Skip Navigation


Japanese Journal of Clinical Oncology Advance Access originally published online on July 22, 2006
Japanese Journal of Clinical Oncology 2006 36(9):564-569; doi:10.1093/jjco/hyl063
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
36/9/564    most recent
hyl063v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Takayama, T.
Right arrow Articles by Ozono, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takayama, T.
Right arrow Articles by Ozono, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© 2006 Foundation for Promotion of Cancer Research

High Levels of Thymidine Phosphorylase as an Independent Prognostic Factor in Renal Cell Carcinoma

Tatsuya Takayama1, Soichi Mugiya1, Takayuki Sugiyama1, Takahiro Aoki1, Hiroshi Furuse1, Hongming Liu1, Yasuhiro Hirano2, Fumitake Kai3, Tomomi Ushiyama1 and Seiichiro Ozono1

1 Department of Urology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, 2 Fujieda Municipal General Hospital, Fujieda, Shizuoka and 3 Haibara General Hospital, Makinohara, Shizuoka, Japan

For reprints and all correspondence: Tatsuya Takayama, Department of Urology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan. E-mail: takayama{at}hama-med.ac.jp

Received April 3, 2006; accepted May 21, 2006


   Abstract
TOP
 Abstract
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Background: We investigated whether thymidine phosphorylase (TP) protein level in renal cell carcinoma (RCC) correlates with clinicopathological characteristics and clinical outcomes.

Methods: TP protein level was measured in 116 RCC specimens and in 90 non-neoplastic kidney tissues using a sandwich-type enzyme-linked immunosolvent assay.

Results: The median TP protein level in RCC tissues was 9.76-fold (range, 3.2–933.9) higher than those in non-neoplastic kidney tissues (P < 0.0001). TP protein level was correlated with T classification, histological grade and mode of infiltration. TP as a prognostic variable was studied using a logistic regression model. TP at higher levels (128 U/mg protein or greater) would play a role as an independent prognostic factor (odds ratio, 13.73; 95% confidence interval, 2.09–90.41; P = 0.0064).

Conclusion: TP at high levels can be regarded as an unfavorable independent prognostic factor. These results may pave a way for a novel approach to effective treatment of RCC.

Key Words: renal cell carcinoma • thymidine phosphorylase • prognostic factor


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Tumor angiogenesis is a complicated multistep process that involves extracellular matrix remodeling, endothelial cell migration and proliferation, capillary differentiation and anastomosis.

Thymidine phosphorylase (TP), known as a platelet-derived endothelial cell growth factor (PD-ECDF) (1–4), may play an important role in both activation of 5-fluorouracil (5-FU) drugs and tumor angiogenesis (5). TP exerts angiogenic activity in vivo while its enzymatic activity is indispensable for its angiogenic effect (6). Many investigators have reported that TP levels are higher in various types of malignant tumor than in the adjacent non-neoplastic tissues, such as renal cell carcinoma (RCC) (7–10).

In this study, we determined TP protein levels in both RCC and non-neoplastic kidney tissues by using ELISA, which has been reported to correlate with the TP activity levels (8,11), and investigated the association of the TP protein levels with clinicopathological characteristics and clinical outcome in patients with RCC.


   PATIENTS AND METHODS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 References
 
PATIENTS
We investigated 116 patients (88 men and 28 women) with RCC. The patient characteristics are shown in Table 1. Tumors were removed surgically in the Department of Urology at Hamamatsu University Hospital and affiliated hospitals between December 1997 and July 2004. None of the patients had received prior chemotherapy, irradiation, or embolization. All investigations were performed after informed consent was obtained according to institutional ethical committee rules.


View this table:
[in this window]
[in a new window]
  		Table 1. Characteristics of the 116 patients and relationship between TP protein level and categories

 
The mean age at surgery was 61.8 years with a range of 35–84. Among 116 patients with RCC, 98 had no metastasis and 18 had distant metastasis at the time of nephrectomy. Survival data were updated in September 2004. The median follow-up duration was 39 months (range, 1.2–97.9). Seventeen patients died of RCC and 5 died of other diseases. Ninety-four patients with RCC in this study remained alive throughout the follow-up period. The tumors were graded according to the criteria of the Japanese Urological Association (12) and were staged according to the TNM system (1997) (13). Moreover, they were also classified into histological subtypes. Tumor size was defined according to the largest diameter of the tumor. T stage was classified into two groups, pT1 + pT2 and pT3 + pT4. The node category was determined by intraoperative and preoperative findings such as ultrasonography, computed tomography scans and magnetic resonance imaging. The metastasis category was based on the results of computed tomography and bone scans. C-reactive protein (CRP) was divided into two groups according to reference interval. Patients and tumor profiles are listed in Table 1.

SPECIMENS AND TISSUE HOMOGENATE
One hundred sixteen tumor specimens and 90 non-neoplastic kidney specimens in the farthest and separable region from the tumor were obtained at surgery from 116 patients with RCC. Non-neoplastic tissues were defined as macroscopically normal tissues with no tumor cells being observed histopathologically. Both tissue specimens were kept at –80°C before use.

An ELISA was used to measure TP levels in RCC and non-neoplastic tissues. Tissue specimens were first homogenized in 10-fold volume of 10 mM Tris–HCl buffer (pH 7.4) containing 15 mM NaCl, 1.5 mM MgCl2 and 50 µM potassium phosphate, and then centrifuged at 10 000x g for 15 min. The supernatant was stored at –80°C until use. The protein concentration of the supernatant extracted from tumor tissues was determined by using a DC protein Assay Kit (Bio Rad Laboratories, Hercules, CA). The TP protein levels in RCC and non-neoplastic kidney tissues were measured by a sandwich ELISA with two monoclonal antibodies (MoAb) specific to human TP (11). The amount of TP was calibrated with the standard solution. TP protein levels in tumor tissues were expressed as U/mg protein, where 1 U is the amount equivalent to 1 µg of 5-FU produced in an hour as an enzyme activity.

STATISTICAL ANALYSIS
All data are presented as the mean (±SD). TP protein levels in tumors and non-neoplastic tissues were compared using the Wilcoxson single rank test. The Mann–Whitney U-test or Fisher's exact test was used for the evaluation of the relationship between TP protein level in tumors and the clinicopathological features. Survival analysis was performed by Kaplan and Meier method. The differences between survival curves were tested by log-rank test. Statistical significance was then chosen for a logistic regression model to determine the prognostic value. All P-values presented are two-sided. Statistical significance was calculated by using package STATVIEW Ver. 5 (Abacus Concepts, CA, USA). P-values of <0.05 were considered to be statistically significant.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 References
 
We examined the enzymatic activity of TP in 116 RCCs and 90 non-neoplastic kidney tissue specimens (Fig 1). The median value of TP protein level in RCCs (128.0 U/mg protein) was 10-fold higher than those in non-neoplastic kidney tissues (11.7 U/mg protein). The median activity ratio (carcinoma/non-neoplastic tissue) was 9.76.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. TP level in tumor and non-neoplastic tissues. Boxes indicate the range of a half of data (between upper and lower quartile). Lines in boxes show median values. Bars at both sides of boxes denote the ranges of lower 10% and upper 90%; and open circles indicate values beyond these ranges.

 
Among patient factors, significant relation could not be evidenced. In contrast, however, TP protein level in tumor was found significantly higher in progressive T classification, histological grade and mode of infiltration among tumor factors (Table 1 and Fig. 2).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. TP protein level according to T classification (A), histological grade (B) and mode of infiltration (C) in renal cell carcinoma. Data are expressed as the mean ± standard deviation.

 
We investigated whether TP protein level would contribute as an independent prognostic variable. To evaluate cause-specific survival based on the TP protein level of RCC, 116 patients were divided into two groups at the median value (high TP, 128.0 U/mg protein ≤ TP value; low TP, TP value < 128 U/mg protein). Five patients with metastasis are included in low TP group, while 13 patients in high TP group. As depicted in Table 2, univariate analysis indicated that CRP, tumor status, lymph node metastasis, distant metastasis, cell type, histological grade, mode of infiltration, venous invasion and TP protein level in tumor would be significant prognostic factors. A logistic regression model was constructed using the aforesaid established prognostic factors. Multivariate analysis showed that both cell type (P = 0.0033) and TP activity (P = 0.0099) were independent significant factors, with the odds ratios being 8.552 and 11.180, respectively. As shown in Fig. 3, the 5-year cause specific survival rate of the patients with low TP was 93.6%, whereas that of patients with high TP was 68.6%. A significantly higher rate of cause-specific survival occurred in the patients with low TP than with high TP (P = 0.0024). In addition, as shown in Fig. 4, when all patients were divided into pT1 + pT2 and pT3 + pT4 or with and without metastasis, high level of TP was significantly unfavorable prognostic factor in the patients with pT1 + pT2. On the other hand, in the patients with pT3 + pT4, metastasis, or without metastasis, TP protein level in tumor was not significantly prognostic factor, but high level of TP appeared to be a trend toward poor prognosis in the each dichotomized subgroup.


View this table:
[in this window]
[in a new window]
  		Table 2. Prognostic factor for survival by univariate and multivariate analysis

 

Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Cause specific survival in the groups of low TP (<128.0 U/mg protein) and high TP (≥128.0 U/mg protein) among all patients with renal cell carcinoma.

 

Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Cause-specific survival in the groups of low TP (<128.0 U/mg protein) and high TP (≥128.0 U/mg protein) among the subgroups of pT (A, B) and M (C, D) classification.

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
References
 
PD-ECGF/TP is a mitogenic and angiogenic factor derived from platelets, while the enzymatic activity of TP is required for angiogenesis (6). TP activity in several types of malignant tumors is higher compared with those in the adjacent non-neoplastic tissue (14–17). Interestingly, it is also evidenced that inhibitors of TP inhibit both invasion and metastasis in vivo model systems (18), suggesting that the angiogenic activity of TP might contribute to progression of some tumors.

Concerning RCC, Imazono et al. (7) and Kinsui et al. (8) reported that the median TP enzymatic activity in RCC was 9-fold higher than those in non-cancerous renal tissue. Our study also revealed that TP protein level in RCC was elevated to become 9.76-fold higher compared to that of paired non-neoplastic tissue.

TP activity correlates significantly with various clinicopathological factors. Imazono et al. (7) demonstrated that tumor cell TP immunoreactivity was related to RCC tumor grade, while Kinsui et al. (8) indicated that the TP expression found by ELISA was significantly higher in patients with venous invasion than in those without invasion. In addition, Wada et al. (19) gave a novel view that TP levels in adjacent non-neoplastic kidney tissue, but not RCC tissue, were associated with histological grade. In the present study, TP protein level in RCC increased in parallel with T classification, histological grade and mode of infiltration (Table 1). This result suggests that TP is more associated with direct invasion rather than distant metastasis.

Viewed together, it is postulated that the value of TP expression might be related to prognosis. In fact, Imazono et al. (7) elucidated by multivariate analysis that TP overexpression was an independent prognostic factor in RCC. Suzuki et al. (20) also reported that there was a significant correlation between survival rates and TP expression, whereas angiogenesis did not correlate with TP expression or survival. On the other hand, Kinsui et al. (8) found no correlation between TP level and clinicopathological findings except for venous invasion and survival curves. The present study indicated that high TP protein level in tumor was an independent prognostic factor, with the odds ratio being 11.180 (P = 0.0099) in multivariate analysis. These results suggest that TP has other effects that influence tumor growth, especially direct invasion and prognosis in addition to angiogenic activity, although it is not known how TP expression is regulated in RCC. Generally, high T stage or distant metastasis leads to the poor prognosis in various cancers. In order to more fully investigate the possibility of TP as unfavorable prognostic factor, we divided pT and M classification into pT1 + pT2 and pT3 + pT4, and with or without metastasis, respectively, resulting in the high level of TP appeared to be a trend toward poor prognosis. We also indicated that not clear cell carcinoma but the other type of RCC was an independent prognostic factor. However, this result may reflect that the ratio of spindle cell carcinoma distinctly known as a poor prognostic factor in the other type of RCC is high.

Thinking about therapeutic strategy, we could find that the up-regulation of TP expression in RCC leads to doxifluridine (5'-DFUrd) because TP is also one of the key enzymes involved in the metabolism of fluorouracil-related drugs, and especially, 5'-DFUR is a prodrug of 5'-fluorouracil (5'-Fura) and TP converts it to 5'-Fura (21,22). Moreover, Morita et al. measured the expression of TP and dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme catabolizing 5-FU, and suggested that a high TP/DPD ratio was the candidate for the possible role as indicator of responsiveness to fluorpyrimidines (17). Although this statement cannot be made from our study protocol, carcinoma cells with high TP activity may be more sensitive to fluorouracil (8). We reported that TP protein level in human RCC and normal kidney tissues exhibited a significant positive correlation with sensitivity to 5-FU and 5'-DFUrd using in vitro histoculture drugs response assay (23,24). Ikemoto at al. also reported that IFN{alpha} enhanced TP expression in high TP expression renal cell lines, and antitumor effects were enhanced by IFN{alpha} for 5-FU and 5'-DFUrd (25). In addition, ELISA method is much more convenient compared with conventional enzyme assay, and enough to measure TP levels even in samples weighting as little as 10 mg (11). On the other hand, a novel, specific TP inhibitor, TPI, inhibited TP-enhanced angiogenesis, tumor growth and metastasis (18). However, treatment of tumors with inhibitors of TP in combination with 5-FU and its products is impossible, because TP is one of the enzymes correlated with activation of 5-FU. To overcome these problems, there is a trend to inhibit the downstream mediators of TP function rather than to directly inhibit TP activity (26).

In conclusion, TP at high levels can be regarded as an unfavorable independent prognostic factor. These results may pave a way for a novel approach to effective treatment of RCC.


   Acknowledgments
 
The present investigation was supported in part by a Grant-in-Aid for Scientific Research (C) (16591591) of The Ministry of Education, Culture, Sports, Science and Technology. The authors thank Kazushige Mori (Chugai pharmaceutical Co. LTD, Kamakura, Japan) for their technical advice concerning the ELISA measurement.

Affiliated hospitals which contributed to the present study: Fujieda Municipal General Hospital, Fujieda; Fujinomiya City General Hospital, Fujinomiya; Iwata City Hospital, Iwata; Haibara General Hospital, Haibara; Kikugawa General Hospital, Kikugawa; Kosai General Hospital, Kosai; National Hospital Organization Tenryu Hospital, Hamamatsu; Seirei Mikatabara General Hospital, Hamamatsu; Social Insurance Hamamatsu Hospital, Hamamatsu; Suzuki Clinic, Kannami; Yaizu City Hospital, Yaizu, Shizuoka, Japan and Shinshiro Municipal Hospital, Shinshiro, Aichi, Japan.


   References
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
1 Furukawa T, Yoshimura A, Sumizawa T, Haraguchi M, Akiyama S, Fukui K, et1 al. Angiogenic factor [letter]. Nature 1992;356:668.[Medline]

2 Usuki K, Saras J, Waltenberger J, Miyazono K, Pierce G, Thomason A, et al. Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity. Biochem Biophys Res Commun 1992;184:1311–16.[CrossRef][Web of Science][Medline]

3 Moghaddam A, Bicknell R. Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity. Biochemistry 1992;31:12141–6.[CrossRef][Medline]

4 Sumizawa T, Furukawa T, Haraguchi M, Yoshimura A, Takeyasu A, Ishizawa M, et al. Thymidine phosphorylase activity associated with platelet-derived endothelial cell growth factor. J Biochem (Tokyo) 1993;114:9–14.[Abstract/Free Full Text]

5 Akiyama S, Furukawa T, Sumizawa T, Takebayashi Y, Nakajima Y, Shimaoka S, et al. The role of thymidine phosphorylase, an angiogenic enzyme, in tumor progression. Cancer Sci 2004;95:851–7.[CrossRef][Medline]

6 Miyadera K, Sumizawa T, Haraguchi M, Yoshida H, Konstanty W, Yamada Y, et al. Role of thymidine phosphorylase activity in the angiogenic effect of platelet-derived endothelial cell growth factor/thymidine phosphorylase. Cancer Res 1995;55:1687–90.[Abstract/Free Full Text]

7 Imazono Y, Takebayashi Y, Nishiyama K, Akiba S, Miyadera K, Yamada Y, et al. Correlation between thymidine phosphorylase expression and prognosis in human renal cell carcinoma. J Clin Oncol 1997;15:2570–8.[Abstract/Free Full Text]

8 Kinsui H, Ueda T, Suzuki H, Isaka S, Sekita N, Komiya A, et al. Expression of thymidine phosphorylase in primary human renal cell carcinoma by ELISA Method. Jpn J Cancer Res 2002;93:340–5.[CrossRef][Web of Science][Medline]

9 Mizutani Y, Wada H, Yoshida O, Fukushima M, Kawaguchi A, Nakao M, et al. The significance of thymidine phosphorylase/platelet-derived endothelial cell growth factor activity in renal cell carcinoma. Cancer 2003;98:739–6.

10 Morita T, Matsuzaki A, Tokue A. Quantitative analysis of thymidine phosphorylase and dihydropyrimidine dehydrogenase in renal cell carcinoma. Oncology 2003;65:125–31.[Web of Science][Medline]

11 Nishida M, Hino A, Mori K, Matsumoto T, Yoshikubo T, Ishitsuka H: Preparation of anti-thymidine phosphorylase monoclonal antibodies useful for detecting the enzyme levels in tumor tissues. Biol Pharm Bull 1996;19:1407–11.[Web of Science][Medline]

12 Union international Contre le Cancer. ‘TNM Atlas’. 4th edn. Berlin: Springer-Verlag 1997.

13 The Japanese Urological Association. ‘The Japanese Pathological Society, and the Japan Radiological Society. General Rules for Clinical and Pathological Studies on Renal Cell Carcinoma’. 3rd edn. Tokyo: Kanehara & Co., Ltd. 1999.

14 Takebayashi Y, Yamada K, Miyadera K, Sumizawa T, Furukawa T, Kinoshita F, et al. The activity and expression of thymidine phosphorylase in human solid tumours. Eur J Cancer 1996;32:1227–32.[CrossRef]

15 Kono T, Nishida M, Inagaki N, Tanaka Y, Yoneda M, Kasai S. Development and characterization of 1C6-203, a new monoclonal antibody specific to human thymidine phosphorylase J Histochem Cytochem 2001;49:131–7.[Abstract/Free Full Text]

16 Mori K, Hasegawa M, Nishida M, Toma H, Fukuda M, Kubota T, et al. Expression levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in various human tumor tissues. Int J Oncol 2000;17:33–8.[Web of Science][Medline]

17 Kamoshida S, Shiogama K, Shimomura R, Inada K, Sakurai Y, Ochiai M, et al. Immunohistochemical demonstration of fluoropyrimidine-metabolizing enzymes in various types of cancer. Oncol Rep 2005;14:1223–30.[Web of Science][Medline]

18 Takao S, Akiyama SI, Nakajo A, Toh H, Kitazono M, Natsugoe S, et al. Suppression of metastasis by thymidine phosphorylase inhibitor. Cancer Res 2000;60:5345–8.[Abstract/Free Full Text]

19 Wada S, Yoshimura R, Naganuma T, Yoshida N, Narita K, Ikemoto S. Thymidine phosphorylase levels as a prognostic factor in renal cell carcinoma. BJU int 2003;91:105–8.[CrossRef][Web of Science][Medline]

20 Suzuki K, Morita T, Hashimoto S, Tokue A. Thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF) associated with prognosis in renal cell carcinoma. Urol Res 2001;29:7–12.[CrossRef][Web of Science][Medline]

21 Ishitsuka H, Miwa M, Takemoto K, Fukuoka K, Itoga A, Maruyama HB. Role of uridine phosphorylase for antitumor activity of 5'-deoxy-5-fluorouridine. Gann 1980;71:112–23.[Web of Science][Medline]

22 Miwa M, Nishimura J, Kamiyama T, Ishitsuka H. Conversion of 5'-deoxyfluorouridine to 5-FU by pyrimidine nucleoside phosphorylases in normal and tumor tissues from rodents bearing tumors and cancer patients. Jpn J Cancer Chemother 1997;14:2924–9.

23 Hirano Y, Takayama T, Kageyama S, Ushiyama T, Suzuki K, Fijita K. Thymidine phosphorylase activity in renal cell carcinoma: relationship between histological parameters and chemosensitivity to fluorouracil-related drugs. Urol Res 2002;30:112–15.[CrossRef][Web of Science][Medline]

24 Hirano Y, Takayama T, Kageyama S, Ushiyama T, Suzuki K, Fijita K. Thymidine phoshorylase and dihydropyrimidine dehyrdogenase in renal cell carcinoma: relationship between histological parameters and chemosensitivity to 5-fluorouracil. Eur Urol 2002;43:45–52.

25 Ikemoto S, Sugimura K, Yoshida N, Kuratsukuri K, Wada S, Nakatani T. Comparative antitumor activity of 5-fluorouracil and 5'-deoxy-5-fluorouridine in combination with interferon-alpha in renal cell carcinoma cell lines. Urol Int 2004;73:348–53.[CrossRef][Web of Science][Medline]

26 Uchiyama H, Furukawa T, Okamoto M, Nakajima Y, Matsushita S, Ikeda R, et al. Suppression of thymidine phosphorylase-mediated angiogenesis and 1tumor growth by 2-deoxy-L-ribose. Cancer Res 2002;62:2834–9.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
36/9/564    most recent
hyl063v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Takayama, T.
Right arrow Articles by Ozono, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takayama, T.
Right arrow Articles by Ozono, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?