84 Folate-methionine cycle and folate transport in developing buffalo embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 168
Author(s):  
S. Ansari ◽  
S. Jamwal ◽  
S. Saini ◽  
R. Singh ◽  
D. Malakar
Keyword(s):  
Folic Acid ◽  
Carbon Metabolism ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Folate Transport ◽  
Nucleotide Synthesis ◽  
Folate Concentration ◽  
Vitamin B9 ◽  
Methionine Cycle ◽  
Folate Transporters

Periconceptional folic acid is known to have a major role in the prevention of neural tube defects, leading to global recommendations for folic acid supplementation before and in early pregnancy. Maternal folate throughout pregnancy may have other roles in offspring health, including neurodevelopment and cognitive performance in childhood. Folate and folic acid (vitamin B9) act as a co-enzyme essential for single carbon metabolism, a network of pathways involved in several biological processes including nucleotide synthesis, DNA repair, and methylation reactions. In general, rapidly growing and multiplying cells require an adequate supply of folate. A primary deficiency of natural folate resulting in an increase of the total homocysteine concentration may be detrimental to the quality of the oocyte, subsequent fertilisation, embryogenesis, implantation, and fetal development. However, to date, folate-methionine metabolism and folate transport have not been studied in developing buffalo embryos. The present study details transcript expression for genes encoding key enzymes in the linked folate-methionine cycles in the ovary tissue, cumulus cells, immature oocytes, IVM oocytes, and pre-implantation embryos and also estimates the folate concentration in follicular fluid (FF) of buffalo. The FF was pooled and collected by aspiration of different sizes of surface follicles (2-8mm diameter). The total number of analysed samples was three, with different dilutions and estimation of folate in FF of buffalo done by chemiluminescence assay. Total RNA was extracted from oocytes, cumulus cells, ovarian tissue, and embryos produced from IVF. RT-PCR was performed to analyse the expression of folate-methionine cycle enzymes and folate transporters. Transcripts for all the enzymes of the folate-methionine cycle (i.e. SHMT, MTR, MTRR, MAT1A, MAT2B, GNMT, AHCY, CBS, and DHFR) and folate transporters (FOLR1, FOLR2) and reduced folate carrier (SLC19A1) were expressed in ovarian tissue, cumulus cells, oocytes, and pre-implantation embryos. Immunocytochemical analysis revealed FOLR2 and SLC19A1 protein expression on the plasma membrane and/or cytoplasm of the oocytes and embryos, and FOLR1 in the nucleus of pre-implantation embryos. The folate concentration in FF was 24ngmL−1. This is the first report to examine the concentration of folate in FF and revealed the identification of transcripts in different samples of buffalo species. The presence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo, therefore indicating that folate from FF is being disseminated through folate receptors within oocytes and embryos to participate in the folate pathway. This study advocates the necessity for examination of the result of folate supplementation throughout invitro embryo production for improving the quality and quantity of transferable blastocysts and subsequently live calf births in buffalo.

scholarly journals Regulatory mechanisms of intestinal folate uptake in a rat model of folate oversupplementation

2010 ◽  
Vol 105 (6) ◽  
pp. 827-835 ◽  
Author(s):  
Som Dev ◽  
Nissar Ahmad Wani ◽  
Jyotdeep Kaur
Keyword(s):  
Folic Acid ◽  
Membrane Vesicles ◽  
Mrna Levels ◽  
Uptake System ◽  
Folate Transport ◽  
Down Regulation ◽  
Ph Optimum ◽  
Nucleotide Biosynthesis ◽  
Simple Diffusion ◽  
Folate Transporters

Folic acid is essential for numerous biological functions, ranging from nucleotide biosynthesis to the remethylation of homocysteine. Folic acid is unable to cross the biological membranes by simple diffusion, so there exists a well-developed epithelial folate transport system for the regulation of normal folate homeostasis in the intestine. Any perturbances in the folate uptake system might lead to a state of folate deficiency, which in turn is strongly associated with the risk of various cancers, birth defects and CVD. Countries with obligatory folate fortification of food (USA and Canada) have documented a significant decrease in neural tube defects in newborns. However, the effect of folate oversupplementation on the intestinal absorption of folic acid has not been studied. We studied the process of folate transport and the expression of folate transporters in the rat intestine after folate oversupplementation. Rats were oversupplemented with tenfold the normal requirement of folic acid for periods of 10 and 60 d. Folate uptake in intestinal brush-border membrane vesicles followed saturable kinetics with pH optimum at 5·5. Acute, but not chronic, folate oversupplementation led to a significant down-regulation in intestinal folate uptake at acidic pH optima and was associated with a decrease in Vmax without any significant change in the Km of the folate uptake process. The decrease in folate uptake was also associated with the down-regulation in the protein levels of major folate transporters, proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC), without altering their mRNA levels. Hence, it was concluded that acute folate oversupplementation results in a significant decrease in intestinal folate uptake by down-regulating the expressions of RFC and PCFT, via some post-transcriptional or translational mechanisms.

scholarly journals Folate Supplementation during Oocyte Maturation Positively Impacts the Folate-Methionine Metabolism in Pre-Implantation Embryos

2021 ◽  
Author(s):  
Sikander Saini ◽  
Vishal Sharma ◽  
Shama Ansari ◽  
Amit Kumar ◽  
Abhishek Thakur ◽  
...  
Keyword(s):  
Folic Acid ◽  
Production Rate ◽  
Oocyte Maturation ◽  
Folic Acid Supplementation ◽  
Folate Metabolism ◽  
Acid Supplementation ◽  
Maturation Medium ◽  
Metabolism Enzymes ◽  
Methionine Cycle ◽  
Folate Transporters

Abstract Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. The present study investigated the effect of folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 µM), were in vitro fertilized and cultured. Cumulus expansion markers (Ptx3 and Ptgs2) in cumulus cells were highly upregulated after 50 µM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 µM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 µM folic acid in maturation medium increases the blastocyst production rate, improves oocyte maturation, reduces ROS production as well as upregulate the expression of Folr1 and folate metabolism enzyme, Mtr.

scholarly journals UV/Vis absorption spectroelectrochemistry of folic acid

2021 ◽  
Author(s):  
F. Olmo ◽  
A. Rodriguez ◽  
A. Colina ◽  
A. Heras
Keyword(s):  
Folic Acid ◽  
Optical Signal ◽  
Oxidation Products ◽  
Basic Medium ◽  
Analytical Technique ◽  
Pharmaceutical Tablets ◽  
Solution Interface ◽  
Vitamin B9 ◽  
Analytical Protocol ◽  
Parallel Configuration

AbstractUV/Vis absorption spectroelectrochemistry is a very promising analytical technique due to the complementary information that is simultaneously obtained from electrochemistry and spectroscopy. In this work, this technique is used in a parallel configuration to study the oxidation of folic acid in alkaline medium. Herein, UV/Vis absorption spectroelectrochemistry has been used to detect both the oxidation products and the folic acid consumed at the electrode/solution interface, allowing us to develop an analytical protocol to quantify vitamin B9 in pharmaceutical tablets. Linear ranges of three orders of magnitude have been achieved in basic medium (pH = 12.9), obtaining high repeatability and low detection limits. The spectroelectrochemical determination of folic acid in pharmaceutical tablets at alkaline pH values is particularly interesting because of the changes that occur in the optical signal during the electrochemical oxidation of FA, providing results with very good figures of merit and demonstrating the utility and versatility of this hyphenated technique, UV/Vis absorption spectroelectrochemistry.

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopusoocytes

1998 ◽  
Vol 274 (1) ◽  
pp. C289-C294 ◽  
Author(s):  
Chandira K. Kumar ◽  
Toai T. Nguyen ◽  
Francis B. Gonzales ◽  
Hamid M. Said
Keyword(s):  
Folic Acid ◽  
Epithelial Cells ◽  
Cdna Clone ◽  
Xenopus Oocytes ◽  
Intestinal Epithelial Cells ◽  
Maximal Velocity ◽  
Mrna Levels ◽  
Acidic Ph ◽  
Intestinal Epithelial ◽  
Folate Transport

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17–20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity ( V max) of the uptake process [the apparent Michaelis-Menten constant ( K m) also changed (range was 0.31 to 1.56 μM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

Viability of ovarian tissue after freezing

1957 ◽  
Vol 147 (929) ◽  
pp. 520-528 ◽  
Keyword(s):  
Systematic Study ◽  
Normal Saline ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Rapid Freezing ◽  
Freezing And Thawing ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Fertilized Egg ◽  
Cells Cultured

The fertilized egg of the rabbit, obtained from the Fallopian tube, is very sensitive to freezing and thawing, even after treatment with glycerol (Smith 1953). By contrast, the cumulus cells of the follicular granulosa which adhere to the egg after ovulation are much more resistant, and a few survive without any special precautions being taken (Smith 1949). A systematic study of the viability of the cumulus cells cultured after freezing and thawing by various methods (Smith 1953) gave the following results: (1) A majority of the cells survived when suspended in homologous serum, cooled slowly to — 79° C and thawed rapidly at + 40° C. Very few survived rapid freezing. (2) Addition of 15 % glycerol to the suspending medium improved survival after slow cooling but not after rapid cooling. (3) Cells suspended in normal saline failed to survive either slow or rapid cooling. Addition of glycerol promoted some survival after slow cooling. These experiments emphasized the need for slow cooling, already demonstrated with bull spermatozoa, and the superiority of serum over saline as a suspending medium. The addition of glycerol to the medium, though conducive to survival after freezing and thawing, was not as necessary as with spermatozoa.

scholarly journals Relationship between Vitamin B9 (Folic Acid), Vitamin B12 (Cobalamin), and Peripheral Neuropathy in Children with Beta-Thalassemia Major

2021 ◽  
Vol 9 (2) ◽  
Author(s):  
Uni Gamayani ◽  
Titin Junaidi ◽  
Nushrotul Lailiyya ◽  
Nur Suryawan ◽  
Nanan Sekarwana
Keyword(s):  
Folic Acid ◽  
Peripheral Neuropathy ◽  
Vitamin B12 ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Control Group ◽  
Case Group ◽  
Beta Thalassemia Major ◽  
Vitamin B9 ◽  
Significant Difference

Vitamin B9 (folic acid) and B12 (cobalamin) are essential vitamins that play roles in the process of hematopoiesis and maintaining the function of peripheral nerves. Therefore, these deficiencies may create a risk for peripheral neuropathy in beta-thalassemia major patients. The purpose of this study is to determine the relationship between vitamin B9 level, vitamin B12 level, and peripheral neuropathy in beta-thalassemia major children. It was an observational analytical study with a case-control design has been conducted at Dr. Hasan Sadikin General Hospital Bandung, Indonesia, in May–July 2019. There were 47 beta-thalassemia major children with peripheral neuropathy (case) and 41 healthy children (control). All subjects completed a general demographic questionnaire, underwent neurological examination, and were tested for vitamin B9 and B12 serum levels. Data were then analyzed using the unpaired t test to compare the vitamin levels between both groups and Spearman’s rank correlation test to investigate the correlation between vitamin levels and the number of affected nerves in the case group. Comparison of folic acid levels in the case group (21.52±6.22 ng/mL) and the control group (23.81±7.51 ng/mL) showed no significant difference (p=0.19). In contrast, cobalamin in the case group (288.57±168.61 ng/mL) and the control group (385.95±197.48 ng/mL) showed a significant difference (p=0.01). In addition, there was a moderate correlation (p=0.004, r=0.41) between folic acid level and the number of motoric nerves affected in the case group. In conclusion, cobalamin level correlates with peripheral neuropathy in beta-thalassemia major patients, and folic acid level correlates with the number of affected nerves, especially motoric nerves. HUBUNGAN ANTARA VITAMIN B9 (ASAM FOLAT), VITAMIN B12 (KOBALAMIN), DAN NEUROPATI PERIFER PADA ANAK DENGAN TALASEMIA BETA MAYORVitamin B9 (asam folat) dan B12 (kobalamin) merupakan vitamin esensial yang berperan dalam proses hematopoiesis dan menjaga fungsi saraf tepi. Defisiensi vitamin ini dapat menimbulkan risiko neuropati perifer pada pasien talasemia beta mayor. Tujuan penelitian ini mengetahui hubungan antara kadar vitamin B9, vitamin B12, dan neuropati perifer pada anak talasemia beta mayor. Metode penelitian ini adalah analitik observasional dengan rancangan studi kasus kontrol yang dilakukan di RSUP Dr. Hasan Sadikin Bandung, Indonesia pada Mei–Juli 2019. Terdapat 47 anak talasemia beta mayor dengan neuropati perifer (kelompok kasus) dan 41 anak sehat (kelompok kontrol). Seluruh subjek penelitian mengisi kuesioner demografi umum, menjalani pemeriksaan fisis neurologis, serta dilakukan tes kadar vitamin B9 dan B12 serum. Uji t test tidak berpasangan digunakan untuk membandingkan kadar vitamin pada kedua kelompok dan uji korelasi Spearman untuk membandingkan kadar kedua vitamin tersebut dengan jumlah saraf yang terkena pada kelompok kasus. Perbandingan kadar asam folat kelompok kasus (21,52±6,22 ng/mL) dan kelompok kontrol (23,81±7,51 ng/mL) menunjukkan perbedaan yang tidak bermakna (p=0,19), sedangkan perbandingan kadar kobalamin kelompok kasus (288,57±168,61 ng/mL) dan kelompok kontrol (385,95±197,48 ng/mL) menunjukkan perbedaan yang bermakna (p=0,01). Selain itu, terdapat korelasi sedang (p=0,004; r=0,41) antara kadar asam folat dam jumlah saraf motorik yang terkena pada kelompok kasus. Kesimpulan, kadar kobalamin berhubungan dengan neuropati perifer pada penderita talasemia beta mayor dan kadar asam folat berhubungan dengan jumlah saraf yang terkena, terutama saraf motorik.

scholarly journals Role of Folate and Folic Acid During Pregnancy

2021 ◽  
Vol 9 (12) ◽  
pp. 1488-1492
Author(s):  
Saniya Sahar
Keyword(s):  
Folic Acid ◽  
Neural Tube ◽  
Maternal Nutrition ◽  
Tissue Growth ◽  
Food Fortification ◽  
Nucleotide Synthesis ◽  
Folic Acid Fortification ◽  
C1 Metabolism ◽  
Pteroylglutamic Acid

Abstract: Pregnancy represents a period of fast tissue growth of maternal and foetal tissues that's related to enhanced energy and nutrient needs. Maternal nutrition throughout gestation period, has being essential for best offspring development, reducing long unwellness burden and for general health throughout life. Maternal Folate throughout pregnancy might have numerous roles in offspring health, as well as neurodevelopment and psychological feature performance in childhood. Folate is crucial for C1 metabolism, a network of pathways concerned in many biological processes as well as nucleotide synthesis, deoxyribonucleic acid repair and methylation reactions. The periconceptional use of pteroylglutamic acid (Folic Acid ) containing supplements reduces the primary incidence, as well as recurrence of neural tube defects. Folic Acid (FA) are artificial form of a necessary vitamin generically considered Folates or B9. It is concerned in one-carbon metabolism, and it's been connected to lowering neural tube Defect (NTD). National programs to mandate fortification of food with Folic Acid have reduced the prevalence of NTDs worldwide . The indisputable protecting role of Folic Acid in the hindrance of NTD, in addition to the low compliance of women to Folic Acid recommendations, has aroused the choice of mandatory Folic Acid fortification, a policy currently in place in over eighty countries worldwide. Mandatory food fortification needs food makers to feature Folic Acid to certain foods (e.g. starch or grain products), whereas voluntary fortification permits Folic Acid to be added to foods at the discretion of manufacturers. Food fortification with Folic Acid because the intervention is likely to achieve increasing Folic Acid intake among populations throughout the world. The objective of this article is to discuss the Role of Folic Acid and Folate during pregnancy and to review the role of Folate and Folic Acid , metabolism , absorption and Folic Acid effects on maternal on the basis of recent findings that are important for implementation of fortified food to design future studies. Keywords: Neurodevelopment, Methylation Reactions, Pteroylglutamic Acid, Bioavailability, Monoglutamates.

scholarly journals Folic acid – role in the body, recommendations and clinical significance

2019 ◽  
Vol 18 (1) ◽  
pp. 50-59 ◽  
Author(s):  
Aneta Myszczyszyn ◽  
Rafał Krajewski ◽  
Monika Ostapów ◽  
Lidia Hirnle
Keyword(s):  
Folic Acid ◽  
Biological Effects ◽  
Folic Acid Supplementation ◽  
The Body ◽  
Leafy Vegetables ◽  
Congenital Defects ◽  
Natural Form ◽  
Vitamin B9 ◽  
Increased Risk

AbstractIntroduction. Folic acid is a compound classified as B group vitamins. In the body it is subject to processes that transfer its inactive form into a form responsible for biological effects of folic acid, i.e. 5-methyltetrahydrofolate (5-MTHF). It is, in particular, responsible for processes of the correct biosynthesis of purine and pyridine bases present in the formation of DNA and RNA molecules. Humans do not synthesize the endogenous form of folic acid; therefore, it is vital to supplement this vitamin in its natural form or multivitamin preparations. The most folic acid is found in the green leafy vegetables (spinach, peas, asparagus) and in offal (liver). An adequate supply of folic acid is especially indicated in pregnant women with a reduced amount of folic acid due to its use by an intensively developing foetus. The recommended dose of folic acid during this period is 0.4 mg/24h and this dose varies depending on the patient’s and her family’s medical history. The updated state of knowledge on the role of vitamin B9 in the body has been presented. The importance of its supplementation in specific clinical cases was analyzed.Summary. Many studies indicate an important role of the folic acid in the prevention of congenital defects of the nervous, cardiovascular and urogenital systems. Its deficiency increases the risk of complications in pregnancy, such as recurrent miscarriages, pre-eclampsia or postpartum haemorrhage. For this reason, a prophylactic folic acid supplementation is recommended, in women with increased risk of its deficiency, in particular.

Role of sodium ion in transport of folic acid in the small intestine

1986 ◽  
Vol 251 (2) ◽  
pp. G218-G222 ◽  
Author(s):  
J. Zimmerman ◽  
J. Selhub ◽  
I. H. Rosenberg
Keyword(s):  
Small Intestine ◽  
Folic Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Test Solution ◽  
Brush Border Membrane Vesicles ◽  
Acid Uptake ◽  
Folate Transport ◽  
Luminal Membrane

The effect of sodium on folate transport across the intestinal luminal membrane was analyzed using two techniques: the "influx" chamber and isolated brush-border membrane vesicles. Preincubation of tissue in Na+-free medium did not have a consistent effect on folic acid influx provided that Na+ was present in the test solution. Replacement of Na+ in the test solution by choline+ resulted in a significant reduction of folic acid influx. However, when intestinal sheets that had been equilibrated in Na+-free solution were exposed to test solution containing either Na+, Li+, K+, Rb+, Cs+, Tris+, or guanidinium+ as main cations, folic acid influx was not significantly decreased. Concentration-dependence studies showed that replacement of Na+ by Rb+ did not affect the saturable mechanism of folate transport. Rather, a decrease in nonsaturable folic acid uptake accounted for the slightly reduced influx observed in the presence of Rb+. Experiments with brush-border membrane vesicles revealed that methotrexate uptake was significantly higher in the presence of external Na+ than in the presence of K+, but was not different from uptake in the presence of K+ plus valinomycin. These data suggest that the saturable component of folate transport is not Na+ dependent, and nonsaturable transport of folic acid across the luminal membrane occurs in part through a conductive pathway that involves a negatively charged species of folate and a cation whose membrane permeability affects the rate of folate transport. The importance of Na+ in this process in vivo derives from the fact that Na+ is the most permeant cation available at the absorptive site in the small intestine.

A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 2055-2061 ◽  
Author(s):  
Inbal Lasry ◽  
Bluma Berman ◽  
Rachel Straussberg ◽  
Yael Sofer ◽  
Hanna Bessler ◽  
...  
Keyword(s):  
Folic Acid ◽  
Fold Increase ◽  
Loss Of Function ◽  
Transmembrane Helices ◽  
Intracellular Loop ◽  
Acid Growth ◽  
Folate Transport ◽  
Stable Transfectants ◽  
Growth Requirement ◽  
Inactivating Mutations

Abstract Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [3H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.

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