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with restriction endonuclease enzymes Tsp45I and Bsu36I (New England Biolabs), respectively, and the samples were electro- phoresed in 2% agarose gels.

The amplification reaction for the polymorphisms in the signal peptide region was performed on a Biometra thermal cycler with initial denaturation at 96.5 °C for 10 min, followed by 30 cycles, each one comprising denaturation at 96.5 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1 min, with a final extension time of 1 min at 72 °C. The amplified PCR fragments for markers +869 and +915 were digested with restriction endonuclease enzymes MspA1 I and BgII (New England Biolabs), respectively, and the samples were electrophoresed in 10% polyacrylamide gels 30% (19 : 1 acrylamide–polyacrylamide).

Immunoassay for bio-active TGF-ββββ 1

Plasma samples were available from 70 young controls (22 males and 48 females) and from 73 centenarians (24 males and 49 females). They were prepared from 15 mL of fresh EDTA- treated venous blood centrifuged at 960 g for 20 min at 4 °C and stored at −70 °C until the assay. Bio-active TGF-β1 plasma levels were quantified with a TGF-β1 Immunoassay (Promega System, Italy). To measure the amount of naturally processed TGF-β1 in plasma samples, we proceeded directly to the ELISA protocol without acid treatment, according to the manufac- turer’s instructions.

Statistical analysis

Allele and genotype frequency distributions were compared between young and centenarian groups by the Monte-Carlo χ2 test. Hardy–Weinberg equilibrium (HWE) was checked by Monte Carlo Markov Chain analysis (Guo & Thompson, 1992). Haplo- type frequencies were estimated by using an Expectation Maximization (EM) algorithm, implemented with the Arlequin package (Schneider et al., 2000). A general linear model (imple- mented in the SPSS Package) was used to assess the age effects on plasma levels.

Acknowledgments

This work was partially supported by grants from the Italian Min- istry of Health Progetto Finalizzato 2002 Studio Multicentrico sui ‘Determinanti Genetici e non Genetici di Salute nell’Età Avanzata’, European Union Grants FUNCTIONAGE & ECHA, T- CIA & Italian Ministry of Education, University & Research PRIN 2003. We thank Drs M. Bonafè and S. Salvioli for helpful discussions.

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