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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

It is known that the hepatitis B virus X protein (HBx) plays a crucial role in the pathogenesis of HCC, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In the present study, HepG2 cell lines were cultured and transfected with pEGFP-N1 and pEGFP-N1-X. Twenty-four hours after transfection, cells were harvested and total RNA was extracted using TRIzol® reagent. The expression of HBx in HepG2 cell line was assayed by real-time polymerase chain reaction and was detected by Western blotting. Moreover, proteomic analysis was performed for the HepG2-pEGFP-X cells and HepG2-pEGFP control cells. The combination of 2DE and MALDI-TOF-MS/MS revealed that SEC13L1 (SEC13-like 1 isoform b), PA28 alpha (proteasome activator REG alpha), serine–threonine kinase receptor-associated protein (STRAP) and nm23/nucleoside diphosphate kinase (NME) were upregulated in HepG2-pEGFP-X cells. STRAP is known to be a WD40 domain-containing protein, which interacts with TβR-I and TβR-II and negatively regulates TGF-β signalling, was also found increased in human cancers. NME is known to be involved in the regulation of cancer cell progression and metastasis. These results would help the understanding of how HBx maintains tumorigenicity and progression of HCC.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Hepatocellular carcinoma (HCC) is one of the most common malignancies in sub-Saharan Africa, and Southeast Asia and China. In China, chronic infection with hepatitis B virus (HBV) is often complicated by cirrhosis and HCC [1, 2]. The pathogenesis of HBV-induced HCC is poorly understood, although HBx, a 17-kDa regulatory protein encoded by HBV is believed to contribute to the development of HCC [3, 4]. Several studies on HBx transgenic mice have investigated the hepatocarcinogenic effects of HBx. Initial reports showed that mice harbouring HBx develop progressive features that are characteristic of malignant transformation of liver cells [5, 6]. Histopathological changes include the formation of foci of altered liver cells, benign adenomas and eventually the development of HCC [5, 6]. HBx is a multifunctional regulatory protein with activities affecting transcriptional regulation, cell cycle control, DNA repair, signal transduction pathways, apoptotic cell death and cellular adhesion. However, the exact molecular mechanisms of HBx in HCC are not well understood.

Hepatitis B virus X protein does not directly bind DNA but functions via protein–protein interaction [7]. To search and identify proteins directly or indirectly interacting with HBX, and elucidate the mechanisms of interaction, we have constructed combine plasmid pEGFP-HBx and transfected into HepG2. To better understand the mechanism of the HBx gene in HCC cell lines, we performed proteomic study of intracellular proteins of HepG2-pEGFP-X and HepG2-pEGFP cells to explore the altered expression of proteins that could contribute to the development of HBx-related HCC.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Construction of the eukaryotic expression vector pEGFP-N1-X carrying HBx gene

To investigate the different expressed proteins between cells which were transfected by pEGFP-N1 and cells transfected by pEGFP-N1-X, the pEGFP-N1-X carrying HBx gene was constructed. For the construction of pEGFP-N1-X, the circular HBV DNA (adr subtype) was used as a PCR template for amplifying the HBx gene. The primer combination used to amplify the region encoding HBx protein was 5′-CCCAAGCTTATGGCTGCTCGGGTGTG-3′ (sense primer) and 5′-GGGGTACCCCGGCAGAGGTGAAAAAG-3′ (antisense primer). The sense-primer included the translation initiation codon for the HBx protein, 5′-HBx sequences, and the HindIII restriction site, and the antisense primer included a complementary sequence to the 3′-end of HBx as well as KpnI restriction site. The PCR product amplified from HBx region was digested with HindIII and KpnI, and subcloned into the HindIII–KpnI sites of the multiple cloning site of the vector pEGFP-N1 (Clontech, Palo Alto, CA, USA).

Cell culture and HBx gene transfection

HepG2 cells and the transfected cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco-BRL) at 37 °C in 5% CO2/air. To transfect pEGFP-N1 (HepG2-pEGFP) and the pEGFP-N1-X (HepG2-pEGFP-X) plasmid-containing HBx into HepG2 cells, transfection was performed using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Following incubation for 24 h, the HepG2-EGFP and HepG2-EGFP-X cells were harvested for further analysis.

RNA isolation and RT-PCR

Total RNA was extracted from HepG2-pEGFP, HepG2-EGFP-X cells, respectively, using TRIzol reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. First-strand DNA synthesis was primed with oligo (dT) and completed using TaKaRa RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa, Dalian, China) followed by polymerase chain reaction (PCR). The amplification programme was as follows: 95 °C, 5 min (95 °C, 30 s; 58 °C, 30 s; 72 °C, 45 s; 30 cycles) and 72 °C, 7 min. PCR products were detected by 1% agarose gel with ethidium bromide and analysed by ChemiImagerTM 5500 (Alpha Innotech, San Leandro, CA, USA). HBx sense primer was: 5′-atggctgctcgggtgtgctgccaac-3′; antisense primer: 5′-ttaggcagaggtgaaaaagttgcatg-3′.

Fluorescence Microscopy and Laser Confocal Microscopy

At 24 h after transfection, the cells were washed twice with PBS and analysed using an Olympus IX70 fluorescence microscope, with an NB filter (Olympus, Tokoyo, Japan), to visualize the expression of EGFP and HBx-EGFP. Images were additionally processed using Adobe Photoshop 7.0.

A 12-well plate with glass slides (14 mm × 14 mm) was seeded with suspensions of cell line HepG2. After overnight, cells were transfected with pEGFP-N1 or pEGFP-N1-X as previously study, and at 24 h after transfection, viewed with a Zeiss LSM 510 confocal microscope equipped with LSM 510 software version 2.02 and Ar/Kr (458 and 488 nm) and He/Ne (543 nm) lasers. The lens used was a Plan–Neofluar oil lens.

Western blot analysis

Cells were lysed with a buffer containing 50 mm Tris–HCl (pH 8.0), 150 mm NaCl, 0.02% NaN3, 100 μg/ml PMSF, 1 μg/ml aprotinin and 1% Triton X-100. The total protein concentration was measured using the Bio-Rad (Biorad, Hercules, CA, USA) protein assay kit. Twenty-microgram total protein was subjected to 12% SDS-PAGE and blotted onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat dry milk in PBS and incubated (gentle agitation overnight at 4 °C) with anti-HBx monoclonal antibody (1:1000) (Alexis, Lausen, Switzerland), anti-GFP monoclonal antibodies (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-β-actin polyclonal antibodies (1:1000) (Cell Signaling, Danvers, MA, USA). The blots were then incubated with HRP-conjugated anti-mouse antibody (1:4000) at room temperature for 2 h. The labelled bands were detected by chemiluminescence using ECL Western blotting detection reagents and exposure to X-ray film.

Sample preparation

The transient transfected cell lines, HepG2-pEGFP and HepG2-pEGFP-X, were harvested by treatment with trypsin, and rinsed two times in cold PBS. They were then solubilized in lysis buffer (7 m urea, 2 m thiourea, 4% CHAPS, 40 mm Tris, 40 mm DTT, 10 mm PMSF, 1 ul nuclease mix). The cell mixtures, 1.25 × 106 cells per 100 μl, were vortexed three to four times within 45 min at 4 °C and then stored at −20 °C for 2DE. Total protein concentration of the mice sera were detected by BCA assay (Shenergy Biocolor, Shanghai, China).

IPG-DALT two-dimensional electrophoresis and image acquisition and analysis

The samples were solubilized in 200 μl of rehydration solution [9.8 m urea (Amersham, Uppsala, Sweden), 4% CHAPS (Amersham), 1% IPG buffer (Amersham), 40 mm DTT (Amersham), and 10 mm PMSF (Sigma-Aldrich)] and then loaded into DryStripTM (13 cm, pH 3–10 NL, Amersham). The IPG strips with the gel-side down were soaked in the solution and rehydrated overnight. Isoelectric focusing was performed on MultiphorTM II (Amersham) at 18 °C. After IEF finished, each IPG strip was incubated for 15 min with equilibration solution 1 [6 m urea (Sinopharm, Beijing, China), 50 mm Tris (Sinopharm), 2% SDS (Sinopharm), 30% glycerol (Sinopharm), 100 mg DTT and 0.002% bromophenol blue (Sigma-Aldrich) and equilibration solution 2 [same as equilibration solution 1 plus 250 mg of iodoacetamide (Amersham) and with the exception of DTT], respectively, then IPG strips were directly loaded onto 15% polyacrylamide (Sinopharm) gels and sealed with 0.5% agarose. Two-dimensional SDS-PAGE gels were run for about 9 h at 18 °C. The protein spots were visualized in gel by ammoniacal silver nitrate staining (analytical) and digitized by an imaging system ChemiImagerTM 5500 (Alpha Innotech). The protein spots were analysed using ImageMasterTM (Amersham) when electrophoresis ended.

Stained 2D gels were captured by an imaging system ChemiImagerTM 5500 (Alpha Innotech). Target gels were analysed with ImageMasterTM software (Amersham) including spot detection, background subtraction, matching, etc.

Quantitative real-time RT-PCR

Primers for SEC13L1 (forward primer: 5′-TCCTGGCATCGTGCTCCTA-3′, reverse primer: 5′-TGCTCGTGGCTCTTCTCC-3′), PA28 alpha (forward primer: 5′-CTGATGACCAGCCTCCAC-3′, reverse primer: 5′-CATTCCCTTTGTTTCTCCC-3′), STRAP (forward primer: 5′-GACCCGTGGTTGATTTG-3′, reverse primer: 5′-TGTATCTCCCTGGCGTAG-3′), NME (forward primer: 5′-GACCGTCCATTCTTTGCCG-3′, reverse primer: 5′-GCCCGTCTTCACCACATTCA-3′), and human β-actin (forward primer: 5′-TGTGTTGGCGTACAGGTCTTTG-3′, reverse primer: 5′-GGGAAATCGTGCGTGACATTAAG-3′) were synthesized by Invitrogen corporation. The RT reaction was performed on 500 ng of total RNA with Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed using Realtime PCR Master Mix (SYBR Green) (TOYOBO, Osaka, Japan). PCR reactions were performed in a total volume of 20 μl (2× Realtime PCR Master Mix) in Rotor Gene Real-Time PCR detection system (Corbett, Australia). The PCR programme was as follows: 1 cycle for 5 min at 95 °C; 40 cycles for 15 s at 95 °C, 15 s at 62 °C, 15 s at 72 °C. The specificity and identity of the PCR product was checked by performing a melting curve test. After quantified with real-time PCR, quantification was performed using the comparative CT method. The target transcript was normalized to an endogenous reference (simultaneous β-actin reactions). All samples were analysed in triplicate.

Statistical analysis

All data represent mean values ±SD from experiments performed at least three times. Student's t-test was used for statistical analysis and the results were considered significant when the value of P ≤ 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

HBx gene expression was confirmed by RT-PCR

To determine whether HBx has the ability to affect the expression of the other correlative intracellular proteins, first, we confirmed whether HBx gene was expressed in HBx-transfected cells. As shown in Fig. 1, HBx mRNA expression of HepG2-pEGFP-X cells was determined by RT-PCR.

image

Figure 1.  Hepatitis B virus X protein mRNA expression levels of transfected cells. HepG2 cells were transfected with cDNA encoding EGFP-X and EGFP in the mammalian expression vector pEGFP-N1. At 24 h after transfection, HBx mRNA expression levels of HepG2, HepG2-pEGFP-X and HepG2-pEGFP cells were analysed by RT-PCR. Total RNA was isolated from HepG2, HepG2-pEGFP-X and HepG2-pEGFP cells (A). To detect HBx mRNA expression, reverse transcription products of these cells were analysed by PCR (B).

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Fluorescence microscopy and laser confocal microphotography

In addition, EGFP fluorescence was observed by a fluorescence microscopy and laser confocal microscopy. When EGFP and EGFP-X were expressed, fluorescence was detected in the transfected cells at 24 h. In cells that expressed EGFP, the fluorescence was distributed throughout the entire cell (Fig. 2). In cells expressed EGFP-X, the fluorescence was observed in both the nucleus and cytosol. Therefore, we conclude that EGFP and EGFP-X were successfully transfected into HepG2 cells.

image

Figure 2.  Establishment of HepG2 cell lines with constitutive HBx expression and the location of HBx protein in the transfected cells. At 24 h after transfection, HepG2-pEGFP-X and HepG2-pEGFP cells were observed with phase contrast microscope, and EGFP fluorescence was observed with a fluorescence microscope (A) and laser confocal microscope (B): a and b, HepG2 cells transfected with pEGFP; c and d, HepG2 cells transfected with pEGFP-X (A). a, b, c and d, HepG2 cells transfected with pEGFP-X; e, f, g and h, HepG2 cells transfected with pEGFP (B).

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Western blot analysis: the expression of HBx in HepG2-pEGFP-X cells

Furthermore, in HepG2-pEGFP-X cells, the expression of HBx-EGFP fusion protein was identified by Western blot analysis with an anti-HBx and anti-GFP antibody. Anti-HBx primary antibody detected 47-kDa fusion protein in the lysates of HepG2-pEGFP-X cells. Anti-GFP antibody also resulted in the detection of a 47-kDa band in HepG2-pEGFP-X cells and a 30-kDa band in HepG2-pEGFP cells (Fig. 3). These results clearly show that the HBx protein is synthesized in transfected cells (HepG2-pEGFP-X).

image

Figure 3.  Analysis of HBx protein expression in HepG2 cells transfected with HBx. At 24 h after transfection, HBx protein expression of HepG2, HepG2-pEGFP-X and HepG2-pEGFP cells were analysed by Western blot. To detect the expression of HBx-EGFP fusion protein, 20 μg samples of total cell lysates were size fractionated by SDS-PAGE and transferred to polyvinylidene fluoride membrane and then analysed by Western blot analysis using specific antibody for HBX and GFP. HBx-EGFP fusion protein expression was detected using HBX MoAb (A). HBx-EGFP fusion protein expression was detected using GFP MoAb (B). β-actin was included as an internal control (C).

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Detection of the differentially expressed proteins of HepG2-pEGFP-X cells on 2DE gels

Each sample of the HepG2-pEGFP or HepG2-pEGFP-X proteins had been repeatedly run at least three times on 2DE gels. More than 500 spots of proteins were visualized on the gels by silver staining. The differences in spot intensities between HBx transient transfected HCC cells (Fig. 4B) and control cells (Fig. 4A) were compared for each gel. Representative gels of each sample were chosen for analysis by ImageMasterTM (Amersham) to determine the differentially expressed spots.

image

Figure 4.  Representative silver-stained 2DE gels of intracellular proteins of HepG2-pEGFP control (A) and HepG2-pEGFP-X (B). Proteins (200 μg) were loaded and separated first on IPG strips with a range of pH 3–10, and then on 14 × 18-cm2 glass plates. The positions of identified proteins are marked by circles. The spot numbers corresponded to those in Table 1.

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The identification of differentially expressed proteins by MADI-TOF-MS/MS

The identified spots were picked out of the gels stained by sliver, digested and subjected to MALDI-TOF-MS analysis. MALDI-TOF-MS/MS analysis provided a good PMF spectrum for each spot. Four spots were identified as SEC13L1, PA28a, STRAP, NME protein (Table 1). The subsection views of these proteins are shown in Fig. 5. The results show that all the four proteins are increased in HepG2-pEGFP-X cells.

Table 1.   Identification of the differentially expressed proteins in HepG2-pEGFP-X.
No.ProteinMr pIAccession no. of NCBI or Swiss-ProtFold change in the HBx cellsScore
  1. The fold change was analysed by ImageMasterTM (Amersham), by comparing the intensity of the spot of HepG2-pEGFP-X with the corresponding spot of the HepG2-pEGFP control.

1SEC13L1 (SEC13-like 1 isoform b)35,5185.22gi:34335134 Q53GB23.7945
2PA28 alpha (proteasome activator REG alpha)28,7055.78gi:5453990 Q063234.8659
3STRAP(serine-threonine kinase receptor-associated protein)38,4284.99gi:20149592 Q9Y3F43.50118
4NME (nm23/nucleoside diphosphate kinase)20,3987.07gi:35068 P155313.52144
image

Figure 5.  Subsection views of the differentially expressed proteins on the two-dimensional gels. The selected spots are circled by ellipses. Number under subsection indicates comparative protein quantity calculated by integration of density to area using ImageMaster. A and B represents proteins from HepG2-pEGFP and HepG2-pEGFP-X respectively. Subsections are from the same gels shown in Fig. 4.

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The increased expression of proteins were confirmed by real-time RT-PCR

To confirm that the expression levels of these four proteins were altered in HepG2-pEGFP-X cells, real-time RT-PCR was carried out using β-actin as an internal control. The results indicated that the expressions of SEC13L1, PA28 alpha, STRAP and NME were upregulated in HepG2-pEGFP-X cells at the transcriptional level (Fig. 6).

image

Figure 6.  Differential expression of SEC13L1, PA28alpha, STRAP and NME mRNA between HepG2-pEGFP-X and HepG2-pEGFP. The expression of SEC13L1, PA28alpha, STRAP and NME mRNA was determined by real-time PCR. The copy number given was corrected for the expression levels of β-actin and is the average of three experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Chronic HBV infection is closely associated with the incidence of HCC [2]. Among the four proteins that originate from the HBV genome, HBx has been reported to be associated with hepatocellular carcinogenesis. HBx induces liver cancer in transgenic mice [5]. Although it does not bind directly to DNA, HBx affects transcriptional activation via its interaction with nuclear transcription factors and the cytoplasmic modulation of signal transfection pathways. It has also been reported that HBx interacts with transcription factors in the nucleus [8]. Furthermore, an interaction between HBx and p53 inhibits p53 function [9], and several studies have demonstrated an inhibitory effect of HBx on DNA repair and apoptosis [10, 11]. Therefore, HBx is thought to be associated with the development of HCC, but the precise function of HBx in the tumourigenic transformation of liver cells remains unclear. Identifying proteins that interact with HBx directly or indirectly will reveal more clearly the role of HBx protein in the development of HCC, as the functions of HBx protein are via protein–protein interaction.

Proteomics is the study of the complete protein complement or the proteome of the cell. The proteome is dynamic and is in constant flux at each stage of the disease. At the protein level, distinct changes occur in pathological processes, including altered expression, differential protein modification, changes in specific activity and aberrant localization, all of which may affect cellular function. Identifying and understanding these changes is the goal of proteomics. Powerful proteomic techniques with high resolution, high throughput and real time express that analysis can provide new ideas for detecting more key proteins in pathological processes.

In this study, pEGFP-N1-X, a eukaryotic expression vector carrying HBx full-length gene, was transfected into HepG2 cells and identified fusion protein HBx-EGFP expressed in transfected cells by RT-PCR and Western blot. In addition, by fluorescence microscopy and laser confocal microscopy, we found that HBx protein was predominantly expressed in the cytoplasm and at the periphery of nucleus, a finding similar to the previous report [12]. Furthermore, to screen more key proteins related to the development of HCC, we used two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF-MS/MS) and found some distinctly different proteins between HepG2-pEGFP and HepG2-pEGFP-X.

SEC13L1 belongs to the SEC13 family of WD-repeat proteins. It has similarity to the yeast SEC13 protein, which is required for vesicle biogenesis from endoplasmic reticulum during the transport of proteins. But its functions are not clearly known.

PA28 alpha (proteasome activator REG alpha), an 11S regulator, 28 kDa, forms ring-shaped heptamer of known crystal structure that bind reversibly to the cylindrical 20S proteasome and activate its pepetide-hydrolysing activity [13]. The proteasome has been reported to be a potential cellular target of HBx [14]. Many previous studies have shown that HBx interacts with the subunits of proteasome [15], impairs the activation of the 20S proteasome by PA28 [16], affects hepadnavirus replication through a proteasome-dependent pathway and suggested that interaction of HBx with the proteasome may contribute to the pleiotropic functions of HBx [17]. Our results reveal that PA28 alpha are increased in HBx-transfected cells, suggesting that the proteasome pathway might be involved in the pathological process of HBx-related HCC.

Serine–threonine kinase receptor-associated protein (STRAP), a novel WD-domain protein, which interacts with transforming growth factor-β (TGF-β) receptor, negatively regulates TGF-β-induced transcription, and ubiquitously expresses and localizes in both cytoplasm and nucleus. STRAP is upregulated in 60% colon and in 78% lung carcinomas. Knockdown of endogenous STRAP by small interfering RNA increases TGF-β signalling, reduces ERK activity, increases p21 (Waf/Cip1) expression and decreases tumorigenicity [18]. P21, a protein encoding by WAF (CIP1) gene, is directly induced by p53 and inhibits the cyclin-dependent kinase (CDK) to regulate cell cycle progression and thus inhibit the growth activity of tumour cells [19, 20]. In the present study, the transient overexpression of HBx protein in HepG2 cells induced STRAP upregulated expression. It suggests that the potential carcinogenesis of HBX is closely correlated with STRAP negative regulation of TGF-β signalling pathways.

NME (nm23/nucleoside diphosphate kinase) gene is located on chromosome 17q21. NME protein has been detected in the membrane, cytoplasmic and nuclear fractions of cells. In addition, NME is secreted by tumour cells, both in vitro and in vivo [21–24]. In a number of cancer patient cohort studies, the correlation between NME expression and metastatic potential of the tumour and/or patient prognosis and survival was addressed [25]. A combination of reduced NME expression and high tumour metastatic potential was observed in several solid carcinomas. By contrast, other reports either show no or a positive correlation between NME expression and tumour metastasis and/or progression [26, 27]. In our study, we also found that the expression of NME protein was increased in HBx transient transfected HepG2 cells. This result suggested, in the early stage of HBx protein overexpression, that the inhibition of tumour metastasis was via upregulated expression of NME and thus performed compensation and protection function.

Summarizing, an application of proteomics is to analyse the proteins related to specific genes or pathways by combining the gene knock-in and proteomic approaches. In our study, we identified several proteins that were involved in the processes of the cellular proteolysis, carcinogenesis and tumour metastasis, and were upregulated in the HBx-transfected cells by a comparative proteomic analysis of the HBx gene knock-in and control hepatoma cells. The present study may provide a clue to understanding the molecular mechanism underlying the HBx-related HCC.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was funded by National High Technology Research and Development Program (863 Program no. 2001AA213111) from the Ministry of Science and Technology, Key Program (no. 30530660) from National Natural Science Foundation of China, and Basic Research Program (no. 04JC14004, 06QA14064) from the Science and Technology Commission of Shanghai.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References