing vector harboring human DHFR gene were successfully constructed.When the up-expression of DHFR gene, the drug-resistant in ovarian cancer cell may be increase, which suggest that there were certain contact between resistance increases with microfilament gathered and the change of the mitochondria.
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Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.
Subject(s)
Humans , Amino Acid Sequence , Bangladesh , Base Sequence , Dihydropteroate Synthase/genetics , Malaria, Vivax/parasitology , Molecular Sequence Data , Nepal , Philippines , Plasmodium vivax/enzymology , Polymorphism, Genetic , Tetrahydrofolate Dehydrogenase/geneticsABSTRACT
Many of the opportunistic infections that occur at this late stage can be fatal and since that Pneumocystis carinii pneumonia (PCP) is a leading opportunistic infection found among immunocompromised (CD4 cell < 200) patients worldwide. DHFR is responsible for the growth and maturation of sporozoites stage (life cycle) in Pneumocystis as reported. Currently, 13 million chemical compounds are available for virtual screening in ZINC database. The biological information of four known drug molecules like TMP/SMX, Dapsone, Atovaquone and Pentamidine were collected from the PubChem compound database. Q-Site Finder online tool was used to determine the active site of DHFR in P. jiroveci. LogP values of chemical compounds were identified with the Atom-additive method. Since, existing drugs are synthetic chemicals that give more side effects in Pneumocystis affected patients. Polar surface area value of oxamide (86.18) was predicted to be in the ranges of existing drug values. Pentamidine was proved to be a more efficient ligand based on the dock score of -26.3398 still could not be considered as the natural compound oxamide also was highly comparable with the value of -20.3173. The binding affinity of the selected molecule was analyzed through Pose View and LigPlot.
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Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. A characteristic set of mutations in this bifunctional enzymatic protein leads to reduced competitive drug binding at the enzyme's active site. The carbon content distribution study at mutational sites and along the amino acid sequence of this important protein was carried out using carbon analysis tool CARBANA. The mutational sites at residues 16, 51, 59, 108, and 164 were investigated. The study reveals that the carbon content and distribution of A16V and S108N mutants is shifting towards a normal distribution of the carbon content which is symmetrical about 0.3145, conforming to the value for stable and ordered protein. The study also reveals that carbon distribution of PfDHFR-TS mutant protein is maintained at 31.45% of carbon all along the sequence. The hydrophobicity of the entire sequences also balances quite well at the optimum position and carbon is the only element contributing towards this stability. Thus, the study of carbon distribution in mutations of PfDHFR-TS is the most significant step towards understanding the biological features which can provide possible approaches for the design of new drugs to overcome antifolate resistance.
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Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99 percent). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5 percent), followed by the wild-type A383/A553 (17.5 percent) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.
Subject(s)
Humans , Dihydropteroate Synthase , Drug Resistance , Malaria, Vivax , Plasmodium vivax/enzymology , Point Mutation , Tetrahydrofolate Dehydrogenase , Alleles , DNA, Protozoan , Endemic Diseases , Malaria, Vivax , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Plasmodium vivax , Plasmodium vivax , ThailandABSTRACT
The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.
Subject(s)
Humans , Amino Acid Substitution/genetics , Antimalarials/pharmacology , Drug Combinations , Drug Resistance , Haplotypes , Iran , Malaria, Vivax/parasitology , Mutation, Missense , Plasmodium vivax/enzymology , Polymorphism, Genetic , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Tetrahydrofolate Dehydrogenase/geneticsABSTRACT
Introducción. La acumulación progresiva de mutaciones en los genes dhfr y dhps lleva al parásito Plasmodium falciparum a evadir la acción de la sulfadoxina-pirimetamina, situación que aumenta el nivel de resistencia del parásito a estos medicamentos y conlleva a la aparición de fallas del tratamiento. Objetivos. Determinar la frecuencia de mutaciones en los genes dhfr y dhps de P. falciparum asociadas con resistencia a sulfadoxina-pirimetamina, en muestras de pacientes de tres zonas endémicas de Colombia: La Carpa, Guaviare; Casuarito, Vichada; Tierralta y Puerto Libertador, Córdoba. Materiales y métodos. Se incluyeron 40 muestras de pacientes con malaria no complicada por P. falciparum. Los alelos 108, 59 y 164 del gen dhfr se analizaron mediante PCR específica de alelo y los alelos 51 del gen dhfr y 436, 437 y 540 del gen dhps por PCR y restricción enzimática. Resultados. En el gen dhfr encontramos en todas las muestras las mutaciones asparagina 108 e isoleucina 51. No se detectaron alelos mutantes en los codones 59 y 164 del gen dhfr, ni en el codón 436 del gen dhps. La mutación glicina 437 estuvo presente en 36 muestras y el alelo silvestre alanina en tres de Tierralta y una de La Carpa. La mutación ácido glutámico 540 sólo se halló en Casuarito. Conclusiones. En las poblaciones de P. falciparum analizadas prevalecen los alelos asparagina 108, isoleucina 51 y glicina 437, lo que indica un efecto acumulativo de mutaciones y la necesidad de vigilar la aparición de nuevos alelos mutantes que puedan conducir a la pérdida total de la eficacia de la sulfadoxina-pirimetamina.
Introduction. Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. Objectives. To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. Materials and methods. Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. Results. The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. Conclusions. The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.
Subject(s)
Mutation , Plasmodium falciparum , Pyrimethamine , Sulfadoxine , Tetrahydrofolate Dehydrogenase , Dihydropteroate SynthaseABSTRACT
Objective To construct a folic acid deficient model in zebrafish and to observe the axial development in folic acid deficient embryos, so as to probe the mechanism by which folic acid deficiency induces abnormal development of axis. Methods We constructed the folic acid deficient zebrafish model by both using the antagonism of dihydrofolate reductase (MTX) and knocking-down dihydrofolate reductase gene. Then we observed the axial excursion of folic acid deficient embryos at 17 hpf under microscope. We labeled and observed the positions of liver, spleen and heart by using whole-mount in situ hybridization with specific antisense RNA probes. The expressions of some genes, which are down stream factors of Nodal signal pathway and important for axial development, were detected by whole-mount in situ hybridization and Real-time PCR. Results Parts of folic acid deficient embryos had axial excursion and abnormal positions of liver, spleen and heart. The expressing intensities of ntl and gsc appeared normal in folic acid deficient embryos, but the expressing spatial patterns were abnormal, which revealed the malformation of axial mesoderm. Conclusions Folic acid deficiency induced the abnormal development of axis and the malformation of axial mesoderm. Folic acid deficiency had no obviouse effect on Nodal pathway.
ABSTRACT
La enzima dihidrofolato reductasa está implicada en la producción de la base pririmidínica timidina, componente esencial de la estructura del ADN. Por tanto, cualquier sustancia que la inhiba tiene como efecto la inhibición de la síntesis del ADN, y es potencialmente útil para el tratamiento de varios tipos de cáncer como leucemias linfoblásticas. En este trabajo se determina el grado de inhibición que los extractos etanólicos obtenidos de las esponjas marinas colombianas Svenzea zeai, Amphimedon compressa, Ircinia campana, Aplysina archeri, Xestospongia proxima y Xestospongia muta, presentan sobre la enzima purificada de origen humano dihidrofolato reductasa. Los resultados muestran que la mayoría de los extractos de estas esponjas inhiben esta enzima. Estos resultados se comparan con los del medicamento usado contra el cáncer, Metotrexate®, el cual se utiliza como control de inhibición de los ensayos y se observa que algunas de las esponjas tienen mayor inhibición que este medicamento.
Dihydrofolate reductase is an enzyme involved in the production of pyrimidinic base timidin, a structural component of DNA, therefore whatever substance that inhibit this enzyme inhibit the DNA synthesis as a consequence and it can be potentially useful as a treatment of several types of cancer like lymphoblastic leukemias. In this work we determinate the inhibition grade that the ethanol extracts from Colombian marine sponges: Svenzea zeai, Amphimedon compressa, Ircinia campana, Aplysina archeri, Xestospongia proxima y Xestospongia muta, over the human purified enzyme dihydrofolate reductase. The results shown that most of marine sponge extracts inhibite the enzyme. Results are compared with methotrexate® a medicament used against cancer which is used as a control for the bioassays. Results demonstrate that some of the analyzed extracts have more inhibition than the control metotrexate®.
ABSTRACT
Foram analisadas a freqüência e distribuição de mutações nos genes dihidrofolato redutase e dihidropteroato sintetase do Plasmodium falciparum, usando a metodologia de reação em cadeia da polimerase e polimorfismos de hidrólise por enzimas de restrição, em amostras de sangue infectado proveniente de crianças moçambicanas, residentes em Maputo. A análise foi feita antes e 7 dias após o tratamento com sulfadoxina-pirimetamina (S/P). Os resultados mostraram a ocorrência de mutações pontuais nos genes estudados e a presença de combinações de três alelos em dhfr (51Ile, 59Arg e 108Asn) e do quintúplo mutante (dhfr 51Ile, 59Arg, 108Asn e dhps 437Gly, 540Glu), ambas situações associadas à falha terapêutica no sétimo dia após tratamento com S/P. Esses achados mostram a importância de se estudar a resistência à S/P em Moçambique, e como os marcadores moleculares de resistência aos antimaláricos podem fornecer dados importantes para a política nacional de controlo da malária.
The frequency and distribution of mutations in Plasmodium falciparum, dihydrofolate reductase and dihydropteroate synthase genes were analyzed, using the polymerase chain reaction and restriction fragment length polymorphism methodology, in infected blood samples from Mozambican children living in Maputo, before and seven days after treatment with sulfadoxine/pyrimethamine (S/P). The results showed the occurrence of point mutations in the genes studied and the presence of combinations of three alleles in dhfr (51Ile, 59Arg and 108Asn) and "quintuple" mutant (dhfr 51Ile, 59Arg, 108Asn and dhps 437Gly, 540Glu). Both of these situations were associated with seven-day therapeutic failure, following treatment with S/P. These findings show the importance of studying S/P resistance in Mozambique, and how molecular markers for antimalarial resistance can provide important data for national malaria control policy.