human lymphocytes and up-regulated by zinc (Vandeghinste et al. 2000), with a task of anti- oxidant agent because zinc–sulphur clusters are sensitive to changes in cellular redox state and oxidizing sites, inducing transfer of zinc by MTs to other proteins, such as antioxidant enzymes (Maret and Vallee 1998). Since high MT gene expression in lymphocytes is associated with zinc deficiency in ageing (Mocchegiani et al. 2003), this transfer may be very limited in ageing leading to suggest a different role of MT: from protective as antioxidant agent in young adult-age (Kelly et al. 1996), to a dangerous role in ageing because of the inability of zinc release by MT (Mocchegiani et al. 2002). Indeed, a lack of zinc ion bioavailability in ageing allows a derangement of the immune response as well as impaired antioxidant activity (Mocchegiani et al. 1998). Because MT induction and expression are also under the control of IL-6 (Jacob et al. 1999), this phenomenon of the limited zinc release by MT in ageing is strictly related to the chronic inflam- matory status due to high circulating levels of IL- 6 (Mocchegiani et al. 2003). On the other hand, abnormal high MTs, enhanced IL-6 and low zinc ion bioavailability are also present in diseases characterised by chronic inflammation (infec- tions) and in syndrome of accelerated ageing (Down’s syndrome) (Mocchegiani et al. 2002, 2003). By contrast, low MTs, limited inflamma- tory condition and satisfactory zinc ion bioavail- ability occur in successful ageing (Mocchegiani et al. 2002). These findings clearly suggest that the limited zinc release by MT in ageing and chronic inflammation is a crucial problem for the efficiency of the immune system and antioxidant

study is to screen these two single nucleotide polymorphisms in the MT1a coding region from healthy Italian old female. This may be an useful tool for better understanding the genetic influ- ences of MT1a on the attainment of successful ageing. Moreover, their allelic frequency distri- bution was studied in association with intracellular zinc ion bioavailability and zinc release by MTs using specific fluorescent zinc probe (Zinpyr-1) and NO donor (Dyethilamine-NOnoate acet- oxymethylated) (AcOM-DEA/NO), because NO is involved in zinc release by MTs via s-nitrosyla- tion (Zangger et al. 2001).

Materials and methods

Subjects

N. 251 healthy old and very old female were enrolled for the study. Since demographic data document a clear-cut prevalence of females compared to males in people of far advanced age (Franceschi and Bonafe 2003), we have analysed old female population. All the subjects, n. 151 old (mean age 73.8 ± 5.9) and n. 100 nonagenarians (mean age 91.4 ± 4.1) were born in Central Italy and still living in the same area at the time of enrolment. They were judged to be in good health based on their clinical history and blood tests. The

healthy old subjects were recruited in accor- dance with immunogerontological ‘‘SENIEUR’’ protocol (Ligthart 2001). Nonagenarians were recruited according to the grade of activities of daily living. They were independent, did not re- quire special daily care and did not take any therapeutic treatment for at least 2 months before the enrolment.

Amplification was accomplished by initial incu- bation at 94(cid:2)C for 5 min; the following 35 cycles were at 94(cid:2)C for 1 min, 60(cid:2)C for 30 s, 72(cid:2)C for 1 min and a final extention at 72(cid:2)C for 7 min. The products of PCR were digested with Pst-I at 37(cid:2)C overnight (Roche, Germany), then separated on 3% agarose gel and stained with ethidium bro- mide. The presence of adenine (A allele) at locus 1,245 within the coding region of MT1A produced one fragment of 390 bp, whereas the presence of guanine (G allele) generated two fragments of 199 bp and 191 bp after Pst-I digestion.

MT-1a SNPs in the coding region

We screened two single-nucleotide polymor- phisms (SNPs) found in dbSNP database (http:// www.ncbi.nlm.nih.gov/SNP/) (PubMed Reference rs 8052394; rs 11640851) corresponding one to a A/G transition at +1245 nt position in the coding region of human MT1a gene and one to A/C transition at +647 nt position in the coding region of the same gene. The first polymorphism corre- sponded to amino acid transition from lysine in the MT1a-A(adenine) to arginine in the MT1a-G (guanosine); while the second one corresponded to amino acid transition from asparagine in the MT1a-A (adenine) to threonine in the MT1a-C (cytosine). We performed a PCR-restriction fragment length analysis (PCR-RFLP) using the following primers: +1245 A/G MT1a sense 5¢-ATCTCCCATCTCCGACAGTG-3¢ antisense 5¢-AAAGAAAACCAGAGCCAG-

Twenty-five microlitres of PCR mixture was composed of genomic DNA samples (200 ng), 300 lmol/l dNTPs, 2 mM MgCl , 1· Taq poly- 2 merase buffer, 10 pmol of each primer, 5% of DMSO, 2.5 U of Taq DNA polymerase (Eppen- dorf, Germany). Amplification was accomplished by initial incubation at 94(cid:2)C for 4 min; the fol- lowing 40 cycles were at 94(cid:2)C for 1 min, 60(cid:2)C for 30 s, 72(cid:2)C for 1 min and a final extention at 72(cid:2)C for 4 min. The products of PCR were digested with Mnl-I at 37(cid:2)C overnight (Roche, Germany), then separated on 3% agarose gel and stained with ethidium bromide. The presence of adenine (A allele) at locus 647 within the coding region of MT1A produced one fragment of 187 bp, whereas the presence of citosine (C allele) gen- erated two fragments of 140 bp and 47 bp after Mnl-I digestion.

Genomic DNA from peripheral blood leuko- cytes taken from old and nonagenarian healthy subjects, extracted by the phenol chloroform method, was amplified with the primers described above. Conditions for amplification were as follows.

Haplotype analysis