HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dutta, B. Articles by Prasad, P. D. Search for Related Content PubMed PubMed Citation Articles by Dutta, B. Articles by Prasad, P. D. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 45, 31925-31929, November 5, 1999

Cloning of the Human Thiamine Transporter, a Member of the Folate Transporter Family^*

Binita Dutta, Wei Huang^§, Mariela Molero^§, Ramesh Kekuda^§, Frederick H. Leibach^§, Lawrence D. Devoe, Vadivel Ganapathy^§, and Puttur D. Prasad^§¶

From the Departments of  Obstetrics and Gynecology and ^§ Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K_t = 2.5 ± 0.6 µM) in a Na⁺-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H⁺ gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Thiamine, as a component of the coenzyme thiamine pyrophosphate, is important for glycolysis and energy production in mitochondria (1). It is an essential nutrient, and its requirement in the developing fetus is met by the transplacental transport from maternal circulation to fetal circulation. The concentration of thiamine is more than 10-fold higher in fetal umbilical vein plasma compared with that present in maternal plasma (2), suggesting the presence of an efficient transport system for the vitamin in placenta. The underlying mechanism of transplacental transport of thiamine has been investigated using in vitro perfusion techniques (3, 4) as well as isolated placental brush border membrane vesicles (5). Thiamine transport has also been functionally characterized in other tissues/cells such as intestine (6-9), erythrocytes (10), hepatocytes (11), and neuroblastoma cells (12). These studies have shown that the transport of thiamine is a carrier-mediated, energy-dependent process.

Thiamine is an organic cation. Depending on the pH of the solution, thiamine can exist either as a monovalent or bivalent cation (6). Absorptive tissues such as placenta (13, 14), kidney (15, 16), and intestine (17) are known to express specific organic cation/H⁺ antiport systems that are capable of transporting both endogenous as well as exogenous organic cations. The presence of an outwardly directed proton gradient was shown to induce concentrative thiamine accumulation in brush border membrane vesicles isolated from human term placenta, suggesting a possible role of an organic cation/H⁺ antiport system in the uptake of thiamine into the placenta (5). Consistent with such an uptake mechanism, the thiamine uptake into placental brush border membrane vesicles was abolished in the presence of proton ionophores and unaffected by the presence of an inside-negative membrane potential. Existence of a similar uptake mechanism for thiamine has also been described in intestine (8) and erythrocytes (10).

In this study, we describe the molecular cloning of a cDNA from human placenta which, when expressed in mammalian cells, induces the uptake of thiamine. This thiamine transporter (ThT1)¹ shares significant homology (40% identity) with the reduced-folate transporter (RFC or FOLT) previously cloned from human placenta (18). Northern analysis indicates that the ThT1-specific transcript (~3.8 kb in size) is widely expressed in human tissues. The human gene of ThT1 (~22 kb), which has already been sequenced as a part of the Human Genome Project, maps to chromosome 1q24 and consists of 6 exons and 5 introns as determined by comparison of sequences of tht1 gene and ThT1 cDNA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- SuperScript Plasmid System for cDNA cloning, Dulbecco's modified Eagle's medium, and Lipofectin were purchased from Life Technologies, Inc. Nitropure transfer membranes were obtained from Osmonics (Minnetonka, MN). [³H]Thiamine (specific activity, 400 mCi/mmol), [3',5',7,9-³H](6S)-5-methyltetrahydrofolic acid (specific activity, 30 Ci/mmol), [³H(G)]riboflavin (specific activity, 30 Ci/mmol), and [3,5,7-³H]methotrexate (specific activity, 19.7 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). [3',5',7,9-³H]Folic acid (specific activity, 30 Ci/mmol), [methyl-³H]choline chloride (specific activity, 85 Ci/mmol), [ethyl-1 ¹⁴C]tetraethylammonium (TEA) bromide (specific activity, 55mCi/mmol), and [methyl-³H]MPP (specific activity, 60 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). L-[carboxyl-¹⁴C]Ascorbic acid (specific activity, 16.6 mCi/mmol), [N-methyl-³H]cimetidine (specific activity, 18.2 Ci/mmol), [-³²P]dCTP (specific activity, 3000Ci/mmol), and the Ready-to-Go oligolabeling kit were procured from Amersham Pharmacia Biotech. The cDNA for rOCT2 (19, 20) was kindly provided by Dr. J. B. Pritchard (NIEHS, National Institutes of Health, Raleigh, NC). The original reports on the cloning of rOCT3 (21) and rOCTN2 (22) from rat placenta were from our laboratory. The cDNA for rOCT1 (23) was obtained by screening a rat liver cDNA library using rOCT3 as the probe. The cDNA for rOCTN1 was obtained by screening a rat placental cDNA library using hOCTN1 as the probe (24).

cDNA Cloning and Sequencing-- Previous studies from our laboratory have led to the cloning of the RFC from human placenta (18). Blast search of the GenBank peptide sequence data base using the amino acid sequence of human RFC showed that a sequence entry of a RFC-like protein has been submitted to the data base (accession CAA15926). This protein, as per the GenBank entry, is 468 amino acids long, truncated at the N terminus lacking the initiator methionine and shared 39% identity and 55% similarity with human RFC. The amino acid sequence of the RFC-like protein was derived by GENSCAN prediction from the nucleotide sequence of a genomic clone isolated from a genomic library of human chromosome 1 (nucleotide sequence data base accession no. AL021068, submitted by C. Bird, Sanger Center Chromosome 1 Mapping Group). Neither the complete amino acid sequence of the protein nor its function was known. In order to get the full-length cDNA of the RFC-like protein, we generated a cDNA probe specific to the protein by RT-PCR and screened a human placental cDNA library. The RT-PCR was done using poly(A)⁺ RNA isolated from human term placenta and the primers 5'-GCCACCAGAAAGTCACAAGTT-3' (upstream primer) and 5'-CAGGAGAGAAAAGAGAGATA-3' (downstream primer). The upper primer had an additional three nucleotides (underlined) added at the 5' end to ensure primer pair compatibility. The expected size of the RT-PCR product was 656 bp, comprising the region coding for Gln¹²⁶ to Leu³⁴³ of the RFC-like protein. The identity of the product obtained was confirmed by sequencing before using it as the probe in library screening.

The screening of the placental cDNA library was done by colony screening of the plasmid cDNA library grown on Nitropure transfer membranes as described previously (25). The cDNA probe was labeled with [-³²P]dCTP by random priming using the Ready-to-Go oligolabeling kit. Positive clones were identified and the colonies purified by secondary screening. The size of the cDNA inserts of the positive clones was determined by restriction digestion using EcoRI/BamHI to release the insert from the vector followed by size fractionation by agarose gel electrophoresis. A single clone with the largest insert size was selected for further characterization.

Both sense and antisense strands of the cDNA were sequenced by primer walking. Sequencing by the dideoxynucleotide chain termination chemistry was performed by Taq DyeDeoxy terminator cycle sequencing using an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analyzed using the BCM Search Launcher server (26) and NCBI server.

Northern Analysis-- Tissue distribution of the ThT1-specific transcript was determined by Northern analysis. A hybridization-ready, commercially available human multiple tissue blot (CLONTECH). The blot was probed sequentially with ThT1 cDNA and then with glyceraldehyde-3-phosphate dehydrogenase cDNA under high stringency conditions.

Functional Expression of the ThT1 cDNA-- The cloned cDNA was functionally expressed in HeLa cells by vaccinia virus expression system as described previously (25, 27). Subconfluent HeLa cells grown on 24-well plates were first infected with the recombinant (VTF_7-3) vaccinia virus encoding T7 RNA polymerase and then transfected with 1 µg of the plasmid carrying the full-length cDNA using Lipofectin. In most experiments, transport measurements were made at 37 °C with a 10-min incubation using [³H]thiamine in the absence of Na⁺ at pH 8.0. The composition of the transport buffer was 25 mM Tris/Hepes, pH 8.0, supplemented with 140 mM NMDG chloride, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. When the influence of Na⁺ on thiamine uptake was investigated, NMDG chloride in the transport buffer was replaced with NaCl. When the influence of pH on transport was investigated, transport buffers of different pH were prepared by varying the concentration of Tris, Hepes, and Mes. Transport was terminated by aspiration of the uptake buffer followed by two rapid washes with 2 ml of ice-cold transport buffer. The cells were then solubilized with 0.5 ml of 1% SDS in 0.2 N NaOH and transferred to vials for quantitation of the radioactivity associated with the cells. Endogenous transport was always measured in parallel in cells transfected with empty vector (pSPORT) and subtracted from the corresponding transport values measured in cells transfected with vector cDNA to obtain the cDNA-specific uptake of the vitamin.

Data Analysis-- Uptake measurements were made either in duplicate or triplicate, and each experiment was repeated two to three times with separate transfections. Results are given as means ± S.E. of these replicate values. Kinetic analyses were carried out by nonlinear as well as linear regression methods using the commercially available computer program SigmaPlot (SPSS Inc., Chicago, IL).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The cloned cDNA (ThT1, for thiamine transporter; GenBank accession no. AF160812) is 3530 bp long and has an open reading frame of 1494 bp including the termination codon. The open reading frame is flanked by a 96-bp-long 5'-noncoding sequence and a 1941-bp-long 3'-noncoding sequence. The open reading frame encodes a 497-amino acid protein (Fig. 1A) with a molecular mass of 55.4 kDa and a pI of 6.35. Hydropathy analysis of the primary amino acid sequence using the Kyte-Doolittle method (28) with a window size of 17-24 amino acids per transmembrane helix predicts a topographical model with 12 putative transmembrane domains (Fig. 1B). The strongly preferred model for the arrangement of the transmembrane domains across the membrane is the one with both the N terminus and the C terminus toward the inside. There are two potential sites for N-linked glycosylation at positions 63 and 314 in putative extracellular domains. The primary amino acid sequence also displays three sites (Ser⁸, Thr²², and Ser²⁹¹) with consensus sequence for protein kinase C-dependent phosphorylation in putative intracellular domains. Interestingly, a 17-amino acid residue sequence, which is a signature of G-protein-coupled receptors (29), is also present in the amino acid sequence of ThT1 (amino acid residues 407-423).

View larger version (60K): [in this window] [in a new window]   Fig. 1.   A, comparison of amino acid sequences of human RFC1 and human ThT1. Regions of identity (dark shading) and similarity (light shading) are indicated. B, topographical model of ThT1 based on hydropathy analysis.

A comparison of the amino acid sequence of human ThT1 with the protein sequences in the SwissProt sequence data base confirmed that ThT1 is identical to RFC-like protein, however, with an additional 29 amino acids at the N terminus. The closest relative of ThT1 is RFC1 (18), the reduced-folate transporter, with a sequence identity of 40% and similarity of 55% at the amino acid level. A comparison of amino acid sequence of ThT1 and RFC1 is presented in Fig. 1A. Thus, ThT1 is the second member of the folate transporter family to be cloned.

The expression of ThT1 in human tissues was investigated by Northern analysis using a commercially available multiple tissue blot containing size-fractionated poly(A⁺) RNA obtained from several tissues of human origin. The ThT1-specific hybridization signal (3.8 kb in size) was widely distributed in human tissues (Fig. 2). The intensity of the hybridization signal obtained was especially high in skeletal muscle, followed by placenta, heart, liver, and kidney.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis of ThT1 expression in human tissues. A commercially available Northern blot containing 2 µg/lane poly(A)+ RNA isolated from various tissues of human origin was probed sequentially with ThT1- and glyceraldehyde-3-phosphate dehydrogenase-specific cDNA probes. The sizes of RNA hybridizing bands were determined using RNA standards run in parallel in an adjacent lane.

The functional expression of the clone was done in HeLa cells by transient transfection followed by vaccinia virus-induced expression of the cDNA. Since the cloned transporter showed significant homology to RFC1, we first investigated the ability of the cloned transporter to transport folate and its analogs. The uptake of [³H]5-methyltetrahydrofolate, [³H]folic acid, and [³H]methotrexate was studied in cells transfected with either empty vector or ThT1 cDNA in the presence (Fig. 3A) or absence (Fig. 3B) of Na⁺. These studies showed that none of these compounds was a transportable substrate for ThT1. We then tested the ability of ThT1 to transport several water-soluble vitamins like riboflavin, ascorbic acid, and thiamine. Of these substrates tested, only the uptake of thiamine was ~2-fold higher in ThT1 cDNA-transfected cells in comparison to vector-transfected cells (Fig. 3). Removal of Na⁺ from the transport buffer did not abolish the transport activity, demonstrating that the transport activity mediated by ThT1 is Na⁺-independent. Subsequent experiments involving the characterization of the transport function of ThT1 were done in the absence of Na⁺.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Functional expression of ThT1 cDNA. HeLa cells were transfected either with pSPORT vector alone or pSPORT-ThT1 cDNA. Functional expression of the cDNA was carried out using the vaccinia virus expression technique. Transport of [3H]5-methyltetrahydrofolate (MTF), [3H]folic acid, [3H]methotrexate (MTX), [3H]riboflavin, [14C]ascorbic acid, and [3H]thiamine (0.25 µCi each) was measured in these cells in the presence (A) and absence (B) of Na+. The cells were incubated with the substrate for 10 min at 37 °C. The pH of the transport buffer was 7.5.

The transport of thiamine in placental and intestinal brush border membrane vesicles was stimulated by an inside-out proton gradient (5, 8). We therefore examined the effect of extracellular pH on thiamine transport in vector- and cDNA-transfected cells (Fig. 4A). The uptake of thiamine at pH 8.0 was almost 3-fold higher than the uptake at pH 6.0. The process of thiamine uptake displayed a distinct pH optimum at about pH 8.0. Increasing the pH from 6.0 to 8.0 stimulated thiamine uptake, but further increase in pH above 8.0 inhibited the uptake. The endogenous thiamine transport measured in empty vector-transfected cells was much less compared with uptake in cells transfected with ThT1, and this endogenous uptake was similarly influenced by pH. The pH optimum of the endogenous transport, however, was not 8.0, and the uptake kept increasing even when the pH was increased to 8.5. 

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, influence of pH on ThT1-induced thiamine transport in HeLa cells. HeLa cells were transfected with either pSPORT vector alone or pSPORT-ThT1 cDNA. Transport of [3H]thiamine (2.5 µM) in these cells was measured for 10 min at 37 °C using NMDG chloride-containing transport buffers of different pH. B, saturation kinetics of ThT1-specific thiamine transport. Transport of thiamine was measured in vector-transfected cells and in ThT1 cDNA-transfected cells in parallel over a thiamine concentration range of 0.25-10 µM. The ThT1-specific transport was calculated by subtracting the transport measured in vector-transfected cells at each concentration of thiamine from the corresponding transport measured in ThT1 cDNA-transfected cells.

Thiamine is an organic cation. Recently, a number of organic cation transporters have been cloned and functionally characterized (30). The members of the organic cation transporter family accept a variety of organic cations as substrates. It was therefore of interest to see if any of these organic cation transporters is capable of transporting thiamine. We expressed all of the known members of the organic cation transporter family (OCT1, OCT2, OCT3, OCTN1, and OCTN2) in HeLa cells and compared the ability of these transporters to mediate the transport of thiamine and TEA, a prototypical organic cation substrate for these transporters. Although all of the organic cation transporters were functionally expressed, as seen by the increased TEA uptake in cDNA-transfected cells compared with vector-transfected cells, none of them, except OCT3, showed significant transport of thiamine (Table I).

The substrate specificity of ThT1 was evaluated by directly measuring the uptake of structurally diverse organic cations as well as by assessing the ability of these organic cations to inhibit the ThT1-mediated [³H]thiamine uptake. First, the uptake of radiolabeled choline, TEA, MPP, cimetidine, and thiamine was compared in cells expressing ThT1 and control cells transfected with empty vector. All these compounds are organic cations and are known to be transported via organic cation/H⁺ antiport process in several tissues. However, there was no difference in the transport of TEA, choline, MPP, and cimetidine between control cells and ThT1-expressing cells (data not shown). Under identical conditions, the transport of thiamine was higher in ThT1-expressing cells than in control cells as expected. Next, the ability of various organic cations (1 mM) to inhibit the ThT1-mediated thiamine transport was investigated. Only unlabeled thiamine (1 mM) was able to inhibit the transport of [³H]thiamine (Table II). Other organic cations (guanidine, cimetidine, and choline) failed to inhibit thiamine uptake. These results provide confirmatory evidence that, unlike other organic cation transporters, ThT1 is very specific for thiamine. The transport process mediated by ThT1 was saturable with a K_t of 2.5 ± 0.6 µM for thiamine (Fig. 4B).

Comparison of the nucleotide sequence of ThT1 cDNA with the nucleotide sequence from which the amino acid sequence of RFC-like protein was derived indicated that the human gene coding for ThT1 has been sequenced in its entirety as a part of the Human Genome Project. The gene (~22 kb) maps to chromosome 1q24. A comparison of the nucleotide sequences of the tht1 gene and ThT1 cDNA has enabled us to deduce the exon-intron organization of the gene (Fig. 5). The gene consists of 6 exons and 5 introns. All exon-intron boundaries conform to consensus donor-acceptor sequences (gt/ag) for RNA splicing. An autosomal recessive disorder called thiamine-responsive megaloblastic anemia has been described (31), and the defect in this syndrome has been localized to chromosome 1, band 1q23.3 (32). Fibroblasts from these patients lack the high affinity transport system for thiamine. The ability of these cells to transport thiamine can be restored by transfecting the cells with the yeast thiamine transporter gene, THI10 (33). These findings suggest that, the primary abnormality in thiamine-responsive megaloblastic anemia is the absence of a functional high affinity thiamine transporter and that the tht1 gene is most likely defective in these patients. Although both ThT1 and THI10 are thiamine transporters and THI10 is able to functionally correct thiamine transport defect, a comparison of the amino acid sequences of the two proteins by Blast search showed no significant homology between the two proteins.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Exon-intron organization of the human tht1 gene and its organizational relationship to the human ThT1 cDNA. Black boxes represent the protein coding regions of the exons, and open boxes represent 5'-untranslated region in exon 1 and 3'-untranslated region in exon 6. The nucleotide positions of the start and stop codons, and the exon junctions in cDNA are indicated.

In summary, we have cloned a human thiamine transporter from the placenta. Its transport function is Na⁺-independent. Structurally this transporter is related to the reduced-folate transporter whose transport function is also Na⁺-independent. Thus, ThT1 represents the second member of the folate transporter gene family. It is to be noted, however, that the substrate for ThT1 is an organic cation whereas the substrate for RFC1 is an organic anion. Even though both transporters are pH-dependent, the function of ThT1 is stimulated by an outward-directed H⁺ gradient, whereas the function of RFC1is known to be stimulated by an inward-directed H⁺ gradient (34). The transport mechanism of these two transporters with respect to the involvement of H⁺ appears to be different. Transporters for other water-soluble vitamins have been previously identified and functionally characterized. The multivitamin transporter SMVT is specific for biotin and pantothenate and its transport function is obligatorily dependent on Na⁺ (25, 35, 36). Two different transporters (SVCT1 and SVCT2) have been identified for the transport of ascorbic acid (vitamin C) in mammalian cells, and both of them are obligatorily dependent on Na⁺ for their function (37). Thus, in addition to the differences in substrate selectivity, the vitamin transporters also appear to differ in Na⁺ dependence.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HD33347 (to V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF160812.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. Tel.: 706-721-1761; Fax: 706-721-6608; E-mail: pprasad@mail.mcg.edu.

	ABBREVIATIONS

The abbreviations used are: ThT, thiamine transporter; RFC, reduced-folate carrier; NMDG, N-methyl-D-glucamine; bp, base pair(s); kb, kilobase(s); TEA, tetraethylammonium, MPP, 1-methyl-4-phenylpyridinium; RT-PCR, reverse transcription-polymerase chain reaction; Mes, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. Vogl, C. M. Klein, A. F. Batke, M. E. Schweingruber, and J. Stolz Characterization of Thi9, a Novel Thiamine (Vitamin B1) Transporter from Schizosaccharomyces pombe J. Biol. Chem., March 21, 2008; 283(12): 7379 - 7389. [Abstract] [Full Text] [PDF]

				V. S. Subramanian, Z. M. Mohammed, A. Molina, J. S. Marchant, N. D. Vaziri, and H. M. Said Vitamin B1 (thiamine) uptake by human retinal pigment epithelial (ARPE-19) cells: mechanism and regulation J. Physiol., July 1, 2007; 582(1): 73 - 85. [Abstract] [Full Text] [PDF]

				V. S. Subramanian, J. S. Marchant, and H. M. Said Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2 Am J Physiol Cell Physiol, November 1, 2006; 291(5): C851 - C859. [Abstract] [Full Text] [PDF]

				B. Ashokkumar, N. D. Vaziri, and H. M. Said Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms Am J Physiol Renal Physiol, October 1, 2006; 291(4): F796 - F805. [Abstract] [Full Text] [PDF]

				R. Mendoza, M. M. Anderson, and J. Overbaugh A putative thiamine transport protein is a receptor for feline leukemia virus subgroup a. J. Virol., April 1, 2006; 80(7): 3378 - 3385. [Abstract] [Full Text] [PDF]

				V. S. Subramanian, J. S. Marchant, and H. M. Said Targeting and Trafficking of the Human Thiamine Transporter-2 in Epithelial Cells J. Biol. Chem., February 24, 2006; 281(8): 5233 - 5245. [Abstract] [Full Text] [PDF]

				S. M. Nabokina, J. C. Reidling, and H. M. Said Differentiation-dependent Up-regulation of Intestinal Thiamin Uptake: CELLULAR AND MOLECULAR MECHANISMS J. Biol. Chem., September 23, 2005; 280(38): 32676 - 32682. [Abstract] [Full Text] [PDF]

				S. M. Nabokina and H. M. Said Characterization of the 5'-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies Am J Physiol Gastrointest Liver Physiol, October 1, 2004; 287(4): G822 - G829. [Abstract] [Full Text] [PDF]

				Y. Wang, R. Zhao, and I. D. Goldman Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier Clin. Cancer Res., September 15, 2004; 10(18): 6256 - 6264. [Abstract] [Full Text] [PDF]

				L. G. Boros, M. P. Steinkamp, J. C. Fleming, W.-N. P. Lee, M. Cascante, and E. J. Neufeld Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) Blood, November 15, 2003; 102(10): 3556 - 3561. [Abstract] [Full Text] [PDF]

				J. C. Reidling and H. M. Said In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2 Am J Physiol Cell Physiol, September 1, 2003; 285(3): C633 - C641. [Abstract] [Full Text] [PDF]

				V. S. Subramanian, J. S. Marchant, I. Parker, and H. M. Said Cell Biology of the Human Thiamine Transporter-1 (hTHTR1). INTRACELLULAR TRAFFICKING AND MEMBRANE TARGETING MECHANISMS J. Biol. Chem., January 31, 2003; 278(6): 3976 - 3984. [Abstract] [Full Text] [PDF]

				K. Oishi, S. Hofmann, G. A. Diaz, T. Brown, D. Manwani, L. Ng, R. Young, H. Vlassara, Y. A. Ioannou, D. Forrest, et al. Targeted disruption of Slc19a2, the gene encoding the high-affinity thiamin transporter Thtr-1, causes diabetes mellitus, sensorineural deafness and megaloblastosis in mice Hum. Mol. Genet., November 1, 2002; 11(23): 2951 - 2960. [Abstract] [Full Text] [PDF]

				K. Balamurugan and H. M. Said Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis Am J Physiol Gastrointest Liver Physiol, July 1, 2002; 283(1): G37 - G43. [Abstract] [Full Text] [PDF]

				H. M. Said, A. Ortiz, V. S. Subramanian, E. J. Neufeld, M. P. Moyer, and P. K. Dudeja Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460 Am J Physiol Gastrointest Liver Physiol, July 1, 2001; 281(1): G144 - G150. [Abstract] [Full Text] [PDF]

				G. Rindi and U. Laforenza Thiamine Intestinal Transport and Related Issues: Recent Aspects Experimental Biology and Medicine, September 1, 2000; 224(4): 246 - 255. [Abstract] [Full Text]

				C. I Onochie, L. M Korngut, J. B Vanhorne, S. M Myers, D. Michaud, and L. M Mulligan Characterisation of the human GFRalpha-3 locus and investigation of the gene in Hirschsprung disease. J. Med. Genet., September 1, 2000; 37(9): 674 - 679. [Abstract] [Full Text]

				X. Wu, W. Huang, M. E. Ganapathy, H. Wang, R. Kekuda, S. J. Conway, F. H. Leibach, and V. Ganapathy Structure, function, and regional distribution of the organic cation transporter OCT3 in the kidney Am J Physiol Renal Physiol, September 1, 2000; 279(3): F449 - F458. [Abstract] [Full Text] [PDF]

				S.-N. Huang and P. W. Swaan Involvement of a Receptor-Mediated Component in Cellular Translocation of Riboflavin J. Pharmacol. Exp. Ther., July 1, 2000; 294(1): 117 - 125. [Abstract] [Full Text]

				R. Zhao, F. Gao, Y. Wang, G. A. Diaz, B. D. Gelb, and I. D. Goldman Impact of the Reduced Folate Carrier on the Accumulation of Active Thiamin Metabolites in Murine Leukemia Cells J. Biol. Chem., January 5, 2001; 276(2): 1114 - 1118. [Abstract] [Full Text] [PDF]

				P.-K. Lo, J.-Y. Chen, P.-P. Tang, J. Lin, C.-H. Lin, L.-T. Su, C.-H. Wu, T.-L. Chen, Y. Yang, and F.-F. Wang Identification of a Mouse Thiamine Transporter Gene as a Direct Transcriptional Target for p53 J. Biol. Chem., September 28, 2001; 276(40): 37186 - 37193. [Abstract] [Full Text] [PDF]

R. Zhao, F. Gao, and I. D. Goldman Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1512 - C1517. [Abstract]