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.
Enzyme Stability ; Enzymes, Immobilized ; Hydrogen-Ion Concentration ; Kinetics ; Ligands ; Magnetite Nanoparticles ; Mycobacterium tuberculosis ; Temperature ; Tetrahydrofolate Dehydrogenase

PURPOSE: The initiation of mandatory folic acid fortification using pteroylmonoglutamic acid (PteGlu) has reduced the rate of congenital malformations. However, it also appears to be responsible for several adverse effects, including increased cancer incidence. This may be related to physicho-chemical characteristics of PteGlu. This study examines the potential effect of high concentrations of PteGlu on a population subjected to mandatory folic acid fortification using an in vitro model. METHODS: Caco-2 (colorectal cancer) and MCF7 (breast cancer) cell lines were cultured at 6 different PteGlu concentrations (0, 0.1, 1, 50, 250, and 500µg/ml) for 6 days. Cell growth was determined using thiazolyl blue tetrazolium bromide assay. The genotype of dihydrofolate reductase 19bp deletion/insertion (DHFR 19-del) was also scored in cell lines using a restriction fragment length polymorphism technique to examine whether genetic variations may factor in cell proliferation. RESULTS: PteGlu exhibited differential growth promoting properties between cell lines. Caco-2 cells did not show a significant growth difference at low concentrations compared to control, however, at higher concentrations, the growth showed a contrasting trend in the early experimental period, while MCF7 showed enhanced cell growth at all concentrations. The DHFR 19-del genotype differed in the two cell lines. CONCLUSION: Altered response to PteGlu by Caco-2 and MCF7 may reflect a tissue specific disease aetiology or genotype specific differential enzyme activity, for example by DHFR, to critical levels of PteGlu. As folic acid fortification is a blanket intervention, and DHFR and other enzyme activities vary between individuals, PteGlu intake may have an as yet undefined effect on health. These findings may be relevant when considering mandatory folic acid fortification for disease prevention.
Caco-2 Cells ; Cell Line* ; Cell Proliferation ; Folic Acid* ; Genetic Variation ; Genotype ; Humans ; Incidence ; Polymorphism, Restriction Fragment Length ; Tetrahydrofolate Dehydrogenase

BACKGROUND: Excessive exposure to fluoride can reduce intelligence. Methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 ( MTHFD1 ) polymorphisms have important roles in neurodevelopment. However, the association of MTHFD1 polymorphisms with children's intelligence changes in endemic fluorosis areas has been rarely explored. METHODS: A cross-sectional study was conducted in four randomly selected primary schools in Tongxu County, Henan Province, from April to May in 2017. A total of 694 children aged 8 to 12 years were included in the study with the recruitment by the cluster sampling method. Urinary fluoride (UF) and urinary creatinine were separately determined using the fluoride ion-selective electrode and creatinine assay kit. Children were classified as the high fluoride group and control group according to the median of urinary creatinine-adjusted urinary fluoride (UF Cr ) level. Four loci of MTHFD1 were genotyped, and the Combined Raven's Test was used to evaluate children's intelligence quotient (IQ). Generalized linear model and multinomial logistic regression model were performed to analyze the associations between children's UF Cr level, MTHFD1 polymorphisms, and intelligence. The general linear model was used to explore the effects of gene-environment and gene-gene interaction on intelligence. RESULTS: In the high fluoride group, children's IQ scores decreased by 2.502 when the UF Cr level increased by 1.0 mg/L (β = -2.502, 95% confidence interval [CI]:-4.411, -0.593), and the possibility for having "excellent" intelligence decreased by 46.3% (odds ratio = 0.537, 95% CI: 0.290, 0.994). Children with the GG genotype showed increased IQ scores than those with the AA genotype of rs11627387 locus in the high fluoride group ( P   <  0.05). Interactions between fluoride exposure and MTHFD1 polymorphisms on intelligence were observed (Pinteraction < 0.05). CONCLUSION Our findings suggest that excessive fluoride exposure may have adverse effects on children's intelligence, and changes in children's intelligence may be associated with the interaction between fluoride and MTHFD1 polymorphisms.
Child ; Creatinine ; Cross-Sectional Studies ; Fluorides/urine* ; Formate-Tetrahydrofolate Ligase ; Humans ; Intelligence/genetics* ; Methylenetetrahydrofolate Dehydrogenase (NADP) ; Methylenetetrahydrofolate Reductase (NADPH2)

Methotrexate is a very potent inhibitor of dihydrofolate reductase and causes bone marrow suppression and megaloblastic anemia. It is widely used in combination with other chemotherapeutic agents in lymphoproliferative disorders. A 63 year old man with ischioneuralgia developed exertional dyspnea and dizziness after he had intentionally taken methotrexate in doses of 5mg per day for 2months. Five months after discontinuation of methotrexate, his bone marrow showed the hypercellular marrow with 90% cellularity, 15% blasts and marked dysgranulopoiesis, suggestive of refractory anemia with excess blasts(RAEB). The hematopoietic cells were not enough aspirated for proper diagnosis in follow up bone marrow after three months. The bone marrow aspirates showed 13% blasts, and marked dysgranulopoiesis. The bone marrow biopsy showed hypercellular marrow with 100% cellularity, but marked fibrosis was developed. The cytogenetic study revealed normal karyotype.
Anemia, Megaloblastic ; Anemia, Refractory ; Biopsy ; Bone Marrow ; Cytogenetics ; Diagnosis ; Dizziness ; Dyspnea ; Fibrosis ; Follow-Up Studies ; Humans ; Intention ; Karyotype ; Lymphoproliferative Disorders ; Methotrexate* ; Middle Aged ; Myelodysplastic Syndromes* ; Tetrahydrofolate Dehydrogenase

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder of unknown etiology. Inflammation may usually extend beyond the joints and involve other organs. Clinically detectable splenomegaly is present in 5~10% of RA. Methotrexate (MTX) is a structural analog of folic acid that inhibits the enzyme dihydrofolate reductase, so cellular proliferation is reduced. MTX has been proven to be effective in treating RA and is believed to be nononcogenic at low, weekly dose employed in the patients with RA. However, recently there has been increased concern about the oncogenic potential of MTX because of several case reports describing the occurrence of non-Hodgkin's Lymphoma (NHL) in the patients with RA treated with MTX. A 65-year-old woman with RA was treated with low dose MTX (i.e. 10 mg/week) for 3 years. Because of prolonged left upper abdominal pain and thrombocytopenia associated with huge splenomegaly, splenectomy was performed. Biopsy revealed splenic B-cell NHL. We report a case of RA with splenomegaly who developed B-cell NHL in spleen during low dose MTX therapy.
Abdominal Pain ; Aged ; Arthritis, Rheumatoid* ; B-Lymphocytes ; Biopsy ; Cell Proliferation ; Female ; Folic Acid ; Humans ; Inflammation ; Joints ; Lymphoma, Non-Hodgkin* ; Methotrexate ; Spleen ; Splenectomy ; Splenomegaly ; Tetrahydrofolate Dehydrogenase ; Thrombocytopenia

Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo*-IRES-k2tPA fusion gene and a DHFR gene, we screened colonies by k2tPA expression level. We selected 7 clones that expressed high level of k2tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene (k2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr- cell line 8-1, the expression level of k2tPA could amount to 17.1 microg/10(6) cell x 24 h.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; DNA Nucleotidyltransferases ; genetics ; Gene Amplification ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics ; Tetrahydrofolate Dehydrogenase ; genetics

BACKGROUNDFolic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.

METHODSMorpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.

RESULTSDHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.

CONCLUSIONSDHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; physiology ; Female ; Heart ; embryology ; Heart Defects, Congenital ; etiology ; Tetrahydrofolate Dehydrogenase ; genetics ; physiology ; Zebrafish ; Zebrafish Proteins ; genetics ; physiology

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.
Cloning, Molecular ; Escherichia coli ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Staphylococcus aureus ; drug effects ; Tetrahydrofolate Dehydrogenase ; genetics ; beta-Defensins ; biosynthesis ; genetics ; pharmacology

BACKGROUNDTo study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression.

METHODSUsing the N-terminal truncated hTPO(T184) gene as target gene, two plasmidsre were constructed: pDC- T184 and pGC-T184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr cells to establish dual gene amplification and selection system of DHFR plus GS gen and respectively transfected to establish single gene amplification and selection system of DHFR or GS gene. Three selective methods in dual selective system to compare expression efficiency of hTPO were designed: the first method (DG) was to use drug pressure of MTX, then use MSX; the second method (GD) was reversed; the third method was simultaneously to use MTX and MSX as drug pressure.

RESULTSDHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure.

CONCLUSIONSMTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.

Animals ; CHO Cells ; Cricetinae ; Gene Amplification ; drug effects ; Gene Expression ; Glutamate-Ammonia Ligase ; genetics ; Methotrexate ; pharmacology ; Plasmids ; genetics ; Tetrahydrofolate Dehydrogenase ; genetics

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.
Amino Acid Substitution/genetics ; Antimalarials/*pharmacology ; Drug Combinations ; *Drug Resistance ; Haplotypes ; Humans ; Iran ; Malaria, Vivax/*parasitology ; *Mutation, Missense ; Plasmodium vivax/*enzymology/genetics/isolation & purification ; Polymorphism, Genetic ; Pyrimethamine/*pharmacology ; Sulfadoxine/*pharmacology ; Tetrahydrofolate Dehydrogenase/*genetics