Department of Veterans Affairs Medical Center, Long Beach, California 90822; University of California School of Medicine, Irvine, California 92697; Children's Hospital-Harvard Medical School, Boston, Massachusetts 02115; INCELL Corporation, San Antonio, Texas 78249; and Westside Department of Veterans Affairs Medical Center- University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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Thiamine (vitamin B1) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na+ independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca2+/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes.
colonic thiamine transport
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INTRODUCTION |
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THIAMINE (vitamin
B1), a water-soluble micronutrient, is essential
for normal cellular functions, growth, and development. Thiamine in its
coenzyme form, thiamine pyrophosphate, plays a critical role in normal
carbohydrate metabolism, in which it participates in the
decarboxylation of pyruvic and -ketoglutamic acids and in the
utilization of pentose in the hexose monophosphate shunt (2). Thiamine deficiency in humans leads to a variety of
clinical abnormalities including cardiovascular disorders (e.g.,
peripheral vasodilatation, biventricular myocardial failure, edema, and
potentially acute fulminant cardiovascular collapse) and neurological
disorders (e.g., confusion, disordered ocular motility, neuropathy, and ataxia of gait) (2, 31, 34). Thiamine deficiency
represents a significant nutritional problem and occurs under variety
of conditions such as in alcoholics (in whom impairment in the
intestinal absorption process of the vitamin is believed to be a
contributing factor; Refs. 17, 30,
33, 34), in patients with thiamine-responsive megaloblastic anemia (in whom impairment in membrane thiamine transport
is believed to be the cause; Refs. 4, 7,
16, 22), and in patients with diabetes
mellitus (27) and celiac diseases (32) and
those on long-term therapy with diuretic medications (28).
Humans and other mammals cannot synthesize thiamine and thus must obtain the vitamin from exogenous sources via intestinal absorption. The intestine, therefore, plays a critical role in regulating body thiamine homeostasis. Thus understanding the mechanism and regulation of the intestinal thiamine absorption process is of significant nutritional importance. The intestine is exposed to thiamine from two sources, a dietary source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine (11, 12, 19). Absorption of dietary thiamine has been the subject of intense investigations over the past two decades (see Refs. 21 and 26 and references therein). With a variety of human and animal small intestinal preparations, these investigations demonstrated the involvement of a specialized carrier-mediated mechanism for thiamine uptake (21, 23, 26). As to the bacterial source of thiamine, previous studies showed that the normal microflora of the large intestine synthesize considerable amounts of thiamine and that up to 50% of this thiamine exists in the free, absorbable form (11, 12, 19). In addition, human and rat large intestine is capable of absorbing thiamine from its lumens (13, 14, 19). Nothing, however, is known about the uptake mechanism involved. Recent studies from our laboratory (5, 15, 24, 25) showed that human colonocytes are able to transport a number of other water-soluble vitamins that are also synthesized by the normal microflora of the large intestine. Specialized carrier-mediated systems were shown to be involved in the uptake of folate, biotin, pantothenic acid, and riboflavin by human colonocytes (5, 15, 24, 25). The aim of this study was, therefore, to determine whether human colonocytes also possess a carrier system for thiamine uptake and, if so, to delineate the characteristics of such a transport system. To do this, we used the human-derived cultured colonic epithelial cell line NCM460 (18) as a model system. The results demonstrated, for the first time, that human colonocytes do indeed possess a specialized, carrier-mediated mechanism for thiamine uptake. This provides further support for the notion that this source of thiamine may contribute to host nutrition and especially toward cellular nutrition of local colonocytes.
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MATERIALS AND METHODS |
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Custom-made 3H-labeled thiamine (sp. act. 555 GBq/mmol; radiochemical purity >98%) was purchased from American Radiolabeled Chemicals (St. Louis, MO). NCM460 cells and M3:10 growth medium were obtained from INCELL (San Antonio, TX). Trypsin, fetal bovine serum, and other cell culture reagents were obtained from Life Technologies (Grand Island, NY). All other chemicals and reagents used in this study were of analytical grade and were obtained from commercial sources.
NCM460 cells were grown and subcultured as described by us previously (15, 24, 25). Cells were used between passages 29 and 39. Uptake studies were performed on confluent monolayers 3-5 days after confluence. Uptake of thiamine was examined in cells incubated in Krebs-Ringer (K-R) buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4, unless otherwise specified) at 37°C. Labeled and unlabeled thiamine was added to the incubation medium at the onset of the uptake experiment. In certain experiments, cells were pretreated with the compound under study for a specific period of time before the addition of [3H]thiamine and the start of uptake experiments. Uptake was examined over a period of 3 min, i.e., initial rate (unless otherwise specified), and the reaction was terminated by the addition of 2 ml of ice-cold buffer followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer and digested with 1 ml of 1 N NaOH, neutralized with HCl, and then counted for radioactivity. Protein contents of cell digest were measured on parallel wells by using a Bio-Rad kit (Richmond, VA).
The metabolic form of the 3H radioactivity taken up by NCM460 monolayers after 3- and 10-min incubation in the presence of 150 nM [3H]thiamine was determined by cellulose-precoated TLC. In this study, cells were washed three times with K-R buffer after uptake, suspended in 75% ethanol, and homogenized. The supernatant was then applied onto the TLC plate and run using a solvent system of isopropanol-acetate buffer (0.5 M, pH 4.5)-water (65:15:20 vol/vol/vol).
Semiquantitative RT-PCR and Northern blot
analysis.
Three micrograms of poly(A)+ RNA isolated from NCM460 cells
were primed with gene-specific primer (to synthesize the first-strand cDNA) by using a SuperScript First-Strand Synthesis for RT-PCR kit
(Life Technologies) as described by the manufacturer. Two specific
primers spanning the entire open reading frame (ORF) of the SLC19A2
cDNA sequence that was cloned from human fibroblasts, skeletal muscle,
placenta, and brain (forward primer, 5'-CGCGCCCCGGATGGATGT-3'; reverse
primer, 5'-GCTGCTGTGAAGTCAAGAAAT-3'; Refs. 4,
6, 7, 16) were used to search
for expression of the SLC19A2 gene (the product of which is the THTR-1
protein) in NCM460 cells. PCR conditions were denaturation at 94°C
for 2.5 min, 9 cycles of 94°C denaturation for 30 s, 56°C
annealing for 30 s, and 68°C extension for 2 min, followed by 30 cycles of 94°C denaturation for 30 s, 56°C annealing for
30 s, 68°C extension for 2 min with an increment of an
additional 5 s for every cycle, and a final 7-min extension at
68°C. The PCR product was separated through 0.7% agarose gel with
images captured by using an Eagle Eye II system (Stratagene, La Jolla,
CA). The amplified RT-PCR product was normalized to the amplified
-actin RT-PCR products. The nucleotide sequence of the identified
PCR product was confirmed by sequencing using a commercial vendor
(Seqwright, Houston, TX).
Western blot analysis. The membranous fraction was isolated from NCM460 cells by homogenization of cells in buffer containing (in mM) 300 mannitol, 5 EGTA, 12 Tris · HCl, pH 7.1, and 1 phenylmethylsulfonyl fluoride and then centrifuged at 3,000 g for 15 min. The supernatant was centrifuged at 20,000 g for 30 min. The resulting pellet (150 µg protein) was treated with Laemmli sample buffer and resolved on a 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were electroblotted onto Immun-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) overnight. The blots were washed twice in PBS-Tween 20 for 10 min, blocked with 5% dried milk in PBS-Tween 20 for 1 h at room temperature, and washed with PBS-Tween 20. They were then probed with anti-human THTR-1 polyclonal antibodies [1:25,000 diluted in 1% dried milk-PBS-Tween 20 (Sigma)] for 1 h at room temperature, washed twice in 1% dried milk-PBS-Tween 20, and reacted with goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma) (1:5,000 diluted in 1% dried milk-PBS-Tween 20) for 1 h at room temperature. The blots were finally washed twice in PBS for 10 min, and color was developed by using an ECL kit (Amersham). Specific bands were quantitated by using Eagle Eye Software (Stratagene). A similar procedure was used in the case of native human colonic apical membrane preparations isolated as described by us previously (5).
The anti-human THTR-1 polyclonal antibodies were raised against a synthetic peptide in rabbits by a commercial vendor (Alpha Diagnostic Intl., San Antonio, TX). The design of the antigenic peptide was as follows. The sequence of the THTR-1 protein was searched for any similarities with other known proteins by using a Blast algorithm and a Swissport database. Because the human reduced folate carrier has been reported to have some degree of homology with THTR-1 (4, 6, 7, 16), the sequence of that protein was therefore aligned alongside the THTR-1 by using a Clustal W algorithm to detect the regions of lowest homology. The regions of THTR-1 protein showing the lowest homology were then analyzed for hydrophilicity (Kyte-Doolittle hydropathy plot), antigenicity (Hopp/Woods and protrusion index antigenicity profiles), and accessibility. A specific region of the THTR-1 protein that corresponds to amino acids 17-35 of the human sequence (TVLLRTARVRRECWFLPTA) was determined to be specific for this protein and was used for raising the polyclonal antibodies. We also confirmed the uniqueness of the selected peptide by using the Swissport database to avoid any cross-reactivity of the resulting antibodies with other proteins. The selected peptide was synthesized as keyhole limpet hemocyanin conjugate and was used for immunization in two rabbits.Data presentation and statistical analysis. Uptake results presented in this paper are means ± SE of multiple uptake determinations and are expressed as picomoles or femtomoles per milligram of protein per unit of time. Statistical differences were analyzed by Student's t-test or ANOVA, with statistical significance being set at P < 0.05. Quantitative variations in the absolute amounts of thiamine uptake were observed in certain experiments, and thus appropriate controls were run simultaneously with each set of experiments. Kinetic parameters of the saturable component of thiamine uptake [i.e., maximum velocity (Vmax) and apparent Michaelis-Menten constant (Km)] were calculated by using a computerized model of the Michaelis-Menten equation as described previously by Wilkinson (35).
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RESULTS |
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General characteristics of thiamine uptake by NCM460
cell: uptake as a function of time and effect of temperature,
pH, and
Na+.
Uptake of low (0.1 µM) and high (10 µM) concentrations of thiamine
by NCM460 cells was investigated as a function of incubation time at
37°C. At both thiamine concentrations, uptake was linear over the
10-min incubation period and occurred at a rate of 0.21 and 14.98 pmol · mg protein1 · min
1,
respectively (Fig. 1). We selected a
3-min period within the linear range as the standard incubation time in
all subsequent studies.
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Uptake as a function of substrate concentration: kinetics of
thiamine uptake process.
In this study, we examined the initial rate of thiamine uptake (3 min)
by NCM460 cells as a function of increasing the substrate concentration
in the incubation medium (0.03-10 µM). Thiamine uptake was found
to include a saturable component. Uptake by this component was
calculated by subtracting uptake by diffusion from total uptake of the
vitamin at each substrate concentration [uptake by diffusion was
calculated from the slope of the line between uptake at high
pharmacological concentration of thiamine (1 mM) and the point of
origin]. Kinetic parameters of the saturable component, i.e., the
apparent Km and Vmax,
were then determined as described in MATERIALS AND METHODS
and found to be 2.33 ± 0.38 µM and 3.76 ± 0.22 fmol/mg
protein for 3 min, respectively (Fig. 2).
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Effect of structural analogs and unrelated organic cations on [3H]thiamine uptake. The effect of the thiamine structural analogs oxythiamine and amprolium (both at 25 µM) on the initial rate of [3H]thiamine (30 nM) uptake by confluent NCM460 monolayers was tested. The results showed that both structural analogs cause a significant (P < 0.01) inhibition in [3H]thiamine uptake (114.5 ± 1.1, 86.3 ± 1.1, and 82.4 ± 1.9 fmol/mg protein for 3 min in controls and in the presence of oxythiamine and amprolium, respectively).
We also examined the effect of the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN) (both at 50 µM) on the uptake of cationic thiamine (30 nM) by NCM460 cells. Neither of these compounds was found to have a significant effect on thiamine uptake (135 ± 1.3, 133 ± 1.0, and 136 ± 1.5 fmol/mg protein for 3 min in controls and in the presence of TEA and NMN, respectively).Effect of metabolic and membrane transport inhibitors on thiamine uptake. In this study, we examined the effect of preincubating the cells for 30 min in buffer containing the metabolic inhibitors fluoride (10 mM), iodoacetate (1 mM), or dinitrophenol (DNP, 10 mM) on the initial rate of thiamine (30 nM) uptake. The results showed significant (P < 0.01 for all) inhibition in the vitamin uptake by all the tested metabolic inhibitors (116.5 ± 2.8, 65.5 ± 7.3, 86.8 ± 5.6, and 77.6 ± 8.3 fmol/mg protein for 3 min for controls and pretreatment with fluoride, iodoacetate, and DNP, respectively).
In another study, we examined the effect of the membrane transport inhibitors DIDS, probenecid, furosemide, and amiloride (all at 1 mM) on the initial rate of thiamine (30 nM) uptake. With the exception of amiloride, which caused significant (P < 0.01) inhibition in thiamine uptake, none of the other tested membrane transport inhibitors significantly affected vitamin uptake (119.5 ± 4.9, 121.3 ± 5.1, 112 ± 3.5, 112.5 ± 4.8, and 82.9 ± 2.3 fmol/mg protein for 3 min for controls and in the presence of furosemide, DIDS, probenecid, and amiloride, respectively).Expression of human thiamine transporter THTR-1 in
NCM460 cells and in native human colonic mucosa.
The SLC19A2 cDNA has been recently cloned from a number of human
tissues including skeletal muscle, brain, and placenta, and its
functionality in transporting thiamine has been established by
transfection assay (4, 6, 7, 16). In this study, we
investigated whether this human thiamine transporter is also expressed
in the human-derived NCM460 cells and in native human colonic mucosa at
both the mRNA and protein levels. Using a RT-PCR procedure described in
MATERIALS AND METHODS, we found clear expression of the
SLC19A2 gene in these cells (Fig.
3A). The identity of the
cloned cDNA was confirmed by sequencing and found to be identical to
that of the SLC19A2 cDNA. Similarly, Northern blot analysis showed
expression of the SLC19A2 mRNA in native human colonic mucosa (Fig.
3B). We also tested for expression of the THTR-1 protein in
NCM460 cells and in apical membrane preparations isolated by an
established procedure (5) from native human colonic mucosa using specific polyclonal antibodies raised in rabbits against a
specific peptide of the THTR-1 protein (see MATERIALS AND
METHODS). A clear band with a molecular mass of ~80 kDa was
observed in the lane containing the membranous fraction of NCM460 cells
and the native human colonic apical membranes (Fig.
4). The specificity of the band was
confirmed by its significant elimination after pretreatment of the
polyclonal antibodies with an excess amount of the antigenic peptide
(Fig. 4).
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Possible role of intracellular regulatory pathways in regulation of thiamine uptake by colonic NCM460 cells. In this study, we examined the possible involvement of Ca2+/calmodulin-, protein kinase C (PKC)- and protein tyrosine kinase (PTK)-mediated pathways in cellular regulation of the thiamine uptake process by NCM460 cells. The study was performed using specific modulators of these pathways. The role of the Ca2+/calmodulin-mediated pathway in the regulation of thiamine uptake by NCM460 cells was investigated by examining the effect of pretreating (for 1 h) NCM460 cells with the Ca2+/calmodulin inhibitor trifluoperazine (TFP; 50 µM) and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13; 100 µM) on the initial rate of uptake of [3H]thiamine (30 nM). The results showed significant (P < 0.01) inhibition in thiamine uptake by these compounds (95.2 ± 6.8, 59.2 ± 3.8, and 60.9 ± 3.9 fmol/mg protein for 3 min for controls and in the presence of TFP and W13, respectively). The inhibitory effect of TFP (50 µM) on thiamine uptake appeared to be mediated via a decrease in the Vmax of the thiamine uptake process [2.27 ± 0.13 and 0.79 ± 0.07 fmol/mg protein for 3 min (P < 0.01) for control and TFP-treated cells, respectively] and the apparent Km [3.61 ± 0.39 and 1.48 ± 0.21 µM (P < 0.01), respectively].
The possible role of a PKC-mediated pathway in the regulation of thiamine uptake by these colonic epithelial cells was also investigated by examining the effect of pretreating NCM460 cells (for 1 h) with modulators of this pathway on the vitamin uptake process. The results showed that neither activating [with the use of phorbol-12-myristate-13-acetate (PMA; 10 µM)] nor inhibiting (with the use of staurosporin and chelerythrine; both at 10 µM) this signaling pathway significantly affects thiamine uptake (103.8 ± 3.3, 113.5 ± 2.4, 103 ± 6.8, and 105.6 ± 4.2 fmol/mg protein for 3 min for controls and after pretreatment with PMA, staurosporin, and chelerythrine, respectively). Similarly, no role for a PTK-mediated pathway was found because pretreatment of NCM460 with modulators of this pathway failed to significantly affect thiamine (30 nM) uptake (97.2 ± 5.7, 89.5 ± 2.5, and 96.6 ± 1.6 fmol/mg protein for 3 min for controls and after pretreatment with 25 µM genistein and 10 µM tyrophostin A25, respectively). ![]() |
DISCUSSION |
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In this study we examined thiamine uptake by human colonocytes using human-derived, nontransformed cultured colonic epithelial NCM460 cells as an in vitro model system. These cells were chosen for the study because they have been demonstrated to possess characteristics similar to those of native colonocytes, including similar uptake mechanisms (5, 15, 18, 24, 25). The results showed thiamine uptake by these cells to be appreciable and to occur with no metabolic alterations in the transported substrate. The latter finding indicates that transport of thiamine into this type of cells is governed mainly by the transmembrane event and not by an intracellular metabolic event of the transported molecule. The latter mechanism has been recently suggested for uptake of another water-soluble vitamin (riboflavin) in isolated rat enterocytes (9).
Uptake of thiamine by NCM460 cells was found to involve a carrier-mediated system as indicated by the saturation in the substrate uptake as a function of concentration and by the inhibition in thiamine uptake by the thiamine structural analogs oxythiamine and amprolium. This system was found to be specific for cationic thiamine because other unrelated organic cations like TEA and NMN failed to affect the vitamin uptake process. Na+ in the incubation medium was found to have no role in thiamine uptake by these cells. This is based on the observation that replacing Na+ in the incubation medium with other monovalent cations failed to affect thiamine uptake. A role for H+ in thiamine uptake, however, was evident, and a thiamine/H+ exchange mechanism may be suggested. Evidence to support the latter suggestion includes the marked increase in thiamine uptake on increase of incubation buffer pH from 5.0 to 8.0 (i.e., on decrease of extracellular H+ concentration) and the significant increase in thiamine uptake on increase of the intracellular H+ concentration (i.e., after cell acidification). The finding that a Na+/H+ exchange inhibitor, amiloride, also inhibits thiamine uptake by NCM460 cells lends further support to this suggestion. The possible involvement of a thiamine/H+ exchange mechanism that is sensitive to inhibition by amiloride has also been suggested for the vitamin uptake in other cell types (3, 8, 10, 23).
Recent studies have reported the cloning of a cDNA of a thiamine transport protein (i.e., the so-called human thiamine transporter-1, THTR-1) from several human tissues including skeletal muscle, brain, and placenta (4, 6, 7, 16). Functional identity of the cloned SLC19A2 cDNA was confirmed by expression in HeLa cells, which showed significant induction in thiamine uptake in cDNA-transfected cells compared with controls (6). It is interesting to note here that the characteristics of the expressed THTR-1 carrier protein with regard to pH profile, apparent Km (2.5 ± 0.6 µM), and lack of effect of unrelated organic cations on thiamine uptake (6) are similar to those observed for the vitamin transport in NCM460 cells reported in this study. For this reason, we decided to investigate whether NCM460 cells also express the mRNA and protein of the SLC19A2 gene. Semiquantitative RT-PCR using specific primers designed from the sequence of the cloned human SLC19A2 cDNA and Western blotting using specific anti-THTR-1 polyclonal antibodies were used. The results showed that these cells do indeed express the mRNA and protein of the SLC19A2 gene. Similarly, native human colonic mucosa was found by Northern and Western blot analysis to express the mRNA and protein of the SLC19A2 gene, respectively. These findings raise the possibility that THTR-1 carrier protein may be involved in thiamine uptake in human colonocytes. Further studies, however, are required to confirm this suggestion.
Possible regulation of the thiamine uptake process of NCM460 cells by specific intracellular regulatory pathways was also investigated. Our findings indicate that modulation of a calmodulin (but not PKC or PTK)-mediated pathway led to a significant inhibition in thiamine uptake by NCM460 monolayers. The effect of the calmodulin-mediated pathway inhibitor TFP was mediated via a decrease in Vmax and the apparent Km of the thiamine uptake process. This suggests that the effect is the net result of alterations in activity (and/or number) and affinity of the thiamine uptake system. Further studies are required to determine the exact mechanism through which this intracellular pathway affects the thiamine uptake process in these cells.
The demonstration of existence of a specialized carrier-mediated mechanism for thiamine uptake in human-derived colonic epithelial NCM460 cells, together with the previous findings of specialized carrier systems for the uptake of other water-soluble vitamins (folate, biotin, pantothenic acid, and riboflavin; Refs. 5, 15, 24, 25) that are also synthesized by the normal microflora of the large intestine (11, 12), further highlight the possible importance of this source of vitamins in host nutrition in general and in cellular nutrition of the local colonocytes in particular.
In summary, the results of this study demonstrate for the first time the existence of a specialized carrier-mediated mechanism for thiamine uptake by a human colonic epithelial cell model. This system appears to involve a thiamine/H+ exchange mechanism and appears to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. Furthermore, NCM460 cells as well as native human colonic mucosa appear to express the mRNA and protein of the SLC19A2 gene.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Department of Veterans Affairs and the National Institutes of Health (DK-56061, DK-58057, DK-54016, and HL-04184).
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. M. Said, VA Medical Center-151, Long Beach, CA 90822 (E-mail: hmsaid{at}uci.edu).
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.
Received 23 January 2001; accepted in final form 14 March 2001.
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