Transcriptional regulation of murine Slc22a1 (Oct1) by peroxisome proliferator agonist receptor-
and -
Wenxian Nie,1
Seth Sweetser,1
Mary Rinella,1 and
Richard M. Green1,2
1Department of Medicine, Division of Hepatology, Northwestern University Feinberg School of Medicine and the 2Chicago Veterans Affairs Medical Center-Lakeside Division, Chicago, Illinois
Submitted 4 February 2004
; accepted in final form 20 September 2004
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ABSTRACT
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The transport and metabolism of organic cationic endobiotics, nutrients, and drugs are essential hepatic functions. Slc22A1 is the basolateral liver transporter mediating the uptake of organic cations; however, little is known about the regulation of this transport protein. Peroxisome proliferator agonist receptor (PPAR)-
and -
agonists are commonly used agents that regulate many hepatocellular transport functions. Thus the purpose of this study was to examine the effects of PPAR agonists on the hepatic regulation and function of Slc22a1. Mice and H35 cells were administered PPAR-
and -
agonists, and the effect on Slc22a1 gene expression was measured. We subsequently cloned the Slc22a1 promoter and employed chimeric constructs to assay Slc22a1 gene transcription. The effects of PPAR-agonist treatment on organic cation uptake was also assayed. Slc22a1 expression was increased by PPAR-
and -
agonist treatment in both murine livers and H35 cells. Gene expression in H35 cells was further increased following transfection with expression vectors of PPAR transcription factors and PPAR agonist treatment. We cloned the promoter region of Slc22a1 and identified a PPAR-response element, and transfections with chimeric Slc22a1; promoter-reporter gene constructs demonstrate that the increased gene expression was transcriptionally regulated. Functional assays confirmed that cells treated with PPAR agonists displayed significant increases in organic cation uptake. PPAR-
and -
agonists transcriptionally increase Slc22a1 gene expression, and the increased Slc22a1 expression results in enhanced cellular organic cation uptake. These studies may have implication for the uptake of organic cationic drugs and for lipid metabolism.
gene expression
THE HEPATIC UPTAKE, biotransformation, and biliary excretion of xenobiotic and endobiotic substances are essential functions of the liver. Many commonly used pharmacological agents (anesthetic, antiarrhythmic, etc.) are organic cationic substances. Expression cloning techniques were previously used to identify Slc22a1 (Oct1) as the first mammalian organic cationic transporter, although several homologous members of this gene family have been subsequently identified (11). Slc22a1 is highly expressed in both human and rodent liver and is the major organic cationic transport protein responsible for hepatocellular organic cationic drug uptake (9, 11, 22). Therefore, Slc22a1-mediated uptake into the liver is an essential step in the biotransformation of many pharmacological substances and nutrients. In addition, Slc22a1 transports the organic cation choline, which is an essential step in the synthesis of phosphatidylcholine via the Kennedy pathway. However, little is known about the regulation of this important hepatic transporter (12, 20, 21).
Peroxisome proliferator agonist receptor (PPAR)-
and -
agonists are pharmacological agents that are commonly employed for the control of dyslipidemias and diabetes mellitus (7, 19). These agents have also been shown to regulate many hepatic transport and metabolic functions. Although several studies have employed Xenopus oocytes or other cell culture expression systems to identify the substrate specificity, kinetic properties, and pharmacological characteristics of Slc22a1, little is known about the regulation of this essential liver transporter (5, 11, 12). Thus, in this study, we have employed both in vivo and in vitro systems to determine the regulation of Slc22a1 expression by PPAR-
and -
.
These studies demonstrate that mice fed PPAR-
and -
agonists have significantly increased gene expression of hepatic Slc22a1 and that similar changes of gene expression also occur in liver-specific H35 cells treated with PPAR-
and -
agonists. We subsequently cloned and sequenced the mouse Slc22a1 promoter region and identified a PPAR regulatory element (PRE) and employed chimeric Slc22a1 promoter-lucerifase constructs to confirm that PPAR-
and -
agonists transcriptionally regulate this gene. Finally, we demonstrated that treatment with PPAR-
and PPAR-
agonists induces an increase in organic cation uptake. These data may have important implications for both hepatic drug metabolism and excretion and hepatobiliary lipid metabolism.
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MATERIALS AND METHODS
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Materials.
Restriction endonucleases and general molecular reagents were purchased from Boehringer-Mannheim (Indianapolis, IN). The Mouse BAC library was obtained from Invitrogen (San Diego, CA). Radionucleotides [32P]dCTP (3,000 Ci/mmol), [14C]tetraethylammonium bromide (3.36 mCi/mmol), and [32P]UTP (800 Ci/mmol) were from New England Nuclear (Boston, MA). Cell culture media and reagents were purchased from Life Technologies (Gaithersburg, MD). Ciprofibrate and Wy14,643 were generous gifts from MS Rao (Chicago, IL), and pioglitazone was from Takeda Pharmaceuticals/Eli Lilly. The expression vector for PPAR-
was a generous gift from J. K. Reddy (Chicago, IL). Male FVB/NJ mice were purchased from Jackson Laboratories (Bar Harbor, ME).
Animals.
Male FVB/NJ mice were fed either standard chow or chow containing Ciprofibrate (0.1%), Wy14,643 (0.1%), or pioglitazone (0.01%) for 10 days. All mice had free access to both chow and water and were housed with 12:12-h light-dark cycling from 6 PM to 6 AM. Mice were killed under deep anesthesia at 8 AM, and the livers were rapidly excised and either immediately used or snap-frozen in liquid nitrogen and kept at 70°C. All animals were treated humanely, and all protocols were approved by the animal care committee.
Cell culture.
Rat hepatoma-derived Reuber H35 cells were from American Type Culture Collection (Manassas, VA). H35 cells were maintained in MEM supplemented with 5% nonessential amino acids, 5%-glutamine, 10% fetal bovine serum, and 10% calf serum and were maintained at 37°C with 5% CO2.
Northern blot analysis.
Total RNA was purified using Ultraspec (Biotecx, TX), and Northern blot analysis was performed as previously described (4). After the verification of RNA quality by ethidium bromide staining, RNA was transferred from the gel to nylon paper and fixed by ultraviolet cross-linking. Filters were hybridized, washed with low- and high-stringency buffers and then subjected to autoradiography. All Northern blot analyses were quantified with densitometry and analyzed relative to gene expression of ubiquitin. Data were compared with densitometry data obtained from chow-fed animals or vehicle alone-treated cells.
Analysis of the Slc22a1 promoter.
A genomic BAC library was screened by hybridization using a cDNA fragment corresponding to the 5'-coding region of Slc22a1. After restriction digestion with Stu1, the fragments were subcloned into pBluescript KS+ (Stratagene, La Jolla, CA) and sequenced using automated sequencing. Ratio of Slc22a1 promoter to luciferase (Slc22a1/LUC) chimeric genes were prepared using an approximately 2.5-kb genomic DNA fragment inserted into the cloning site of the pGL3 basic vector (Promega, Madison, WI). 5'-Deleted constructs were prepared using restriction digested fragments of this chimeric construct. For transient transfections, plasmid DNA was amplified in Escherichia coli and purified using Qiagen columns (Chatsworth, CA). Primer extension was performed using a Primer Extension System employing the instructions of the manufacturer (Promega). 5'-Rapid amplification of cDNA ends (5'-RACE) of Slc22a1 was performed using the instructions of the manufacturer (Clontech, Franklin Lakes, NJ).
DNA transfections and dual luciferase assay.
H35 cells were seeded in six-well plates and grown to densities of 1x106 cells/cm2 for 24 h before transfection. DNA transfections were accomplished by lipofection employing Lipofectamine Plus (Invitrogen, Carlsbad, CA). All experiments were performed in triplicate using 0.98 g Slc22a1/LUC plasmid constructs per well together with 0.02 g pRL-TK (Promega). A control reporter plasmid consisted of a thymidine kinase promoter from herpes simplex virus upstream to a Renilla luciferase. Cotransfection with pRL-TK permitted normalization for transfection efficiency. After a 3-h period for transfection, liposomal suspensions were replaced with 3 ml of complete media and incubated for 24 h. Activities of Slc22a1/LUC constructs were determined using a dual luciferase assay system. Briefly, relative light units attributable to firefly luciferase activity reflected transcriptional activity of Slc22a1/LUC constructs and were measured first using a Optocomp 1 model luminometer (MGM Instruments, Hamden, CT). This was followed by the addition of Stop and Glow buffer, which served to quench luminescence from firefly luciferase and to provide the appropriate substrate for determination of Renilla luciferase activity. After background luminescence subtraction, the ratio of firefly luciferase to Renilla luciferase activity was used to represent the activity of the Slc22a1/LUC promoter constructs normalized for transfection efficiency.
Tetraethylammonium bromide uptake in H35 cells.
[14C]tetraethylammonium (TEA) bromide uptake by H35 cells was performed as previously described (23). Briefly, uptake was performed in buffered uptake solution (Hanks' balanced salt solution and 10 mM HEPES buffer with respective organic cation, pH 7.4) containing TEA (50 µM). Cells were grown to 7080% confluence in a 35-mm2 dish, washed with 3 ml of Tris-buffered saline (TBS; pH 7.4), and uptake was performed at either 37°C or 4°C over 15 min in 750 µl of uptake solution. Uptake was terminated with 3 ml of TEA stop solution [TBS-1 mM TEA (pH 7.4) at 4°C], and the cells were further washed twice with 3 ml of stop solution. H35 cells were solubilized with 0.1 N NaOH for 5 min, followed by neutralization with 0.1 N HCl and 1 M Tris (pH 7.4). Radioactivity was measured by liquid-scintillation counting, using 10 ml of Scintivert scintillation cocktail (Fisher Scientific, Fairlawn, NJ). Proteins were measured in duplicate by the method of Bradford (Bio-Rad, Hercules, CA), with bovine serum albumin used as a standard. All H35 cells were grown in standard media in the presence of Wy14,643 (100 µM), pioglitazone (500 nM), or ethanol vehicle.
Statistical analysis.
All comparisons between two groups were performed using Students t-test, with a P value <0.05 deemed to be statistically significant.
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RESULTS
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We initially fed male FVB/NJ mice either a normal chow diet or a chow diet supplemented with either the PPAR-
agonist Wy14,643 (0.1%) or PPAR-
agonist pioglitazone (0.01%) for 10 days. Figure 1 reveals Northern blot analysis demonstrating the hepatic gene expression of Slc22a1 (Oct1) from these mice. Gene expression of Slc22a1 is significantly increased in a response to dietary treatment with either the PPAR-
(Fig. 1A) or PPAR-
(Fig. 1B) agonists, with a 2.9 ± 0.4 and 3.1 ± 0.4 increase of hepatic Slc22a1 expression from the respective pharmacological agent (P < 0.05). The livers were grossly and microscopically normal by hematoxylin and eosin staining, although there was an increase in hepatic mass induced by treatment with not only ciprofibrate, but also WY14,463 (see Table 1).
We subsequently treated H35 cells with PPAR-
and -
agonists to determine whether similar PPAR-induced effects occurred in this liver-specific cell line. Clofibrate treatment of H35 cells induced an increase in Slc22a1 expression (P < 0.05; see Table 2). Treatment of H35 cells for 24 h with the PPAR-
agonist ciglitazone (200 ng/ml) also results in a comparable increase in Slc22a1 gene expression (Fig. 2B) (P < 0.05). Because PPARs form heterodimers with retinoid-X receptor (RXR) to transcriptionally activate genes, we subsequently treated H35 cells with the RXR agonist 9-cis-retinoic acid (9-CRA). 9-CRA (100 ng/ml) treatment alone did not alter gene expression of Slc22a1, nor did the retinoic acid receptor (RAR) agonist all-trans-retinoic acid (ATRA) (2.5 ng/ml).

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Fig. 2. Effects of PPAR, retinoid X receptor (RXR), and retinoid acid receptor (RAR) agonists on transcriptional activity of Slc22a1. Chimeric Slc22a1 promoter-luciferase constructs were transfected into H35 cells and treated with varying concentrations of clofibrate (02,000 ng/ml; A), ciglitazone (01,000 ng/ml; B), 9-cis-retinoic acid (9-CRA; 0100 ng/ml; C), or all-trans-retinoic acid (ATRA; 025 ng/ml; D). E: chimeric Slc22a1 promoter-luciferase constructs with deletions of the PPAR regulatory element (PRE) were unaffected by treatment with clofibrate (2,000 ng/ml) or ciglitazone (1,000 ng/ml). All data is expressed as firefly luciferase activity, normalized for transfection efficacy using Renilla luciferase activity. *P < 0.05; +P < 0.01.
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To further define the role of PPARs in regulating Slc22a1 gene expression, we cotransfected H35 cells with expression vectors for PPAR-
or -
and treated them with their respective pharmacological agonist. Transfecting H35 cells with vectors expressing either PPAR-
or -
without the PPAR agonists resulted in only an insignificant increase in Slc22a1 gene expression. Although 9-CRA treatment did not increase Slc22a1 expression in H35 cells, expression did increase when 9-CRA was administered following cotransfection with RXR expression vectors (see Table 2).
Because PPAR/RXR heterodimers transactivate genes by binding to PRE in the promoter region, we subsequently screened a mouse genomic BAC library, isolated, subcloned, and sequenced
2.5 kb of the 5'-upstream region of the Slc22a1 gene. We employed 5'-RACE and primer extension to identify the transcriptional start site of Slc22a1 68 bp upstream of the ATG translation site of the coding region. We were able to identify only a sole putative PRE in the 5'-upstream region, at 2392 bp from the ATG start codons.
To study the transcriptional regulation of Slc22a1 by PPAR-
or -
agonists, we developed Slc22a1 promoter-firefly luciferase chimeric constructs and transfected the constructs into the H35 cells. Figure 2A shows that with the use of the 2.55-kb construct containing the PRE sequence, clofibrate treatment for 24 h results in a concentration-dependent (02000 ng/ml) increase in firefly luciferase activity. This concentration-dependent increase in luciferase activity, indicative of increased transcriptional activation of the promoter, is maximally increased fourfold compared with untreated H35 cells. Similarly, treatment with ciglitazone also revealed a concentration-dependent increase in luciferase activity up to a concentration of 1,000 ng/ml, which is maximally increased eightfold (Fig. 2B). Higher concentrations of the PPAR agonists had no further effect. In contrast, treatment with the RXR agonist 9-CRA induces only an insignificant increase in luciferase activity (Fig. 2C). ATRA administration (025 µg/ml) had no effect on luciferase activity, indicating that it is unable to transcriptionally activate the Slc22a1 promoter (Fig. 2D). Deletion of the PRE at 2.35 kb resulted in loss of the response to PPAR agonist stimulation (Fig. 2E). Thus, consistent with the findings of the Northern blot analysis examining steady-state RNA levels, treatment with either PPAR-
or -
agonists resulted in increased transcriptional activation of the Slc22a1 promoter, whereas the RXR agonists resulted in insignificant increases in transcriptional activation of the Slc22a1 promoter, and the RAR agonist ATRA had no effect.
To determine whether these PPAR-induced increases in gene expression of Slc22a1 resulted in increased transport function, we assayed cellular uptake of the model organic cation TEA. Murine methodologies for isolating purified basolateral membrane (bLPM) vesicles have not been previously described (in contrast to rat bLPM vesicles), and we were unable to successfully modify rat liver methodologies to isolate highly purified mouse bLPM. Thus we were unable to assay organic cation vesicular uptake in the livers of these animals. We therefore employed PPAR responsive liver-specific H35 cells for organic cation uptake studies.
H35 cells were treated for 24 h with either PPAR-
or -
agonists, and organic cation uptake of TEA was performed (23). Initial studies determined that uptake of [14C]TEA (25 µM) was linear for 15 min (Fig. 3) and in this time frame at TEA concentrations up to 100 µM. Figure 4 demonstrates that [14C]TEA uptake was significantly increased in response to treatment with either the PPAR-
agonist WY14,643 or the PPAR-
agonist pioglitazone (P < 0.05). [14C]TEA uptake is increased 41% by WY14,463 (100 µM), being 7.16 ± 0.53 pmol·ug protein1·min1 in Wy14,463-treated cells vs. 5.07 ± 0.28 protein1·min1 in controls treated with vehicle alone (P < 0.05). Similarly, pioglitazone (500 nM) treatment results in a 46% increase in [14C]TEA uptake, being 7.40 ± 0.44 vs. 5.07 ± 0.42 protein1·min1 in pioglitazone or vehicle alone-treated cells, respectively (P < 0.05). No additional TEA uptake was noted when H35 cells were treated with the PPAR agonists for 48 h. Thus the alterations of Slc22a1 expression induced by either PPAR-
or -
agonists result in a concomitant increase in organic cation uptake.

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Fig. 3. [14C]tetraethylammonium (TEA) uptake by H35 cells. [14C]TEA uptake (25 µM) was measured in H35 cells for up to 60 min. Uptake at 37°C remained linear for 15 min (dark dashed line) but was not linear at 30 min (solid line). Uptake did not occur at 4°C (light dashed line).
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DISCUSSION
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The cellular uptake and metabolism of endobiotic and xenobiotic substances is an essential function of the liver (24). Many commonly used pharmacological agents are organic cation drugs that are taken up in the liver before biotransformation and excretion. Slc22a1 is the major organic cationic transporter in the human liver, and this gene is responsible for the hepatocellular uptake of numerous organic cationic drugs. In addition, Slc22a1 mediates the uptake of the organic cationic nutrient choline, which is an essential substrate for the hepatic synthesis phosphatidylcholine via the Kennedy pathway. Although numerous studies have examined the kinetic and pharmacological properties of both Slc22a1 and other members of the organic cationic transporter gene family, little is known about the regulation of this essential hepatic transport protein (21, 23, 24).
We initially treated mice with the PPAR-
agonists ciprofibrate and Wy14,643 and with the PPAR-
agonist pioglitazone via dietary supplementation, and these mice exhibited significant increases in gene expression of Slc22a1. PPAR-
agonists have previously been shown to regulate several other hepatic metabolic and transport genes (13, 14, 16, 18). Because treatment with PPAR-
agonists can induce hyperplasia and hypertrophy of hepatocytes, which may potentially diminish expression of hepatic Slc22a1 (data not shown), these data may underestimate the magnitude of the PPAR-
effect. Nonetheless, they clearly demonstrate an increase in expression of Slc22a1 in response to the PPAR agonist drugs.
We subsequently employed the liver-derived H35 cell line to determine whether similar changes in Slc22a1 gene expression occur in response to PPAR-
and -
agonists. H35 cells retain many liver-specific functions, and in fact, they demonstrate similar increases in Slc22a1 gene expression in response to treatment with either the PPAR-
agonist clofibrate or the PPAR-
agonist ciglitazone. 9-CRA is a potent agonist for RXR, and PPARs form heterodimers with RXR to transactivate PPAR-responsive genes (6, 17). However, an effect of 9-CRA occurred only when RXR expression was increased by cotransfection with RXR expression vectors; and PPAR agonists and 9-CRA were coadministered. Thus the lack of effect of RXR agonist treatment can potentially be explained by a relatively low level of endogenous RXR in our cell culture system. Because RXR can form heterodimers with other nuclear transcription factors, treatment with 9-CRA could also reflect a pharmacological response of heterodimer formation with other nuclear transcription factors besides PPARs that may have potentially negated an RXR stimulatory effect.
We then screened a mouse BAC library and sequenced 2.55 kb of the 5'-upstream region of Slc22a1. We identified the transcriptional start site at 68 kb and identified a sole PRE at 2392 bp. 5'-Deletional analysis indicates that the deletion of this PRE region results in the loss of the PPAR-induced stimulatory response, confirming its physiological import in Slc22a1 gene transcription.
We subsequently treated H35 cells with PPAR-
, PPAR-
, RXR, and RAR agonists and transfected the chimeric Slc22a1 promoter-luciferase construct vector to determine the transcriptional regulation of Slc22a1 by these pharmacological agents. Ciglitazone caused a concentration-dependent increase in luciferase activity that was maximally increased eightfold at a concentration of 1,000 ng/ml. In addition, clofibrate induced a concentration-dependent induction of luciferase activity, which was maximally increased fourfold compared with cells treated with vehicle alone. Treatment with the RXR agonist 9-CRA had only a minimal and statistically insignificant increase of luciferase activity, whereas the RAR agonist ATRA had no effect (2). These agonist concentrations are similar to the concentrations that we also found to increase steady-state Slc22a1 mRNA levels and are similar to or below the concentrations that have been reported to be biologically active in hepatic and other cellular systems.
Fatty acids are natural ligands for PPAR receptors, and we have previously demonstrated that Slc22a1 encodes for a high-affinity choline transporter (23). Choline serves as the essential substrate for phosphatidylcholine synthesis via the Kennedy pathway (8). During fasting or food-restricted states, free fatty acids are mobilized and may act as PPAR ligands. The PPAR-mediated effects on Slc22a1 may potentially allow for more efficient hepatic uptake of the phosphatidylcholine precursor choline at times when portal blood choline concentrations may be lower. This could potentially alter hepatic lipid metabolism or secretion into bile. Several previous studies (1, 3, 8, 10, 13, 15, 16, 18) have demonstrated that PPARs regulate other genes involved in hepatic lipid metabolism, and our data are consistent with previously reported data examining the effects of PPARs on other hepatic genes. Thus PPAR-mediated regulation of Slc22a1 and other transporters may have important physiological implications on lipid metabolism (in addition to drug uptake and metabolism) during the fasting state.
Unfortunately, membrane preparations for the isolation of highly purified mouse bLPM vesicles have not been described, and we were unable to successfully modify rat bLPM preparations for the isolation of mouse bLPM vesicles. Nonetheless, we were able to use liver-specific H35 cell cultures to demonstrate that both PPAR-
and -
agonists increase cellular uptake of the model organic cation TEA. Thus the increased gene expression of Slc22a1 results in a functional change in organic cation transport.
In summary, these data indicate that the administration of PPAR-
and -
agonists to mice causes an increase in gene expression of the hepatic organic cationic transporter Slc22a1. Similar induction of gene expression is noted in liver-specific H35 cells. Furthermore, we have cloned and sequence the promoter region of Slc22a1 and have identified a PPAR cis-regulatory element (PRE). Chimeric Slc22a1 promoter-luciferase transfection assays indicate that the PPAR agonists clofibrate and ciglitazone transcriptionally activate the Slc22a1 promoter, and this effect is concentration dependent. Finally, the administration of PPAR-
and -
agonists results in enhanced cellular uptake of the model organic cation TEA. These data may have important implications for the biotransformation and excretion of numerous endobiotics and endobiotics, including several commonly employed pharmacological agents. In addition, these data may be important for the regulation of hepatic choline uptake and the resultant synthesis of phosphatidylcholine. Finally, although the pharmacological and kinetic characteristics of Slc22a1 have been well characterized, these studies are among the first to characterize the regulation of this important cationic transporter.
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GRANTS
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This work was supported by National Institutes of Health Grants R01-DK-59580, R01-HD-4002703 and a Veteran's Administration Merit Review Award.
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ACKNOWLEDGMENTS
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Portions of the manuscript were presented in abstract form at meetings of the American Association for the Study of Liver Diseases in 1998 (Chicago, IL) and 2000 (Dallas, TX) and for Digestive Diseases Week (New Orleans, LA) in 2004.
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FOOTNOTES
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Address for reprint requests and other correspondence: R. M. Green, Northwestern Univ. Feinberg School of Medicine, Searle 10555, 303 E. Chicago Ave., Chicago, IL 60611 (E-mail address: r-green2{at}northwestern.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.
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