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regulatory subunit of PI3-kinase and its role in survival of EREB2.5 cells
1 Department of Virology, Imperial College Faculty of Medicine, Norfolk Place, London W2 1PG, UK
2 Ludwig Institute for Cancer Research, University of Tokyo, Tokyo 113-8655, Japan
3 Department of Physiological Chemistry and Metabolism, University of Tokyo, Tokyo 113-8655, Japan
4 Institute of Pharmacology, Medical School Hannover, Carl Neuberg Strasse 1, D-30625 Hannover, Germany
5 Department of Biochemistry and Molecular Biology, University College London, UK
Correspondence
Paul J. Farrell
p.farrell{at}imperial.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunit of PI3-kinase) was induced were confirmed, but it is now shown that it is the p55 regulatory subunit that is induced. Several EBV-immortalized lymphoblastoid cell lines were shown to express p55. Expression of PI3-kinase p85 regulatory and p110 catalytic subunits was not regulated by EBNA-2. Proliferation of EREB2.5 lymphoblastoid cells was inhibited by RNAi knock-down of p55 protein expression, loss of p55 being accompanied by an increase in apoptosis. p55 is thus a functional target of EBNA2 in EREB2.5 cells and the specific regulation of p55 by EBV will provide an opportunity to investigate the physiological function of p55 in this human cell line.
Present address: Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Class I PI3Ks are composed of a catalytic subunit (p110) and a regulatory subunit (p85, p55 or p50). It is the regulatory subunit that binds to activated growth-factor receptors, bringing the catalytic subunit in proximity to its lipid substrates; the regulatory subunit is thus required for activation of the catalytic subunit in response to growth factor-receptor activation. The conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 by PI3Ks activates signalling via the protein kinase Akt/PKB to several cell-survival mechanisms.
Three different genes give rise to the three class I catalytic subunits p110, -
and -
. The p85, p55 and p50 regulatory subunits are all derived from the same gene (PIK3R1). Different transcription promoters express the separate first exons of p85, p55 and p50, which are then spliced to common additional exons to make the complete mRNAs. The C-terminal protein sequences of p85, p55 and p50 are therefore identical, but each has a unique N terminus (Fig. 1a). Separate genes express the other regulatory subunits, p85 and p55
. The expression and function of PI3Ks in lymphocyte development, differentiation and activation have recently been reviewed in detail by Okkenhaug & Vanhaesebroeck (2003).
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). Infection of primary resting human B lymphocytes by EBV induces cell proliferation very efficiently, and lymphoblastoid cell lines (LCLs) are readily grown out in culture. In LCLs, the virus exhibits a latent infection, expressing only 11 of the viral genes, including the EBNA-2 gene. By replacing the EBNA-2 gene of EBV with a conditionally active oestrogen receptor (ER)–EBNA-2 fusion gene, the EREB2.5 cell line (Kempkes et al., 1995) was created to investigate the function of EBNA-2 in the context of a normal EBV infection. Continued activity of EBNA-2 was shown to be essential for proliferation of LCLs (Kempkes et al., 1995). The EBNA-2 protein is a transcription factor that coordinates latent viral gene expression and induces several cellular genes that are important for proliferation, including c-MYC and RUNX-3 (Kaiser et al., 1999; Spender et al., 2002, 2005). Our previous microarray expression profiling that identified EBNA-2 target genes in EREB2.5 cells reported induction of the p85 mRNA, but no change in p85 protein was detected in response to EBNA-2 (Spender et al., 2002). Use of protein-synthesis inhibitors in those experiments demonstrated that no intermediate protein expression was required between EBNA-2 and induction of the PIK3R1 mRNA.
Early studies using wortmannin to inhibit PI3K activity indicated a role for these enzymes in the immortalization of primary human B cells by EBV (Sinclair & Farrell, 1995). The PI3K inhibitor LY294002 was subsequently shown to prevent growth of established LCLs, causing an accumulation of cells in the G1 phase of the cell cycle (Brennan et al., 2002). Signal-transduction effects of EBV proteins on PI3K activity are thought to be mediated mainly by EBV LMP-2A and LMP-1. The LMP-2A protein can mediate B-lymphocyte or epithelial-cell survival through activation of the PI3K pathway (Fukuda & Longnecker, 2004; Portis & Longnecker, 2004; Scholle et al., 2000; Swart et al., 2000) and LMP-1 can also activate PI3K to promote cell survival and induce actin-filament remodelling (Dawson et al., 2003). As the LMP1 and LMP-2A genes are induced directly by EBNA-2 in EBV LCLs, signal transduction from LMP-1 and LMP-2A to PI3Ks is controlled indirectly by EBNA-2. In contrast to that signal-transduction control, in this paper we focus on the direct regulation of expression of a specific PI3K regulatory subunit by EBNA-2.
Here, we show that it is in fact the p55 regulatory subunit of PI3K that is induced by EBNA-2 in EREB2.5 cells, not p85. Not all EBV-infected cell lines have p55 expression, but RNA interference (RNAi) of the p55 subunit in EREB2.5 cells reduces proliferation and is accompanied by apoptosis. The regulation of p55 expression by EBNA-2 thus provides a valuable and novel system to study the physiological regulation of expression of this PI3K regulatory subunit in human cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) contain a conditional EBNA-2 protein regulated by oestrogen and were maintained in RPMI 1640 medium without phenol red (Gibco-BRL) supplemented with 10 % heat-inactivated fetal calf serum, antibiotics (100 U penicillin ml–1 and 100 µg streptomycin ml–1) and 1 µM -oestradiol. For oestrogen-withdrawal experiments, cells were washed twice in serum-free medium before being resuspended at 5x105 ml–1 in phenol red-free RPMI 1640 medium without -oestradiol. Cells were then incubated for 5 days prior to EBNA-2 induction by addition of 1 µM -oestradiol. -Oestradiol-independent cell lines (EREB/E2) were also generated from EREB2.5 cells by Amaxa transfection with the p554 plasmid expressing wild-type EBNA-2 (Kempkes et al., 1995). These cells were selected for growth in the absence of -oestradiol.
Microarray analysis.
Microarray analysis was conducted on Agilent G4122A 44K HD arrays using total RNA from EREB2.5 cells. Cells were starved of oestrogen, then oestrogen was added back in the presence of protein-synthesis inhibitors and cells were harvested 4 h later as in our previous studies (Spender et al., 2002). RNA preparations from cell cultures with and without oestrogen were transcribed into Cy3- or Cy5-labelled cRNA, respectively, and cohybridized onto the same microarray. Samples derived from three independent experiments were analysed separately on three arrays, including one dye-swap experiment. Arrays were scanned on an Affymetrix 428 instrument and data were extracted by using Imagene 5.0. The values were filtered for genes that showed regulation by at least twofold in each individual experiment. The results were curated for flagged spots and for probes without GenBank accession number and were expressed as a ratio of values plus oestrogen divided by values minus oestrogen. The mean and SD are shown in Table 1. Several of the genes identified were represented on the array multiple times and these values have been included in the computation of the mean and SD.
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) was used; anti-EBNA-2 (PE-2; Dako) was used at 1/500. Antibodies specific for cleaved PARP (Asp 214) were from Cell Signaling Technology. Anti-PIK3R1 p85 (06-195 rabbit antiserum; Upstate) is a pan-p85 antibody recognizing p85, p55 and p50 and was used at a final concentration of 1 µg ml–1. Antisera to p110 (1/250), p110 (1/2000) and p110 (1/5000) (Ali et al., 2004), anti-p55 (1/1000) and anti-p55 (Inukai et al., 1996) and p85-specific antibody U2 (End et al., 1993) have been described previously. Anti-p85 (T15, ab252) was from Abcam and the mouse monoclonal anti--actin (AC-15; Sigma) was used at 1/10 000. The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) (Dako), peroxidase-conjugated rabbit anti-rat (Sigma) and HRP-conjugated sheep anti-mouse Ig (Amersham Biosciences). Bound immunocomplexes were detected by enhanced chemiluminescence (ECL; Amersham Biosciences).
In vitro translation.
In vitro translation was carried out by using a Mammalian Gene Collection (MGC) clone containing the cDNA for p55 (GenBank accession no. BC030815
[GenBank]
). In vitro translation was performed by using 1 µg plasmid linearized with XbaI in the TNT T7 coupled wheatgerm extract system (Promega).
[3H]Thymidine-incorporation assay.
Cells were seeded at a density of 5x104 per well in 96-well plates and were pulse-labelled for 2 h with 1 µCi (37 kBq) [3H]thymidine per well before being harvested onto glass-fibre filters with a cell harvester (Skatron Ltd). The amount of [3H]thymidine incorporated into DNA was measured by scintillation counting and the results were displayed as the mean and SD of at least three separate determinations.
Co-immunoprecipitation assay (IP).
PI3K regulatory and catalytic subunits were immunoprecipitated from cell lysates after pre-clearing with protein A–Sepharose (Amersham Biosciences). Cell lysate was mixed overnight at 4 °C with 1 µg antibody or 1 µg control Ig. Protein A–Sepharose was added for 3 h at 4 °C to bind the immunocomplexes. The Sepharose was washed three times in lysis buffer followed by two washes with PBS before being pelleted and resuspended in SDS sample buffer. The solution was heated to 95 °C for 5 min, the Sepharose was pelleted and the supernatant was analysed by SDS-PAGE and Western blotting.
RNase-protection assay (RPA).
In RPA experiments to identify direct targets of EBNA-2 transcription, protein synthesis was inhibited by pre-treating oestrogen-starved EREB2.5 cells for 2 h with 50 µg cycloheximide ml–1 and 100 µM anisomycin (Sigma) prior to the addition of oestrogen. Total cellular RNA was extracted by using RNAzol B (Biogenesis) and quantified by measuring A260. An RPA probe for detection of p55 RNA was generated by PCR from human genomic DNA using the primers 5'-TTTTCTGACTTGATTGGCTGGG-3' and 5'-CAGTATTACCTGGTGGGTCCATTTC-3' to generate a protected fragment of 168 bp. The PCR product was cloned into pCR2.1-TOPO, sequenced and subcloned into pBS II SK. A 32P-labelled antisense RNA RPA probe was then generated by using T7 RNA polymerase in in vitro transcription of 1 µg plasmid DNA linearized with NotI. RPAs were performed by using an RPA III RNase protection assay kit (Ambion). Cellular RNA was hybridized overnight at 42 °C with 50 000 c.p.m. probe. An equivalent amount of yeast RNA was included in a hybridization reaction as a negative control. Single-strand RNA was digested with an RNase A/T1 mixture for 30 min at 37 °C. Protected fragments were precipitated, fractionated on a polyacrylamide gel and the sizes were compared with 32P-labelled, MspI-digested pBR322 markers. The gel was analysed on a phosphorimager.
Generation of p55 short interfering RNA cells.
Oligonucleotides were cloned into pHEBo-SUPER (Spender et al., 2005) and plasmid DNA was Amaxa electroporated into EREB2.5 cells. Transfected cells were grown in 150 µg hygromycin ml–1 (400 µg ml–1 for the first 3 days) and viable cell numbers were determined 12 days later. Samples were also taken for Western blotting for p55 expression. The p55 oligonucleotides cloned for RNAi were 5'-GATCCCCGACCTGGATTTAGAATATGTTCAAGAGACATATTCTAAATCCAGGTCTTTTTA-3' and 5'-AGCTTAAAAAGACCTGGATTTAGAATATGTCTCTTGAACATATTCTAAATCCAGGTCGGG-3'.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These genes are direct targets of EBNA-2 regulation in the sense that no intermediate protein synthesis is required for the induction of their RNAs. In Table 1, the list is truncated arbitrarily at about twofold induction and genes with no annotated function have been omitted. Notable novel genes in the list include members of the Notch signalling pathway (HES1, HEY1 and DTX1), the secreted DNase 1L3, a protein phosphatase (PPEF1), various chemokines, an orphan receptor CMKOR1 and the Ets-1 transcription factor. The complete list of regulated genes is available from the authors and functional characterization of some of the additional target genes shown in Table 1 will be published separately. In addition to the induction of PIK3R1 that we reported previously, the BCAP (PIK3AP1) RNA is also induced by EBNA-2 (Table 1). BCAP is a tyrosine kinase substrate that connects the B-cell receptor to PI3K activation via the SH2 domains that are present in all of the PIK3R1 regulatory subunits (Okada et al., 2000). Bearing in mind our previous results on PIK3R1 (Spender et al., 2002), its prominent position in the complete-genome list suggested further investigation of this target.
EBNA-2 induces p55 without the need for intermediate protein synthesis
In our previous study of p85 regulation by EBNA-2 in EREB2.5 cells (Spender et al., 2002), the microarray probe used to detect p85 RNA induction was in the 3' end of the gene, but the epitope of the antibody used to detect p85 protein was in the N terminus of the protein, marked as AB6 in Fig. 1(a). Western blotting with a different pan-p85 antibody that recognizes epitopes present in p85, p55 and p50 revealed that it is p55 that is induced by EBNA-2, not p85 (Fig. 1b). The timing of expression of p55 in response to reactivation of EBNA-2 was similar to the timing of expression of LMP-1, which is known to be a direct target of EBNA-2 regulation (Spender et al., 2002). The protein induced in response to EBNA-2 activation was confirmed to be p55, as it was detected (Fig. 1c) by an antibody specific for the unique N terminus of p55. The protein also co-migrated on SDS-PAGE with in vitro-translated p55 protein expressed from an authentic p55 cDNA (Fig. 1d). The induction of p55 was a consequence of EBNA-2 expression and not an artefact of adding oestrogen to the cells, as EREB2.5 cells in which the ER–EBNA-2 fusion gene was replaced with wild-type EBNA-2 expressed p55 constitutively in the absence of oestrogen (Fig. 1e). An RPA specific for the unique 5' exon of p55 (Fig. 2a) showed that induction of the mRNA could be detected as early as 2 h after addition of oestrogen to reactivate EBNA-2 (Fig. 2b). The p55 RNA induction was not prevented by protein-synthesis inhibitors (Fig. 2b), confirming that the p55 promoter is regulated by ER–EBNA-2 without the need for any intermediate protein expression. The RPA probe spanned the transcription start site and the length of the protected fragment (164–168 nt) was consistent with initiation of transcription as in the RefSeq cDNA of p55 (GenBank accession no. BC030815
[GenBank]
).
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is complexed with catalytic subunits in EREB2.5 cells). Regulation of EBNA-2 activity in the EREB2.5 cells by oestrogen had no substantial effect on the protein-expression levels of any of the catalytic subunits (Fig. 3a). For the regulatory subunits, modulation of EBNA-2 activity with oestrogen did not affect the level of p85 or p55 substantially (Fig. 3b). p85 was generally unaffected by modulation of EBNA-2 activity although, in some experiments, there was a slight reduction upon induction of p55. No antibody monospecific for p50 is available (the unique first exon only encodes 6 aa), so this could not be tested directly. Several other LCLs were found to express p55 (Fig. 3c), but not all LCLs have it. We have not yet identified the additional factors that may determine whether p55 is induced by EBNA-2 in LCLs.
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expressed in EREB2.5 cells was complexed with catalytic subunits, p55 was immunoprecipitated from EREB2.5 cell extract and the precipitate was immunoblotted for p110 and p110. Both types of catalytic subunit were bound to the p55 induced in the EREB2.5 cells treated with oestrogen (Fig. 4a). The reciprocal immunoprecipitation experiment, with an antibody to p110 followed by Western blotting to detect all variants of the PIK3R1 regulatory subunit, showed that both p55 and p85 bound to p110 (Fig. 4b). There were similar proportions of p55 and p85 in the complex; in fact, there was slightly more p55.
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is required to prevent apoptosis in EREB2.5 cells is not expressed in all LCLs, depletion of p55 in EREB2.5 cells by an RNAi plasmid (Fig. 5a) expressing a sequence from the unique first exon of p55 greatly reduced the proliferation of the cells over a 12 day period (Fig. 5b), indicating that p55 is required for optimal growth or survival of these cells. Two different assays for apoptosis, production of cleaved PARP (Fig. 5c) and accumulation of sub-G1 DNA content in flow cytometry of propidium iodide-stained cell nuclei (Fig. 5d), were used. Both showed a substantial increase in the amount of apoptotic cells in response to p55 RNAi, indicating that p55 plays a significant role in the survival of these cells. By 12 days of p55 RNAi, 35 % of the cells had a sub-G1 DNA content (gate M1, Fig. 5d). Inactivation of EBNA-2 by withdrawal of oestrogen initially caused an arrest of the cell cycle in both G1 and G2 (Kempkes et al., 1995), but then progressive accumulation of cells with a sub-G1 DNA content over this longer time course (Fig. 5d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) used a conditionally functional EBNA-2 allele similar to that used in this study, but did not distinguish direct targets of EBNA2 from genes that might be regulated downstream of the direct EBNA-2 target genes. In our array analysis of EBNA-2 target genes (Table 1), we have used a strategy involving protein-synthesis inhibitors to identify functionally direct targets. It remains to be determined whether these genes are all regulated by EBNA-2 acting as a transcription factor at their promoters, but our strategy provides a set of genes that is likely to include the direct targets of EBNA-2 transcriptional regulation. Some of the newly identified target genes have clear connections with known functions of EBNA-2, for example, in the Notch signalling pathway (HES1 and DTX1).
In this paper, we focus on the PIK3R1 regulation and show that the p55 regulatory subunit of PI3K is induced by EBNA-2 in EREB2.5 lymphoblastoid cells at the RNA level, resulting in expression of the p55 protein. The overlap of the 3' end of the p55 mRNA with p85 mRNA accounts for the previous data that had been interpreted as an induction of p85 RNA (Spender et al., 2002). The p55 forms complexes with p110 catalytic subunits of PI3K, including p110 and p110. Although there are mechanisms by which oestrogen can activate PI3K signalling from membrane ER (Castoria et al., 2001; Tsai et al., 2001) that could potentially complicate the use of an ER–EBNA-2 fusion protein, our experiments show that the induction of p55 in this cell system was clearly mediated by EBNA-2.
Specific depletion of p55 by RNAi in EREB2.5 cells caused apoptosis, indicating that p55 plays a role in maintaining cell survival in this LCL. Several other LCLs also expressed p55, but some did not, for example C2+Obaji and BM+Akata (Fig. 3c). There was no simple correlation between p55 expression and A or B type EBNA-2 or cord/adult-derived B cells (these details of the cell lines are given in the legend to Fig. 3), so further investigation will be required to understand why only some LCLs have p55. The variation in size of EBNA-2 and LMP1 in the cell lines is a consequence of variation in copy numbers of repeat sequences in these proteins and is a normal feature of the different virus strains represented.
The specific induction of p55 by EBNA-2 in EREB2.5 cells, with p85 present constitutively, makes it particularly interesting to consider what effects on the cell may be mediated uniquely by p55. The unique N terminus of p85 contains the bcr region, which binds to cdc42H, a small G protein of the Rho family. Signalling from cdc42H has major effects on the cytoskeleton and cell polarity (Raftopoulou & Hall, 2004; Wilkinson et al., 2005), so this is presumably avoided in signal transduction via p55. The SH3 domain in the N terminus of p85 is also able to bind to dynamin (Gout et al., 1993). In contrast, the unique N terminus of p55 has been reported to bind to -tubulin (Inukai et al., 2000), perhaps allowing localization of the activation of downstream signalling to a particular site in the cell. In previous work, overexpression of the individual isoforms and analysis of association with various tyrosine kinase receptors showed some differences and revealed that p55 can be phosphorylated on tyrosine (Inukai et al., 2001). Several studies have examined the role of p50 and p55 in insulin signalling (Inukai et al., 1997; Terauchi et al., 1999), but these point more to a specific function for p50 than p55. Knockout of all three PIK3R1 regulatory subunits showed a clear role in B-cell survival, an increased proportion of pro-B cells in the bone marrow and a reduced B-cell proliferative response to lipopolysaccharide, but a normal response to interleukin-4 and CD40-L. These effects were, however, not ascribed to any individual regulatory subunit (Fruman et al., 1999a, b). Expression of individual regulatory subunits by transfection into differentiated myotube cells and measurement of PI3K activity in the context of insulin signalling indicated that p85, p55 and p50 all inhibited PI3K activity (Ueki et al., 2000), but the level of expression of p85 was found to be the important determinant of the resulting signal transduction, partial reduction of p85 levels favouring cell survival but complete depletion causing cell death (Ueki et al., 2002). An increase in the level of p55 RNA and protein has also been reported in response to injury of motor nerves in mice, suggesting a role for PI3K containing p55 in the process of nerve regeneration (Okamoto et al., 2004). Our observation that p55 is induced specifically in human B-cell lines by normal levels of EBNA-2 will thus provide an important opportunity to investigate the physiological significance of p55 in human B cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 April 2006;
accepted 19 June 2006.
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 W. Lucchesi, G. Brady, O. Dittrich-Breiholz, M. Kracht, R. Russ, and P. J. Farrell Differential Gene Regulation by Epstein-Barr Virus Type 1 and Type 2 EBNA2 J. Virol., August 1, 2008; 82(15): 7456 - 7466. [Abstract] [Full Text] [PDF]
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H. J. Martin, J. M. Lee, D. Walls, and S. D. Hayward Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus J. Virol., September 15, 2007; 81(18): 9748 - 9758. [Abstract] [Full Text] [PDF]
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