[Frontiers in Bioscience E3, 194-200, January 1, 2011]
Alterations of respiratory chain complexes in sporadic pheochromocytoma
Rene G. Feichtinger1, Franz A. Zimmermann1, Johannes A. Mayr1, Daniel Neureiter2, Manfred Ratschek3, Neil Jones1, Wolfgang Sperl1, Barbara Kofler1
1
TABLE OF CONTENTS
1. ABSTRACT
Succinate dehydrogenase (SDH) has been associated with carcinogenesis in hereditary pheochromocytoma (PC) and paraganglioma. We investigated if a similar association applies to sporadic pheochromocytoma. No genetic alteration was found in the SDHB, SDHC or SDHD genes of sporadic PC. However, in eight of nine sporadic PCs the SDH activity was, on average, reduced by 40%; moreover, the activities of the other oxidative phosphorylation (OXPHOS) complexes and citrate synthase were significantly lower compared to normal kidney tissue. Furthermore, immunohistochemical staining revealed a significant down-regulation of respiratory chain complexes. Since no pathogenic mutations were detected in the von Hippel-Lindau (VHL) gene, we can rule out that VHL deficiency is causing the general reduction of OXPHOS enzymes observed in the PCs investigated. In contrast to the single enzyme defects found in a subset hereditary PCs, a more generalized reduction of mitochondrial respiration seems to be present in most sporadic PCs. Strikingly, one of the nine PCs showed specific loss of complex I and a compensatory up-regulation of complexes II-V, which is a phenotype usually characteristic of oncocytic tumors.
2. INTRODUCTION
Pheochromocytomas (PCs), paragangliomas (PGLs), neuroblastomas (NBs), ganglioneuroblastomas (GNBs) and ganglioneuromas (GNs) derive from neural-crest cells. Their common origin is underlined by reports of composite PGL-GN, PGL-NB, PGL-GNB and PC-NB (1-3). PCs generally arise from the adrenal medulla, and PGLs from either the sympathetic nervous system or parasympathetic ganglia (4). In contrast to neuroblastic tumors, PCs and PGLs contain mature chromaffin cells. They are usually benign, catecholamine- secreting tumors. Plasma catecholamines, serum chromogranin, urinary free catecholamines, and plasma metanephrins are used in the diagnosis of PC.
PCs are associated with several familial syndromes, including multiple endocrine neoplasia (MEN) type 2A, von Recklinghausen's neurofibromatosis and other endocrinopathies (5). Mutations in the von Hippel Lindau (VHL) gene predispose to a variety of malignant and benign neoplasms, including clear cell renal carcinoma and PC (6-8). Hereditary PGL and PC frequently exhibit germline mutations in the succinate dehydrogenase (SDH) subunits SDHB, SDHC and SDHD, indicating that these SDH subunits act as tumor suppressors in neuroendocrine tissues (9). Carriers of SDH mutations usually develop a tumor earlier in life and have a more severe phenotype than do patients with no germline mutations in these genes. SDHB mutations are often identified in patients with no family history whereas patients with SDHD mutations often have a paternal family history of PC (10). The SDHA subunit is mutated in patients with Leigh syndrome and late-onset optic atrophy, but has not as yet been identified as a factor in hereditary cancer.
However, the majority (~70%) of PCs do not have a detectable mutation in any of the known susceptibility genes and are considered as sporadic tumors (11). Several reports in the past five years have established that approximately 12-16% of apparently sporadic PCs or PGLs carry a germline mutation in one of the well-known susceptibility genes (11-14). Recent biochemical evidence suggests that PC-susceptibility mutations (RET, VHL, SDHB, SDHC, SDHD, NF1) converge into a common signaling defect that abrogates the prolyl hydroxylase Egln3/PHD3-mediated apoptotic response of sympathetic neurons to limiting amounts of nerve growth factor (NGF) during development (15). This model predicts that cells that escape this physiological developmental death would later give rise to PC and PGL.
The use of anaerobic fermentation of glucose as an energy source through glycolysis is a feature common to many solid tumors, in turn leading to a lesser dependence on oxidative phosphorylation (OXPHOS), which is called the Warburg effect (16, 17). The down-regulation of OXPHOS in tumor cells seems to be achieved by different mechanisms. First, profound hypoxia can be the cause of compensatory up-regulation of glycolysis in most tumors. Second, it is becoming more and more evident that the loss of tumor suppressor genes such as VHL and p53 or activation of oncogenes results in down-regulation of OXPHOS (18, 19). Finally, as indicated above, direct inactivation of components of OXPHOS has been detected in a minority of tumors (PC, renal oncocytomas, thyroid oncocytomas) (20-23).
In light of the known alterations of SDH in hereditary PC, the aim of the present study was to elucidate specific alterations of aerobic energy metabolism in sporadic cases of PC.
3. MATERIAL AND METHODS
3.1. Ethics Statement
The study was performed according to the Austrian Gene Technology Act. Experiments were performed in accordance with the Helsinki Declaration of 1975 (revised 1983) and the guidelines of the Salzburg State Ethics Research Committee, being no clinical drug trial or epidemiological investigation. All patients gave informed written consent concerning the surgical removal and therapy of the tumors. Furthermore, the study did not extend to examination of individual case records. The anonymity of the tumor samples has been ensured.
DNA was isolated using a NucleoSpin® Tissue Kit (Macherey-Nagel). 100 ng genomic DNA was used for the PCR amplification. PCR products were incubated with Exo SAP IT 500 (USB Corporation), and the CEQ DTCS Quickstart Kit (Beckman Coulter) was used for the sequencing reaction using the PCR primers listed in Table 1, followed by separation with a CEQ 2000 DNA Analysis System (Beckman Coulter).
3.5. Quantitative PCR for the detection of LOH in SDHB, SDHC or SDHD
Quanta Biosciences B-R SYBRÒ Green SuperMix was used for the quantitative PCR to detect loss of heterozygosity (LOH) in SDHB, SDHC or SDHD. The following PCR protocol was used: 1 cycle: 95�C 3 min; 40 cycles: 95�C 2 sec, 65�C 20 sec, 72�C 10 sec; 1 cycle: 95�C 1 min; 1 cycle: 55�C 1 min. Primers are listed in Table 1.
3.6. Sequencing of mitochondrial complex I and tRNA genes
The primers used for amplification and sequencing are listed in Table 1.
3.7. Western blot analysis
Western blot analysis of 600g homogenates was performed as described previously (24).
3.8. Immunohistochemical staining and analysis
Immunohistochemical staining was performed as described previously (22) using the following antibodies: anti-complex I subunit NDUFS4, anti-complex II subunit 70kDa Fp, anti-complex III subunit core protein 2, mouse monoclonal anti-complex IV subunit I, mouse monoclonal anti-complex V subunit alpha, and mouse monoclonal anti-porin 31HL.
4. RESULTS
Loss of heterozygosity (LOH) in combination with germline SDH mutations are found in a subset of hereditary PC but not sporadic PC. To rule out that the PC tissues used in this study do not also have LOH in SDHB, SDHC and SDHD we analyzed DNA isolated from the tumor tissue by quantitative PCR. None of the tumor tissues had LOH in SDHB, SDHC and SDHD.
Although there was no genetic evidence for specific alteration of SDH, we analyzed the enzymatic activity of SDH in nine tissues of sporadic PC (Table 2). The SDH activity was significantly down-regulated in 8 of 9 PCs (P<0.0001) compared to control kidney tissues. Western blot analysis also revealed a reduction of SDH in eight of nine cases (Figure 1). To test if sporadic PCs are characterized by an isolated SDH defect, we determined the enzymatic activity of the other OXPHOS complexes. A significant down-regulation of the total activity of complex I was found in all cases. Complex III and complex IV activity was also significantly lower in eight of the nine PCs compared to normal adrenal gland and kidney tissue (Table 2). Complex V was less affected. In addition, a significant reduction of citrate synthase activity was observed in eight of the nine PCs compared to normal cortical kidney tissues (Table 2).
Case 8 showed down-regulation of complex I activity with concomitant enormous up-regulation of citrate synthase and the other OXPHOS complex activities (Table 2; Figure 1). Because of the unique biochemical signature we decided to exclude this case from further statistical analysis. The biochemical features of case 8 seem to resemble those of oncocytic tumors (21, 22). Accordingly, case 8 showed a 21-fold increase in citrate synthase activity, a 14-fold increase in complex II activity, a 2-fold increase in complex III activity, a 17-fold increase in complex IV activity, and a 6.5-fold increase in complex V activity compared to the other PCs. In addition, immunoblot analysis showed a severe reduction of the complex I subunit NDUFS4 in case 8, whereas the SDHA subunit was massively up-regulated (Figure 1). Mitochondrial DNA-encoded complex I and tRNA genes were sequenced in case 8, but no pathogenic mutations were found. However, we cannot rule out that a mutation in one of the numerous nuclear complex I genes causes the observed deficiency in case 8.
To substantiate our biochemical analysis we also analyzed further sporadic cases of PC by immunohistochemistry (Figure 2). In agreement with the enzymatic analysis, immunohistochemical staining also revealed a significant reduction of OXPHOS complexes I to IV in comparison to adjacent adrenal gland tissues (P=0.0005). This finding was further underlined by Western blot analysis of complex I and complex II subunits (Figure 1). No significant alterations were found in PC regarding complex V through immunohistochemical staining compared to adjacent adrenal gland (Figure 2; Table 3). This is also in accordance with the observed enzymatic activity of complex V in PC, which is also less severely affected than the activities of the other complexes.
Because only one of the analyzed PCs showed an isolated OXPHOS enzyme defect, we tested if the down-regulation of OXPHOS complexes is due to a general reduction of mitochondria. Therefore, we stained for the mitochondrial membrane protein porin, which can be used as marker for mitochondrial mass. The score values for porin revealed no significant difference between the PC tissues and adjacent adrenal gland (Table 3).
Detailed examination of the immunohistochemical staining at the cellular level revealed that sustentacular cells show strong positive staining for all OXPHOS complexes and porin. In contrast, a reduction or loss of the OXPHOS complexes is observed in the hormone-secreting cells (Figure 2 A - F).
VHL mutations are found in a subset of hereditary PCs. Because VHL is known to regulate degradation of hypoxia-inducible factor (HIF)-1α, which is a master regulator for induction of the glycolytic switch in tumor cells, we sequenced the VHL gene in seven of our sporadic PC cases but did not identify any pathogenic VHL mutations.
5. DISCUSSION
All but one of the nine investigated PCs showed a significant reduction in SDH activity compared to normal cortical kidney tissues. No LOH of SDH subunits was found in the examined samples, which indicates that the observed down-regulation of SDH activity in sporadic PC is not due to pathogenic mutations in genes encoding SDH. Our data are in agreement with those of Asturi et al., who also did not find an association of sporadic PC with SDHB inactivation (25-27). Because those studies did not find inactivation of SDH genes by promoter methylation, we decided not to analyze the SDH genes in more detail.
Like complex II, complexes I, III and IV were also significantly reduced in sporadic PC while the tumor mitochondria showed normal amounts of porin. Therefore, down-regulation of the respiratory chain is not due to a more general decrease in mitochondrial mass. The expression of the OXPHOS complexes is significantly reduced in the hormone-secreting cells of the PC, whereas the sustentacular cells show a strong staining for all OXPHOS complexes (Figure 2). Because absence of S-100 positive sustentacular cells was demonstrated in the majority of malignant PCs (28), the low presence of these cells should not influence the overall metabolic phenotype of PC.
We performed an immunohistochemical analysis on normal adrenal tissue and were able to show that cells of the adrenal cortex and medulla, including chromaffin cells, have substantial staining of porin and OXPHOS complexes. This indicates clearly the down-regulation of OXPHOS complexes during tumor transformation and progression.
Although somatic inactivation of VHL occurs in most sporadic renal cell carcinomas it has been reported that this event is infrequent in sporadic PC (27). Accordingly, we could not detect VHL mutations in our samples. Therefore, VHL mutations can be excluded as being responsible for the low OXPHOS in our samples. Recently, it was shown that p53 gene mutations are frequently found in multiple and malignant PCs (29). p53 is an important regulator of mitochondrial energy metabolism and glycolysis (30). Lebedeva et al. found that p53 null mice and p53 knockdown human primary fibroblasts exhibit mitochondrial DNA depletion and reduced mitochondrial mass (31). However, this is unlikely to be the mechanism of OXPHOS down-regulation in the PCs analyzed in the present study, because we did not observe a substantial down-regulation of mitochondrial mass. The down-regulation of the OXPHOS complexes without reduction of mitochondrial mass has also been observed in undifferentiated NB, another neural crest-derived tumor (24).
One case stood out biochemically by exhibiting a phenotype characteristic for oncocytic tumors (Zimmermann 2010, this issue). Interestingly, histologically, no signs of oncocytic features could be found in this case (data not shown), which is in
contrast to what has been observed in other tumors with specific complex I deficiency and compensatory up-regulation of the citrate synthase and OXPHOS complexes II-V (Zimmermann 2010, this issue). This is, to our knowledge, the first case of a PC with a specific defect in complex I of aerobic energy metabolism.
In summary, in most sporadic PCs, low aerobic energy metabolism does not stem from a defect of a single complex of the respiratory chain, as it does in a subset of hereditary PCs. However, in the majority of sporadic PCs the activity of the respiratory chain on the whole is reduced. Notwithstanding the clinical uniformity of PCs, impairment of aerobic energy metabolism in PC seems to be achieved via a variety of routes, in view of the miscellaneous genetic and biochemical alterations found in hereditary and sporadic PCs.
6. ACKNOWLEDGMENTS
This work was supported by the Children's Cancer Foundation Salzburg and the "Vereinigung zur Foerderung Paediatrischer Forschung und Fortbildung".
7. REFERENCES
1. P. Dundr, D. Dudorkinova, C. Povysil, M. Pesl, M. Babjuk, J. Dvoracek and T. Zelinka: Pigmented composite paraganglioma-ganglioneuroma of the urinary bladder. Pathol Res Pract, 199(11), 765-9 (2003)
doi:10.1078/0344-0338-00495
2. F. Donadio, V. La Ganga, M. Vajo, G. Campanella and S. Coverlizza: (Ganglioneuroblastoma with areas of pheochromocytoma of the adrenal gland. Case report). Minerva Chir, 37(6), 551-8 (1982)
PMid:7088362
3. D. W. Franquemont, S. E. Mills and E. E. Lack: Immunohistochemical detection of neuroblastomatous foci in composite adrenal pheochromocytoma-neuroblastoma. Am J Clin Pathol, 102(2), 163-70 (1994)
PMid:8042583
4. J. Bryant, J. Farmer, L. J. Kessler, R. R. Townsend and K. L. Nathanson: Pheochromocytoma: the expanding genetic differential diagnosis. J Natl Cancer Inst, 95(16), 1196-204 (2003)
PMid:12928344
5. Y. Perel, M. Schlumberger, G. Marguerite, N. Alos, Y. Revillon, D. Sommelet, L. De Lumley, F. Flamant, J. F. Dyon, P. Lutz, H. Heloury and J. Lemerle: Pheochromocytoma and paraganglioma in children: a report of 24 cases of the French Society of Pediatric Oncology. Pediatr Hematol Oncol, 14(5), 413-22 (1997)
doi:10.3109/08880019709028771
6. H. Kanno, K. Kondo, S. Ito, I. Yamamoto, S. Fujii, S. Torigoe, N. Sakai, M. Hosaka, T. Shuin and M. Yao: Somatic mutations of the von Hippel-Lindau tumor suppressor gene in sporadic central nervous system hemangioblastomas. Cancer Res, 54(18), 4845-7 (1994)
PMid:8069849
7. E. R. Maher, L. Iselius, J. R. Yates, M. Littler, C. Benjamin, R. Harris, J. Sampson, A. Williams, M. A. Ferguson-Smith and N. Morton: Von Hippel-Lindau disease: a genetic study. J Med Genet, 28(7), 443-7 (1991)
doi:10.1136/jmg.28.7.443
PMid:1895313 PMCid:1016952
8. T. Shuin, K. Kondo, S. Torigoe, T. Kishida, Y. Kubota, M. Hosaka, Y. Nagashima, H. Kitamura, F. Latif, B. Zbar and et al.: Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res, 54(11), 2852-5 (1994)
PMid:8187067
9. J. J. Briere, J. Favier, A. P. Gimenez-Roqueplo and P. Rustin: Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumor formation. Am J Physiol Cell Physiol, 291(6), C1114-20 (2006)
doi:10.1152/ajpcell.00216.2006
PMid:16760265
10. A. P. Gimenez-Roqueplo, H. Lehnert, M. Mannelli, H. Neumann, G. Opocher, E. R. Maher and P. F. Plouin: Phaeochromocytoma, new genes and screening strategies. Clin Endocrinol (Oxf), 65(6), 699-705 (2006)
doi:10.1111/j.1365-2265.2006.02714.x
PMid:17121518
11. A. P. Gimenez-Roqueplo, N. Burnichon, L. Amar, J. Favier, X. Jeunemaitre and P. F. Plouin: Recent advances in the genetics of phaeochromocytoma and functional paraganglioma. Clin Exp Pharmacol Physiol, 35(4), 376-9 (2008)
doi:10.1111/j.1440-1681.2008.04881.x
PMid:18307724
12. B. Bausch, C. C. Boedeker, A. Berlis, I. Brink, M. Cybulla, M. K. Walz, A. Januszewicz, C. Letizia, G. Opocher, C. Eng and H. P. Neumann: Genetic and clinical investigation of pheochromocytoma: a 22-year experience, from Freiburg, Germany to international effort. Ann N Y Acad Sci, 1073122-37 (2006)
13. F. M. Brouwers, A. G. Elkahloun, P. J. Munson, G. Eisenhofer, J. Barb, W. M. Linehan, J. W. Lenders, R. De Krijger, M. Mannelli, R. Udelsman, I. T. Ocal, B. L. Shulkin, S. R. Bornstein, J. Breza, L. Ksinantova and K. Pacak: Gene expression profiling of benign and malignant pheochromocytoma. Ann N Y Acad Sci, 1073541-56 (2006)
14. H. Brauch, W. Hoeppner, H. Jahnig, T. Wohl, D. Engelhardt, F. Spelsberg and M. M. Ritter: Sporadic pheochromocytomas are rarely associated with germline mutations in the vhl tumor suppressor gene or the ret protooncogene. J Clin Endocrinol Metab, 82(12), 4101-4 (1997)
doi:10.1210/jc.82.12.4101
15. S. Lee, E. Nakamura, H. Yang, W. Wei, M. S. Linggi, M. P. Sajan, R. V. Farese, R. S. Freeman, B. D. Carter, W. G. Kaelin, Jr. and S. Schlisio: Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. Cancer Cell, 8(2), 155-67 (2005)
doi:10.1016/j.ccr.2005.06.015
PMid:16098468
16. P. L. Pedersen: Tumor mitochondria and the bioenergetics of cancer cells. Prog Exp Tumor Res, 22190-274 (1978)
17. E. Racker and M. Spector: Warburg effect revisited: merger of biochemistry and molecular biology. Science, 213(4505), 303-7 (1981)
doi:10.1126/science.6264596
PMid:6264596
18. S. Matoba, J. G. Kang, W. D. Patino, A. Wragg, M. Boehm, O. Gavrilova, P. J. Hurley, F. Bunz and P. M. Hwang: p53 regulates mitochondrial respiration. Science, 312(5780), 1650-3 (2006)
doi:10.1126/science.1126863
PMid:16728594
19. H. Zhang, P. Gao, R. Fukuda, G. Kumar, B. Krishnamachary, K. I. Zeller, C. V. Dang and G. L. Semenza: HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell, 11(5), 407-20 (2007)
doi:10.1016/j.ccr.2007.04.001
PMid:17482131
20. D. Astuti, F. Douglas, T. W. Lennard, I. A. Aligianis, E. R. Woodward, D. G. Evans, C. Eng, F. Latif and E. R. Maher: Germline SDHD mutation in familial phaeochromocytoma. Lancet, 357(9263), 1181-2 (2001)
doi:10.1016/S0140-6736(00)04378-6
21. J. A. Mayr, D. Meierhofer, F. Zimmermann, R. Feichtinger, C. Kogler, M. Ratschek, N. Schmeller, W. Sperl and B. Kofler: Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma. Clin Cancer Res, 14(8), 2270-5 (2008)
doi:10.1158/1078-0432.CCR-07-4131
PMid:18413815
22. F. A. Zimmermann, J. A. Mayr, D. Neureiter, R. Feichtinger, B. Alinger, N. D. Jones, W. Eder, W. Sperl and B. Kofler: Lack of complex I is associated with oncocytic thyroid tumours. Br J Cancer, 100(9), 1434-7 (2009)
doi:10.1038/sj.bjc.6605028
PMid:19352385 PMCid:2694433
23. V. Maximo, J. Lima, P. Soares and M. Sobrinho-Simoes: Mitochondria and cancer. Virchows Arch, 454(5), 481-95 (2009)
doi:10.1007/s00428-009-0766-2
PMid:19343360
24. R. G. Feichtinger, F. Zimmermann, J. A. Mayr, D. Neureiter, C. Hauser-Kronberger, F. H. Schilling, N. Jones, W. Sperl and B. Kofler: Low aerobic mitochondrial energy metabolism in poorly- or undifferentiated neuroblastoma. BMC Cancer, 10149
25. D. E. Benn, M. S. Croxson, K. Tucker, C. P. Bambach, A. L. Richardson, L. Delbridge, P. T. Pullan, J. Hammond, D. J. Marsh and B. G. Robinson: Novel succinate dehydrogenase subunit B (SDHB) mutations in familial phaeochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic phaeochromocytomas. Oncogene, 22(9), 1358-64 (2003)
doi:10.1038/sj.onc.1206300
PMid:12618761
26. D. Astuti, F. Latif, A. Dallol, P. L. Dahia, F. Douglas, E. George, F. Skoldberg, E. S. Husebye, C. Eng and E. R. Maher: Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet, 69(1), 49-54 (2001)
doi:10.1086/321282
PMid:11404820
27. D. Astuti, M. Morris, C. Krona, F. Abel, D. Gentle, T. Martinsson, P. Kogner, H. P. Neumann, R. Voutilainen, C. Eng, P. Rustin, F. Latif and E. R. Maher: Investigation of the role of SDHB inactivation in sporadic phaeochromocytoma and neuroblastoma. Br J Cancer, 91(10), 1835-41 (2004)
doi:10.1038/sj.bjc.6602202
PMid:15505628 PMCid:2410049
28. P. Unger, K. Hoffman, D. Pertsemlidis, S. Thung, D. Wolfe and M. Kaneko: S100 protein-positive sustentacular cells in malignant and locally aggressive adrenal pheochromocytomas. Arch Pathol Lab Med, 115(5), 484-7 (1991)
PMid:1673596
29. T. Yoshimoto, M. Naruse, Z. Zeng, T. Nishikawa, T. Kasajima, H. Toma, S. Yamamori, H. Matsumoto, A. Tanabe, K. Naruse and H. Demura: The relatively high frequency of p53 gene mutations in multiple and malignant phaeochromocytomas. J Endocrinol, 159(2), 247-55 (1998)
doi:10.1677/joe.0.1590247
PMid:9795365
30. W. Ma, H. J. Sung, J. Y. Park, S. Matoba and P. M. Hwang: A pivotal role for p53: balancing aerobic respiration and glycolysis. J Bioenerg Biomembr, 39(3), 243-6 (2007)
doi:10.1007/s10863-007-9083-0
PMid:17551815
31. M. A. Lebedeva, J. S. Eaton and G. S. Shadel: Loss of p53 causes mitochondrial DNA depletion and altered mitochondrial reactive oxygen species homeostasis. Biochim Biophys Acta, 1787(5), 328-34 (2009)
doi:10.1016/j.bbabio.2009.01.004
PMid:19413947 PMCid:2680458
Key Words: Respiratory Chain, Oxidative Phosphorylation, Energy Metabolism, Succinate Dehydrogenase
Send correspondence to: Barbara Kofler, Department of Pediatrics, University Hospital Salzburg, Paracelsus Medical University, Muellner Hauptstr. 48, A-5020 Salzburg, Austria Tel: 43-662-4482-4741, Fax: 43-662-4482-4765, E-mail:b.kofler@salk.at