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Keywords:

  • Nanog;
  • NANOGP8;
  • pseudogene;
  • retrogene;
  • tumorigenesis

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Nanog is a transcription factor that plays key roles in the self-renewal and maintenance of pluripotency in human embryonic stem (ES) cells. Among Nanog's 11 pseudogenes, NANOGP8 theoretically could be a retrogene, but was considered unlikely as it has not been identified in any expressed sequence tags (ESTs). In this study, we found that NANOGP8 was expressed in several cancer cell lines and in all cancer tissues tested. The complete coding sequence was cloned and the sequence is highly homologous to that of Nanog. We were also able to detect its protein expression using anti-Nanog antibody in recombinant Escherichia coli and some cancer cell lines tested. In addition, expression of NANOGP8 in NIH3T3 cells can promote cell proliferation. The expression of NANOGP8 in cancer cell lines and cancer tissues suggests that NANOGP8 may play important roles in tumorigenesis. This work not only has potential significance in stem cell and cancer research, but it also raises the possibility that some of the human pseudogenes may have regulatory functions.

Abbreviations
ES

embryonic stem

ESTs

expressed sequence tags

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

Nanog is a recently identified transcription factor that plays key roles in self-renewal and maintenance of pluripotency in inner cell mass and embryonic stem (ES) cells [1–3]. It is generally believed that the Nanog gene is specifically expressed in human ES cells and germ lineage cells and its expression may be controlled by an interaction between OCT4 and other proteins through an adjacent pair of highly conserved Octamer- and Sox-binding sites of 5′-flanking region of Nanog [4,5]. Nanog expression is rapidly down-regulated during ES cell differentiation, and constitutive expression of Nanog gene inhibits ES cell differentiation [3]. The regulation of Nanog gene expression and the nature of its target genes emerged as central issues in pluripotent stem cell biology [2].

Pseudogenes are common in mammalian genomes such as human and mouse and are defined as inactive versions of functional genes, i.e. they do not produce functional, full-length protein [6]. There are about 20 000 pseudogenes in human genomes [7]. It has been hypothesized that pseudogenes, especially those that are transcribed, have regulatory roles because of their high level of sequence similarities and conservation [8–10].

Recently, pseudogenes have been speculated to be involved in the epigenetic regulation of gene activities [11]. It is interesting that human Nanog has unusually high number of pseudogenes, 11 in total [12,13]. Among them, NANOGP8 is theoretically unique since it has a complete open reading frame and an Alu element in the 3′-UTR which is homologous to that of Nanog. The possibility that NANOGP8 may be a retrogene rather than a pseudogene was entertained, but was considered unlikely as NANOGP8 has not been identified in any expressed sequence tags (ESTs)[12].

In the current study, we found NANOGP8 expressed in several cancer cell lines and all the cancer tissues tested. No expression was detected in primarily cultured fibroblasts. At the same time, NANOGP8 was shown to prompt cell proliferation. These data indicated that NANOGP8 might play important roles in tumorigenesis.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

NANOGP8 is transcribed in cancer cell lines and tumor tissues

Human Nanog pseudogenes comprised 10 processed pseudogenes and one tandem duplicate [12]. Certain regions of these pseudogenes were highly conserved. The coding regions of 70% of Nanog pseudogenes share about 90% sequence identity [13]. Sequence comparison of the mRNA revealed the presence of the 5′-UTR sequence in Nanog, which is absent from other Nanog pseudogenes. We used this feature to discriminate Nanog and its pseudogenes.

NANOGP8 lied in chromosome 15 and shares the greatest similarity to Nanog. It contains a complete open reading frame, which might be transcribed in physiological conditions. To determine whether NANOGP8 could be transcribed, we designed human Nanog specific primers based on its 5′-UTR, and the expected PCR product is 444 basepairs. We also designed a pair of universal primers that would amplify NANOGP8, Nanog, and Nanog's other pseudogenes due to their homology, and the expected PCR product is 403 basepairs. Figure 1 showed the lack of expression of Nanog and its pseudogenes in normal human primarily cultured fibroblasts; Nanog was expressed in ovary teratocarcinoma cell line PA-1 and testicular embryonic carcinoma NTERA-2, and sequencing the 444 basepairs PCR product confirmed the findings (Table 1). Furthermore, when the PCR products were cloned and sequenced, the results indicated that NANOGP8 was expressed in human osteosarcoma cell line OS732, human hepatoma cell line HepG2 and human breast adenocarcinoma cell line MCF-7 (Table 1). At the same time, other pseudogenes were found in OS732, HepG2 and MCF-7, while Nanog gene was also expressed in HepG2 and MCF-7 but not in OS732 (Table 1 and Fig. 1). In this study, we found that NANOGP8 was transcribed in all of the human cancer tissues tested (Table 1 and Fig. 2) as well as in several cancer cell lines. In contrast, Nanog and NANOGP8 were not expressed in normal primarily cultured fibroblast cell line or fetal liver epithelium cells (data not shown). It is interesting that only NANOGP8 was expressed in OS732 cell line, uterine cervix and breast tumor tissues, while Nanog gene was undetectable (Table 1). Thus, it is true that NANOGP8 was transcribed in cancer cell lines and tumor tissues.

image

Figure 1. Detection of Nanog, NANOGP8 and other pseudogenes in tumor cell lines. RT-PCR analysis the expression of Nanog, NANOGP8 and other pseudogenes in human fibroblasts and tumor cell lines OS732, HepG2, MCF-7, THP-1, HeLa, PA-1 and NTERA-2. Sequencing analysis of PCR products (Table 1) confirmed that NANOGP8 was expressed in cell lines OS732, HepG2, and MCF-7 while Nanog was expressed in HepG2, MCF-7, PA-1 and NTERA-2. No-RT data were also shown.

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Table 1.  The sequencing results of RT-PCR product (403 basepairs) clones with the universal primers.
Cells or tissues testedTotal sequenced clonesSequenced clones of NanogSequenced clones of NANOGP8Others
Normal cells
 Human fibroblasts0000
Tumor cell lines
 OS73213056(NANOGP4)
   2(NANOGP5)
 HepG212236(NANOGP4)
   1(NANOGP5)
 MCF-715223(NANOGP4)
   8(NANOGP5)
 THP-10000
 HeLa0000
Teratocarcinoma cell lines
 PA-17700
 NTERA-26600
Tumor tissues
 Uterine cervix5011(NANOGP2)
   2(NANOGP4)
   1(NANOGP7)
 Breast13081(NANOGP4)
   1(NANOGP5)
   3(NANOGP7)
 Urinary bladder8141(NANOGP2)
   2(NANOGP7)
image

Figure 2. Detection of Nanog, NANOGP8 and other pseudogenes in tumor tissues. RT-PCR analysis of Nanog, NANOGP8 and other pseudogenes expression in human fibroblasts and human carcinoma tissues of the uterine cervix, breast and urinary bladder is shown. NANOGP8 was expressed in all of the human cancer tissues that were tested. NANOGP8 was not expressed in human fibroblasts. Nanog was expressed in urinary bladder tumor tissues and PA-1 cell lines. No-RT data were also shown.

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NANOGP8 complete coding sequence was obtained from urinary bladder cancer

Using RT-PCR, the complete coding sequence of NANOGP8 was cloned from urinary bladder cancer tissues and its sequence was found to be highly homologous to Nanog. There are six alternations over 918 sites (Fig. 3 and Supplementary material Fig. 1) and only one change in the inferred amino acid sequence from 253 Gln in Nanog to His in NANOGP8. Thus, it is likely that NANOGP8 and Nanog genes have similar functions.

image

Figure 3. Sequence alignment of human Nanog and NANOGP8 genes. The differences in nucleotides and their positions are shown. The translational start site is defined as +1. There is only one change in the inferred amino acid sequence from Gln (CAG759) in Nanog to His (CAC759) in NANOGP8.

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NANOGP8 and/or Nanog protein was detected in cell lines

The expression levels of NANOGP8 appear relatively low in the cancer cell lines and neoplastic tissues compared to Nanog in EC cells (Figs 1 and 2). Given the very high degree of homology between Nanog and NANOGP8 on the basis of nucleotide sequences, it is likely that the commercially available Nanog antibodies could recognize NANOGP8-translated protein. To confirm this hypothesis, we first constructed GST-NANOP8 fusion protein expressed in recombinant Escherichia coli and then detected NANOP8 using anti-Nanog antibody. Nanog antibody could recognize the fusion protein (∼60 kDa) (Fig. 4B). To directly examine NANOGP8 protein expression, nuclear protein extracts were used for western blot assay. According to Table 1 and Fig. 1, both NANOGP8 and Nanog were transcribed in HepG2, while Nanog was not transcribed in OS732. Thus, it is likely the protein of NANOGP8 and/or Nanog was detected in cancer line HepG2 and the protein of NANOGP8 was found in cancer line OS732 using anti-Nanog antibody and the observed molecular weight (∼34 kDa) of the protein was consistent with its predicted full-length sequence (Fig. 4A). Therefore, the above findings showed that NANOGP8 was translated and it suggested NANOGP8 is a retrogene but not a pseudogene.

image

Figure 4. Western blot detection of NANOG and/or NANOGP8 protein. Detection of NANOGP8 protein in (B)  E. coli (1) and E. coli with NANOGP8 (2) and NANOGP8 in (A) OS732 and Nanog or NANOGP8 in MCF-7 and HepG2 using anti-Nanog antibody. The lack of expression of both Nanog and NANOGP8 in human fibroblasts is also shown. Actin (43 kDa) was used as loading control.

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NANOGP8 was localized in the nuclei of transfected cells

Human Nanog is a transcriptional regulator and is localized in the nucleus [2,3]. Due to the high homology between Nanog and NANOGP8, NANOGP8 is likely a nuclear protein as well. To confirm this, we constructed a NANOGP8 and GFP fusion protein. As shown in Fig. 5, the fusion protein was localized in the nuclei of transfected NIH3T3 (Fig. 5 A and 5B), while GFP in the control group was present diffused in the cytoplasm (Fig. 5C,D). Therefore, NANOGP8 is also a nuclear protein.

image

Figure 5. Nuclear location of NANOGP8-GFP. The NANOGP8-GFP (A,B) and GFP (C,D) vector were introduced into NIH3T3, respectively, and photographed in bright fields and fluorescent field (B,D) and merged photos (A,C).

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NANOGP8 promotes cells to enter into S phase

Cell cycle analysis was performed in NANOGP8 transfected NIH3T3 (pQCXIN-NANOGP8 vector) and mock control (pQCXIN vector) by flow cytometry (Fig. 6A). The percentage of S phase in NANOP8-transfected cells was 53.3%, which was higher in comparison with the mock control (46.5%). The difference was statistically significant (P < 0.05). The results were obtained from three independent clones. These results indicate that expression of exogenous NANOGP8 gene promotes cells to enter into S phase of the cell cycle.

image

Figure 6. FACS analysis results. FACS analysis of cells transfected with NANOGP8 and the mock ones (A) and the MTT assay (B). The percentage of NANOGP8 transfected cells at S phase is 53.3 ± 2.4 and of the mock cells is 46.5 ± 1.3. We examined the effect of NANOGP8 gene expression on NIH3T3 cells growth by MTT assay. NIH3T3 transfected with NANOGP8 gene versus the mock ones, P < 0.05 (except day 1); there are no significant differences between the different clones (data not shown). Data are presented as mean ± SD. The experiments were repeated for three times. Data were analyzed using the Student's t-test.

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NANOGP8 promotes cell proliferation

The increase in the percentage of cells in S phase suggests that NANOGP8 may promote cell proliferation [14]. To confirm this, we examined the effect of NANOGP8 overexpression on NIH3T3 cell growth. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Fig. 6B) indicated that the proliferation rate of NANOGP8-transfected cells was significantly increased.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Pseudogenes are thought to be ‘molecular fossils’, which can be used as a model to study the rate of nucleotide substitution, insertion and deletion in genome [15]. Recent experimental data have indicated that some pseudogenes were transcribed and might be functionally active [9,10]. On the other hand, great care must be taken in validation of some assays for intervention of pseudogenes [16].

Nanog is a recently identified transcriptional factor that plays important role in regulating pluripotency and self-renewal of ES cells [2,3]. Recently, Nanog's 11 pseudogens were identified, including 10 processed pseudogenes and one tandem duplicate [12]. They share sequence homology to the Nanog coding region, but lack the potential to produce a functional protein except the NANOGP8 because of critical mutations [12]. NANOGP8, bearing close similarity to Nanog, theoretically could be a retrogene, but this was considered unlikely as it has not been identified in any ESTs. The EST database provides tremendous gene expression information from many types of tissues or cells. Theoretically any gene transcriptions could be found. We carried out an EST search, where we found it is difficult to distinguish Nanog from NANOGP8 in some ESTs (Supplementary material Fig. 2). This maybe because of the limitation of each clone's sequence information in EST database and the high similarity between NANOGP8 and Nanog, with only six nucleotides alternations and one amino acid mutation being found between them. In addition, NANOGP8 expression is extremely low compared with that of Nanog and this might contribute at least partially to why NANOGP8 ESTs have not been detected from human cancers and cancer cell lines.

To investigate whether NANOGP8 could be transcribed, we designed human Nanog-specific primers and also designed a pair of primers that would amplify NANOGP8, Nanog, and Nanog's other pseudogenes due to their homology. By using these primers for PCR analysis and DNA sequencing, we would be able to identify the transcribed pseudogenes. Our results showed the lack of expression of NANOGP8 and Nanog in normal human primarily cultured fibroblasts, fetal liver epithelium cells (data not shown). Furthermore, when all the PCR products were sequenced, the results confirmed that NANOGP8 was expressed in HepG2, MCF-7, OS732 as well as all the human cancer tissues tested.

In addition, we have obtained the complete NANOGP8 coding region from cancer tissues by RT-PCR excluding the genomic DNA contamination. Analysis of the nucleotide sequences in Nanog and NANOGP8 demonstrated that there are six alternations, which resulted in only one amino acid change in the predicted protein sequence from (Gln, 757CAG) in Nanog and (His, 757CAC) in NANOGP8. Since NANOGP8 has an intact open-reading fragment, so it has the potential to encode a full protein. We then expressed NANOGP8 in E. coli, which could be detected by western blot after GST affinity purification. Further, anti-Nanog antibody can recognize translated NANOGP8 protein in OS732. These data suggested that NANOP8 is a retrogene rather than a pseudogene.

Because cancer cells and stem cells share many common characters, such as unlimited proliferation, it has been suggested that similar mechanisms might be involved in regulating cancer cells and stem cells [17]. Our preliminary results showed that forced expression of Nanog gene in 3T3 fibroblasts could greatly increase cell proliferation rate [18]. When NANOGP8 was stably transfected into NIH3T3 cells, the cells were promoted to enter into the S stage and, at the same time, MTT growth assay showed increased cell proliferation.

Our findings that NANOGP8 is expressed in cancer cell lines and cancer tissues suggest that NANOGP8 may play important roles in tumorigenesis. Oct4, another stem cell self-renewal gene, was found to be up-regulated in breast, pancreatic and colon cancers and expressed in several cancer cell lines such as HeLa and MCF-7 [19–21]. These findings suggested that this type of gene might be involved in tumorigenesis. Nanog was reported recently to be expressed only in some germ cell tumors and breast carcinoma [22–24]. Our results showed the transcription of NANOGP8, but not Nanog, in the human osteosarcoma cell line OS732, so it is likely that only NANOGP8 was translated in this cell line detected by the western blot experiment. Our future goal is to analyze how the NANOGP8 gene is regulated in various cancers. We hypothesize that NANOGP8 may function in tumor cell self-renewal due to the high homology between Nanog and NANOGP8 genes. This work not only has potential significance in stem cell and cancer research, it also raises the possibility that some of the human pseudogenes may in fact be retrogenes and may have important functions in gene regulation.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Total RNA extract and RT-PCR

Total RNA was extracted from cell lines and tissues using Trizol (Invitrogen, Carlsbad, CA, USA) reagent following the manufacturer's instuctions. Total RNA was digested with RNAase-free DNase I (TaKaRa Carlsbad, CA, USA) at 37°C for 30 min and inactivated at 60°C for 10 min. With total RNA (2 µg) as the template and oligo(dT) as the primer, the first cDNA was synthesized in 25 µL reaction system with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA). First-strand cDNA and RNA without reverse transcription were amplified with β-actin primers to confirm the success of RT reaction and no genomic DNA contamination. At the same time, no-RT control (RT reaction without reverse transcriptase) was carried out to further exclude the DNA contamination. cDNA template (3 µL) was used in a 25 µL reaction volume with rTaq DNA polymerase or LA TaqTM DNA polymerase with GC buffer (TaKaRa). Human NANOGP8 mRNA was amplified by RT-PCR using total RNAs extracted from urinary bladder cancer tissue. For NANOGP8, the sense primer 5′-ATGAGTGTGGATCCAGCTTGTCC-3′ and antisense primer 5′-CACGTCTTCAGGTTGCATGTTCA-3′, for Nanog-403 (and/or its pseudogenes), the sense primer 5′-ATGCCTGTGATTTGTGGGCC-3′ and antisense primer 5′-GCCAGTTGTTTTTCTGCCAC-3′, for Nanog-444, the sense primer 5′-ATTATAAATCTAGAGACTCC-3′ and antisense 5′-TTGTTTGCCTTTGGGACTGGT-3′, for β-actin, the sense primer 5′-TCACCACCACGGCCGAGCG-3′ and antisense 5′-TCTCCTTCTGCATCCTGTCG-3′ were used. DNA was amplified with an initial enzyme activation step at 94°C for 5 min, followed by 30 cycles (β-actin) or 34 cycles (Nanog-403; Nanog-444; NANOGP8) of 94°C for 40 s, 55°C for 40 s and 72°C for 40 s (β-actin; Nanog-403; Nanog-444) or 90 s (NANOGP8). The PCR products were analyzed by 1.2% agarose gel electrophoresis and the bands were extracted using gel extraction kit (Omega, Bio-Tek, Doraville, GA, USA). DNA fragments extracted were ligated into T vector (Promega) and sequencing analysis was carried out on positive clones identified.

Cell culture, cancer and normal tissues

All cancer cell lines including OS732, HepG2, MCF-7, THP-1, HeLa, PA-1, NTERA-2 were cultured in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 2 mm l-glutamine (Gibco-BRL, Gathersburg, MD, USA), 100 × nonessential amino acid solution (Hyclone), 100 mm sodium pyruvate (Hyclone), penicillin-streptomycin solution (Hyclone), and 10% fetal bovine serum (Hyclone). For primarily cultured fibroblast and fetal liver epithelium cells, 20% fetal bovine serum was added. All cancer tissues used were obtained from the tissue bank at ZhaoYang Hospital (Beijing, China) and patients gave written consent for the use of these tissues for research purposes.

Nuclear protein extraction

Nuclear protein extraction was performed as described [25]. In brief, cells were subsequently rinsed with ice-cold NaCl/Pi (Hyclone), NaCl/Pi containing 1 mm Na3VO4 and 5 mm NaF, and hypotonic buffer (NaCl/Pi including 20 mm Hepes, 20 mm NaF, 1 mm Na3VO4, 1 mm Na4P2O7, 0.4 µm microcystin, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1 µg·mL−1 each leupeptin, aprotinin and pepstatin). They were lysed with ice-cold hypotonic buffer with 0.2% NP-40. The nuclear pellets were collected by centrifuge at 16 000 g for 20 s and then resuspended in 150 µL high salt buffer (hypotonic buffer containing 420 mm NaCl and 20% glycerol). The pellets were rocked gently on ice for 30 min and centrifuged at 16 000 g for 20 min to separate the nuclear proteins. Protein concentration was determined by Bradford method [26].

Fusion protein expression and GST purification

GST fusion proteins were expressed and prepared according to the manufacture's instructions from Amersham Biosciences (Piscataway, NJ, USA). In brief, E. coli BL21 cells carrying NANOGP8 were grown in LB medium and induced by the addition of 0.1 mm(isopropyl thio-β-d-galactoside). The pellet obtained from 500 mL of solution was suspended and sonicated for 30 s in 70 mL of 10 mm Tris/HCl. The resulting supernatant was subjected to affinity chromatography on a glutathione-Sepharose column (Amersham Biosciences) and then eluted with 50 mm Tris and 10 mm glutathione (pH = 8.0).

Western blot

For western blot analysis, equal protein (30 µg) was examined by 10% (w/v) SDS/PAGE. Proteins on the gel were transferred onto a nitrocellulose membrane in 1.44% glycine, 0.3% Tris (pH = 8.4), 20% methanol at 80 V for 1 h, and the membrane was then blocked with NaCl/Pi, 5% milk, 0.3% Tween-20. The membrane was probed with polyclonal goat anti-human Nanog (1 : 1000, AF1997, R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human Actin (1 : 500, SC-8432, Santa Cruz, Santa Cruz, CA, USA). Results were detected using the WesternBreeze® kit (Invitrogen). X-ray films were scanned with a GDS8000 Gel Image Analysis System (Ultra-Violet Products, Cambridge, UK).

Expression constructs and cell transfection

The GFP cDNA was cloned from pEGFP-N1 vector and inserted into pQCXIN between the BamHI and EcoRI sites. The NANOGP8 was amplified by RT-PCR and inserted into pQCXIN between AgeI and PacI sites. The GFP and(or) NANOGP8 were(was) ligated into the pQCXIN vector to produce the pQCXIN-GFP, pQCXIN-NANOGP8, and pQCXIN-NANOGP8-GFP. NIH3T3 cells were transfected with the expression vector pQCXIN, pQCXIN-NANOGP8 and pQCXIN-NANOGP8-GFP using LipofectamineTM 2000 according to the manufacturer's instructions. Stable clones were selected and isolated in media containing 500 µg·mL−1 G418 (Invitrogen) for cell cycle analysis or MTT assays. For nuclear localization, NIH3T3 cells were performed using LipofectamineTM 2000 and then were taken photos with Zeiss 200 inverted fluorescent microscopy (Carl Zeiss).

Cell cycle analysis and MTT assay

Cell cycle was measured by the propidium iodide (PI) staining method. In brief, cells (1 × 106) were washed twice with cold NaCl/Pi, fixed in 5 mL 70% ethanol at 4°C overnight. Cells were rinsed twice with NaCl/Pi and resuspended in 500 µL NaCl/Pi with 50 µg·mL−1 RNaseA solution at 37°C for 30 min 50 mg·mL−1 PI was added to the incubated solution. Percentages of 15–20thousand cells in G0/G1, S and G2/M phase of the cell cycle were analyzed on a FACScalibur and by Modifit software.

For the MTT assay, cells were plated at 1 × 104 per 24-well plates and were cultured for 1–4 days. Viable cells were evaluated by adding 100 µL of 2.5 mg·mL−1 MTT cultured in 37°C for 4 h. After the removal of MTT solution, 100 µL dimethyl sulfoxide were added to each well and gently shaken for 10 min. The absorbance was determined at 492 nm with plate reader (Sunrise, Tecan, Gr¨odig, Austria).

Data analysis

Data were analyzed by Student's t-test. A value of P < 0.05 was considered statistical significance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by grants from Chinese Academy of Sciences (KSCW2-SW-205; KSCW2-SW-218), from NSFC (30428017) and from The Chinese 973 Program (2004CB117404; 2005CB522603).

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  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
FEBS5186_figS1.pdf19KSupporting info item
FEBS5186_figS2.pdf119KSupporting info item

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