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Published as doi: 10.1096/fj.07-8128com.
 (The FASEB Journal. 2007;21:2352-2358.)
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Improved maturation of CFTR by an ER export signal

Markus W. Wendeler1, Oliver Nufer1,2 and Hans-Peter Hauri3

Biozentrum, University of Basel, Basel, Switzerland

3Correspondence: Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. E-mail: hans-peter.hauri{at}unibas.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel in the plasma membrane of several epithelial cells. Maturation of CFTR is inefficient in most cells, with only a fraction of nascent chains being properly folded and transported to the cell surface. The most common mutation in CFTR, CFTR-{Delta}F508, leads to the genetic disease cystic fibrosis. CFTR-{Delta}F508 has a temperature-sensitive folding defect and is almost quantitatively degraded in the endoplasmic reticulum (ER). Here we tested whether a strong ER export signal appended to CFTR improves its transport and surface expression. We show that a single valine ER export signal at the C terminus of the cytoplasmic tail of CFTR improves maturation of wild-type CFTR by 2-fold. This conservative mutation interfered with neither plasma membrane localization nor stability of mature CFTR. In contrast, the valine signal was unable to rescue CFTR-{Delta}F508 from ER-associated degradation. Our finding of improved maturation of CFTR mediated by a valine signal may be of potential use in gene therapy of cystic fibrosis. Moreover, failure of the valine signal to rescue CFTR-{Delta}F508 from ER degradation indicates that the inability of CFTR-{Delta}F508 to leave the ER is unlikely to be due to a malfunctioning ER export signal.—Wendeler, M. W., Nufer, O., Hauri, H-P. Improved maturation of CFTR by an ER export signal.


Key Words: cystic fibrosis • membrane traffic • transport signal


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized to the apical plasma membrane of numerous epithelia including those found in lung, pancreas, intestine, and kidney (1 2 3) . A deletion of phenylalanine 508 of CFTR, resulting in CFTR-{Delta}F508, leads to a severe form of cystic fibrosis (4 , 5) . Although >1300 different mutations have been identified in the CFTR gene, ~90% of cystic fibrosis patients carry the {Delta}F508 mutation (6) . The F508 deletion prevents proper folding of CFTR in the ER, and its trafficking to the plasma membrane is inhibited (7 8 9 10) . Both wild-type CFTR and CFTR-{Delta}F508 can be detected in ER and ERGIC, but only wild-type CFTR is readily expressed at the cell surface (1 , 11 12 13) . Newly synthesized CFTR-{Delta}F508 is trapped in the ER, recognized by the ER quality control machinery, and eliminated by ER-associated degradation (ERAD) (14 15 16) . The quality control machinery that selects CFTR-{Delta}F508 for degradation and the mechanism for its misfolding are still incompletely understood although several key interactions have come to light, including multiple molecular chaperons on both sides of the ER (17 18 19 20 21 22 23) .

CFTR is a polytopic membrane protein with 12 transmembrane domains, and both its N and C terminus face the cytosol. Transport-competent CFTR is exported from the ER by recruitment into COPII-coated vesicles in a process that is facilitated by a diacidic ER export motif present in the first nucleotide binding domain facing the cytosol (24 , 25) . CFTR transport between ER and late Golgi/endosomal compartments involves conventional as well as a novel pathway dependent on the cell type studied (24) . After reaching the cell surface, CFTR proteins undergo repeated rounds of vesicular traffic in the endocytic-exocytic cycle and eventually reach the lysosomes, where they are degraded (12 , 26) .

Maturation of CFTR is rather inefficient in most cell types tested (8 , 9) . Typically, only 20% to 30% of wild-type CFTR nascent chains become properly folded and are subsequently transported to the plasma membrane, although folding efficiency can vary in different cell types (27) . To develop therapeutic approaches for the treatment of cystic fibrosis it is important to understand the mechanisms that control ER export of CFTR and CFTR-{Delta}F508. Of particular interest is the tug-of-war between chaperone-mediated ER retention and signal-mediated ER export. We wondered whether appending a strong ER export signal, such as a C-terminal valine (28) to the cytoplasmic tail of CFTR, improves maturation of wild-type CFTR and rescues CFTR-{Delta}F508 from ERAD. Here we report studies with wild-type and CFTR-{Delta}F508 proteins in which the most C-terminal leucine was substituted by a valine. The valine signal improved maturation of wild-type CFTR by 2-fold. Valine-modified mature CFTR was equally stable as unmodified mature CFTR. In contrast, the valine signal did not promote maturation of CFTR-{Delta}F508 and failed to prevent its degradation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Mouse mAb M3A7 against human CFTR was kindly provided by J. R. Riordan and also purchased from Upstate Biotechnology (Lake Placid, NY, USA).

Recombinant DNA
Oligonucleotides were purchased from Microsynth (Balgach, Switzerland). Plasmids (pcDNA3.1) containing cDNAs encoding wild-type or mutant ({Delta}F508) CFTR were kindly provided by J. R. Riordan. Substitution of the most C-terminal leucine at position 1480 by valine was carried out by QuikChange mutagenesis (Stratagene, San Diego, CA, USA) using complementary oligonucleotides for changing codon CTT to GTT. All constructs were confirmed by sequencing using standard methods and an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, Rotkreuz, Switzerland).

Cell culture and transfection
HEK293 cells were cultured in DME medium (4.5 g/L glucose) supplemented with 10% FCS and 100 i.u./ml penicillin, 100 µg/ml streptomycin and 1 µg/ml fungizone. HEK293 cells stably expressing CFTR constructs were prepared by electroporation with PvuI-linearized constructs. Briefly, 6 Mio cells were resuspended at 1.2 Mio cells/ml in serum-free medium supplemented with 20 mM HEPES (pH 7.5) and electroporated with 20 µg DNA in 0.4 cm cuvettes using one pulse of 0.25 kV (960 µF capacitance). Cells were then seeded onto gelatin-coated 10 cm dishes and selection was started 48 h after transfection with 0.8 mg/ml G418 (Gibco BRL, Paisley, UK). All cultures were grown at 37°C with 5% CO2 in humidified air.

Metabolic labeling and immunoprecipitation
Stably transfected HEK293 cells were seeded onto gelatin-coated 6-well plates and grown to 80% confluence. For metabolic labeling, the cells were washed twice with D-PBS (PBS containing 0.9 mM CaCl2, 0.45 mM MgCl2), starved in labeling medium (MEM without methionine, supplemented with 10% dialyzed FCS), and pulsed for 20 min with 100 µCi/ml [35S]methionine/cysteine mixture (EasyTagTMEXPRE35S35S Protein Labeling, PerkinElmer Life Sciences, Norwalk, CT, USA). Cells were immediately processed or chased for the indicated times with complete DME medium containing 10 mM L-methionine. For immunoprecipitation, the cells were washed twice with ice-cold PBS and resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 20 mM Na2MoO4, 0.09% Nonidet P-40; pH 7.4) supplemented with 0.2 mM PMSF, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml aprotinin, 1 µg/ml antipain, 17.5 µg/ml benzamidine, and 20 mM iodoacetamide. Cells were lysed by passing them five times through a 25-gauge needle. After 1 h on ice, the lysate was cleared by centrifugation at 100,000 g for 1 h. Cleared lysates were added to protein G-Sepharose beads (Sigma, Geneva, Switzerland) to which antibodies had been prebound. After incubation for at least 1 h on a rotary shaker in the cold, the beads were washed four times with lysis buffer and once with 50 mM Tris (pH 7.5). Immunoprecipitates were incubated in 1 x Laemmli buffer at 60°C for 30 min and separated by 7% SDS-PAGE. Radioactivity was visualized by fluorography and quantified by PhosophorImaging using ImageQuantTM software (Molecular Dynamics, Sunnyvale, CA, USA).

Cell surface labeling of CFTR
Nontransfected or stably transfected HEK293 cells were seeded onto gelatin-coated 10 cm dishes and grown to 90% confluence. The cells were then placed on ice for 30 min and all subsequent steps were performed in the cold. After three washes with PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 (pH 7.5), cells were labeled using 3 ml of 1 mg/ml EZ-Link sulfo-NHS-SS-biotin (Pierce, Rockford, IL, USA) in PBS-Mg/Ca for 30 min. Free reagent was then quenched by two 10 min incubations with serum-free DME medium. After two washes with PBS-Mg/Ca, the cells were scraped and collected by centrifugation at 1200 g for 5 min. The cell pellets were lysed in Nonidet P-40 buffer as above. One-fourth of the cleared lysate was used for immunoprecipitation and the biotinylated proteins in the remaining sample were collected by incubation with 50 µl of ImmunopureSteptavidin beads (Pierce) for 2 h on a rotary shaker in the cold.

ImmunopureSteptavidin beads were then pelleted and washed four times with RIPA buffer I (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS; pH 7.5), once with RIPA buffer II (50 mM Tris, 150 mM NaCl, 0.1% Nonidet P-40; pH 7.5), and once with 50 mM Tris (pH 7.5). Immunoprecipitates and Streptavidin-precipitated biotinylated proteins were incubated in 1 x Laemmli buffer at 60°C for 30 min and separated by 6% SDS-PAGE. For Western blotting, proteins were transferred to nitrocellulose B85 (45 µm, Schleicher & Schuell, Dassel, Germany) at 15 V for 1 h in a semidry transfer cell (Bio-Rad, Hercules, CA, USA) using transfer buffer containing 48 mM Tris, 35 mM glycine, 0.0375% SDS, and 20% (v/v) methanol (pH ~9.2). The nitrocellulose was rinsed with 50% methanol and stained by Amido Black (Serva, Heidelberg, Germany). All subsequent incubations were in PBS containing 5% nonfat dry milk and 0.05% Tween-20 (Serva), and included blocking for 1 h, incubation with the first antibody (M3A7) for 60 min, rinsing three times 10 min, incubation with peroxidase-coupled secondary antibody for 60 min, and rinsing 10 min. After three final 10 min washes with PBS containing 0.05% Tween-20, the nitrocellulose was processed using enhanced chemiluminescence (ChemiGlow ECL reagent, AlphaInotech Corp., San Leandro, CA, USA) and exposed to BioMax MR-1 films or directly analyzed in a ChemImagerTM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Export of many secretory proteins from the endoplasmic reticulum (ER) relies on signal-mediated packaging into ER-derived transport vesicles (29) . The best-studied signaling motifs are diacidic (30) or hydrophobic (28 , 31) . A particularly striking case is a single valine at the C terminus of type I membrane proteins that operates as a potent ER export signal (28) . This signal is recognized by the COPII coat, which drives vesicle budding from the ER. The valine signal also functions in polytopic membrane proteins, including N-methyl-D-aspartate (NMDA) receptors and sialic acid transporter (32 , 33) .

To test whether a strong ER export signal can increase the maturation of CFTR, we substituted the most C-terminal leucine at position 1480 of CFTR by a valine resulting in the construct designated CFTR-V (Fig. 1 A). This construct was stably expressed in HEK293 cells under the cytomegalo virus promoter. Unlike what was reported earlier (9) , transient expression of CFTR proteins in HEK293 cells resulted in the accumulation of aggresomes, and CFTR did not mature to its complex glycosylated form (not shown). In stably transfected cells, however, the expression levels were more favorable and CFTR maturation was comparable to that obtained in most cell lines. To study the maturation of the CFTR constructs with different tail ends in stable transfectants, the transport from ER to Golgi was monitored in pulse-chase experiments using 35S-methionine and conversion of the high-mannose form (also known as B form) to the mature complex glycosylated form (known as C form) was visualized by SDS-PAGE/autoradiography (Fig. 1B ). In the case of CFTR, the B form rapidly disappeared and only a small fraction of it was converted to the C form, which started to appear after ~60 min chase. These results are in line with the transport kinetics and degradation features reported for wild-type CFTR (8 , 9) . In pulse-chase experiments with cells expressing CFTR-V, a greater fraction of the B form was converted to the C form than with wild-type CFTR, indicating improved maturation (Fig. 1B ). Quantification of the pulse-chase experiments showed a 2-fold improved maturation of CFTR-V compared to the maturation of unmodified CFTR (compare Fig. 1C with Fig. 1D ).


Figure 1
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  		Figure 1. C-terminal valine improves maturation of CFTR. A) Partial amino acid sequence of the CFTR C-terminal tail in single letter code. The wild-type tail and the modified tail with L1480V mutation (underlined) are shown. B) HEK293 cells stably expressing wild-type or L1480V mutated CFTR constructs were pulse-labeled with [35S]methionine for 20 min, chased for the indicated times, and subjected to immunoprecipitation with anti-CFTR antibodies. Immunoprecipitates were then separated by SDS-PAGE and visualized by fluorography. The lower band (~140 kDa) represents the core glycosylated (band B) and the upper band (~160 kDa) the complex glycosylated (band C) form of CFTR. C, D) Quantification of fluorograms as shown in panel B. Values of CFTR with C) wild-type tail (HEK clone 6) and D) L1480V modified tail (HEK293 clone 1). Squares indicate core glycosylated form and triangles the complex glycosylated form. Mean values ± SE of at least three independent experiments are shown.

To further characterize CFTR-V, we tested whether its mature, complex glycosylated C form reaches the plasma membrane. This was probed by the accessibility of CFTR-V at steady state to the membrane-impermeable, amino-reactive biotinylation reagent sulfo-NHS-SS-biotin added to intact cells. After cell lysis, an aliquot was used for immunoprecipitation of CFTR proteins; the biotinylated surface proteins in the remaining lysate were affinity-isolated by means of Streptavidin beads and CFTR was visualized by Western blotting after SDS-PAGE. Figure 2 shows that the mature C form of CFTR was accessible to the biotinylation reagent irrespective of the presence or absence of the valine signal. This indicates that the valine signal at the C terminus did not negatively interfere with targeting of the mature protein to the plasma membrane. Cells expressing CFTR-{Delta}F508 were included as a control for the biotinylation assay. In these cells no C form was detectable and no biotinylation signal was observed (Fig. 2 , clone c2), consistent with earlier observations that CFTR-{Delta}F508 fails to acquire complex glycans and does not reach the cell surface (34) .


Figure 2
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  		Figure 2. Plasma membrane localization of CFTR is not affected by a C-terminal valine. Mock-transfected HEK293 cells (mock) or individual clones (c) stably expressing the indicated CFTR constructs were labeled with a membrane-impermeable biotinylation reagent. One-fourth of the lysates were then subjected to immunoprecipitation with anti-CFTR antibodies (total, T); the remaining lysates were incubated with Streptavidine beads to capture biotinylated proteins (surface, S). Precipitates were separated by SDS-6% PAGE and proteins were transferred to nitrocellulose for immunodetection using anti-CFTR antibodies. The lower band represents the core glycosylated (band B) and the upper band the complex glycosylated form (band C) of CFTR. A representative example of three independent experiments is shown.

Next we assessed whether the valine-modified C terminus interferes with the cycling of CFTR between plasma membrane and endosomes. A change of trafficking can be expected to result in altered protein turnover since CFTR is known to cycle between plasma membrane and to be degraded ultimately in lysosomes. To address this, we studied the half-life of mature CFTR and CFTR-V by a pulse-chase approach. Cells were pulsed with 35S-methionine for 1 h and chased for up to 50 h in the presence of excess cold methionine. The CFTR proteins were immunoprecipitated, analyzed by SDS-PAGE/autoradiography (Fig. 3 A), and quantified (Fig. 3B ). The data clearly show that the turnover of CFTR and CFTR-V is indistinguishable, indicating that the valine signal does not affect the stability of mature CFTR. We conclude that post-Golgi trafficking most likely is not affected by a valine signal.


Figure 3
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  		Figure 3. Turnover of CFTR is not changed by a C-terminal valine. A) HEK293 cells stably expressing wild-type or L1480V mutated CFTR constructs were pulse-labeled with [35S]methionine, chased for the indicated times, and subjected to immunoprecipitation with anti-CFTR antibodies. Immunoprecipitates were then separated by SDS-PAGE and visualized by fluorography. Note that only the complex glycosylated mature form (band C) of CFTR constructs is detected. B) Quantification of fluorograms as shown in panel A. Black data points indicate the complex glycosylated form of wild-type CFTR and white data points the complex glycosylated form of the CFTR L1480V mutation. Each data point represents the mean value ± SE of at least three independent experiments.

Exit of CFTR from the ER is blocked by mutation of a consensus diacidic ER export motif present in the first nucleotide binding domain, and it was proposed that the inability of CFTR-{Delta}F508 to leave the ER may be due to a defect of decoding this signal (25) . If this proposal is correct, a potent ER export signal engineered into a cytosolic domain of CFTR that is not affected by the {Delta}F508 mutation should override its retention in the ER, which would express itself by the appearance of mature, complex glycosylated protein. To directly test this, we mutated the most C-terminal leucine of CFTR-{Delta}F508 to valine resulting in CFTR-{Delta}F508-V. The protein was stably expressed in HEK293 cells and its maturation was assessed by pulse-chase. Figure 4 A clearly shows that the valine signal was unable to rescue CFTR-{Delta}F508 from degradation in the ER. No mature C band appeared, and quantification of the B band showed identical degradation kinetics for CFTR-{Delta}F508 and CFTR-{Delta}F508-V (Fig. 4B ).


Figure 4
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  		Figure 4. Maturation of CFTR-{Delta}F508 is not improved by a C-terminal valine. A) HEK293 cells stably expressing CFTR-{Delta}F508 or CFTR-{Delta}F508-V were pulse-labeled with [35S]methionine, chased for the indicated times, and subjected to immunoprecipitation with anti-CFTR antibodies. Immunoprecipitates were then separated by SDS-PAGE and visualized by fluorography. Note that only the immature core glycosylated form (band B) of the CFTR mutants is detected. B) Quantification of fluorograms as shown in panel A. White circles represent the core glycosylated form of CFTR-{Delta}F508 and black circles represent the core glycosylated form of CFTR-{Delta}F508-V. Each data point represents the mean value ± SE of at least three independent experiments.

Collectively, the results demonstrate that a strong ER export signal such as a C-terminal valine improves the maturation of wild-type CFTR. By contrast, a valine signal cannot override the retention of misfolded by the quality control machinery of the ER.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present work demonstrates that a strong ER export signal appended to the C-terminal tail of wild-type CFTR can improve its maturation. This effect is not due to clonal variation. Although we have mainly presented the data of two individual cell clones (i.e., clone 6 expressing wild-type CFTR and clone 1 expressing CFTR-V), other clones pairs were also compared, including wild-type CFTR clone 1 and CFTR-V clone 5 (see Fig. 2 ), and the results were similar.

The L1480V substitution not only needs to be discussed in the context of ER export but also because L1480 is part of a PDZ binding motif. The C-terminal end of CFTR carries the PDZ binding tetrapeptide motif DTRL that binds to PDZ domain-containing proteins (35 , 36) . This interaction may serve several functions, including the positioning of CFTR within multimolecular regulatory complexes (36 37 38 39) , which contributes to CFTR activity and its control by protein kinase A (23 , 40) and enables CFTR to influence, or respond to, other proteins involved in epithelial salt transport such as the epithelial sodium channel (41) . PDZ domain proteins also regulate the endocytic recycling of CFTR (42) . Moreover, mutations near the C terminus precluding PDZ domain binding result in mild disease (2) . The substitution of leucine 1480 by a valine reported here, however, is a conservative mutation not expected to interfere with PDZ binding, since it meets the PDZ type I consensus sequence S/T-X-V/I/L/F/Y (43) . Accordingly, protein labeling of intact adherent cells by a membrane-impermeable biotinylation reagent showed that the L1480V substitution did not interfere with surface localization of mature CFTR (Fig. 2) . In addition, the turnover of wild-type CFTR and CFTR-V was indistinguishable, suggesting that the cycling in the endocytic system was unchanged.

How does the C-terminal valine improve the maturation of CFTR? Recent evidence suggests that the export of wild-type CFTR from the ER requires the COPII machinery, as it is sensitive to Sar1 mutants that disrupt coat assembly and disassembly (25) . Consistent with this finding, mutations of a conserved diacidic ER export motif in the first nucleotide binding domain of CFTR impairs the maturation of the B to the C form. The diacidic motif, originally detected in the cytoplasmic domain of VSV-G protein (30) , recognizes the Sec24 subunit of COPII (44 , 45) and thereby promotes export of CFTR from the ER (25) . This signal-mediated ER export is inefficient, as the majority (up to 80%) of CFTR is degraded in heterologous expression systems. Obviously only a fraction of total CFTR is sorted into ER export vesicles. The valine signal might positively influence this sorting process by directing more molecules into the export-competent pool. Such a pool likely is associated with ER exit sites since the C-terminal valine can interact with COPII components (28) . An alternative CFTR model for maturation proposes that CFTR harbors positive sorting signals that are masked in immature molecules. Only molecules that acquire correct conformation display the sorting determinants. This would be similar to the need for correct folding and oligomerization of VSV-G or ERGIC-53 (30 , 46) . The endogenous ER export signals of these proteins are decoded by the COPII machinery only when presented in oligomeric form. In contrast, the valine ER export signal does not depend on oligomerization (28) . Moreover, the valine signal located at the most extreme C terminus is most likely freely accessible for sorting by COPII. This interaction may remove immature CFTR from the degradation pathway and thus prolong the time for correct folding.

The principal consequence of the F508 deletion in CFTR was proposed to be due to the inability of this molecule to reach the correct conformation for presenting the acidic ER export signal to the COPII coat subunit Sec24 (25) . Our results argue against this hypothesis since the valine signal was unable to rescue CFTR-{Delta}F508 from ERAD. It appears unlikely that the F508 negatively affects the decoding of the valine signal by COPII. A more likely scenario is that the valine/COPII interaction occurs normally, but the F508 deletion leads to permanent misfolding, which favors the interaction of CFTR-{Delta}F508 with ER-associated chaperones. This is in line with a recent study showing that siRNA-mediated down-regulation of the Hsp90 cochaperone Aha1 rescues the ER export of CFTR-{Delta}F508 in HEK293 cells (47) . A negative sorting mechanism for CFTR-{Delta}F508 was proposed based on the finding that removal of multiple arginine frames in CFTR results in transport and maturation of CFTR-{Delta}F508 (34) . It was suggested that only unfolded molecules expose these frames. Similarly, RXR motifs have also been proposed to operate in ER retention/retrieval of unassembled plasma membrane K(ATP) potassium channels, {gamma}-amino butyric acid B (GABAB), and NMDA receptors (for a review, see ref. 48 ). However, our results showed that the L1480V substitution on CFTR-{Delta}F508 was not able to overcome the retention of misfolded molecules. This is in line with the established model that only correctly folded proteins are able to leave the ER.

Although gene therapies for cystic fibrosis have been evaluated, effective treatment for cystic fibrosis airway disease has been limited by inefficient gene delivery and by a lack of persistent gene expression (49 50 51) . There has been significant progress of gene therapy in cystic fibrosis (52 53 54 55) , but there clearly is a requirement for newer approaches to improve efficiency and duration of expression. Delivery of CFTR with improved maturation mediated by a valine signal may contribute to further progress.


   ACKNOWLEDGMENTS
 
We thank Käthy Bucher for excellent technical assistance and John R. Riordan for providing wild-type and {Delta}F508 CFTR constructs as well as antibodies against CFTR.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Present address: Molecular Biology, AstraZeneca R&D Charnwood, Leics LE11 5RH, UK.

Received for publication January 18, 2007. Accepted for publication March 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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