Involvement of Na+-Ca2+ exchanger in intracellular Ca2+ increase and neuronal injury induced by polychlorinated biphenyls in human neuroblastoma SH-SY5Y cells.

In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 microg/ml) intracellular calcium concentration ([Ca2+]i) increase. Two rather specific sodium-calcium (Na+-Ca2+) exchanger (NCX) inhibitors, bepridil (10 microM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 microM), reduced A1254-induced [Ca2+]i increase. A 24-h exposure to 30 microg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca2+]i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca2+]i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca2+]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca2+]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells.

The G/C915 polymorphism of transforming growth factor beta1 is associated with human longevity: a study in Italian centenarians.

Sequence variations in a variety of pro- or anti-inflammatory cytokine genes have been found to influence successful aging and longevity. Because of the role played by the transforming growth factor beta1 (TGF-beta1) cytokine in inflammation and regulation of immune responses, the variability of the TGF-beta1 gene may affect longevity by playing a role in inflamm-aging. Two polymorphisms, G/A -800 and C/T -509, located in the 5' region, and two missense polymorphisms, T/C 869 and G/C 915 which change (Leu > Pro)10 and (Arg > Pro)25, respectively, located in the signal peptide, were analysed in 419 subjects from Northern and Central Italy, including 172 centenarians and 247 younger controls. In addition, the effects of the TGF-beta1 genetic variability on plasma levels of the biologically active form (naturally processed) of this cytokine were studied in 143 randomly selected subjects, including 73 centenarians. Significant differences were found at the +915 site as far as the C allele and GC genotype were concerned, both of them being lower in centenarians than in young controls (P=0.034 and 0.028, respectively), but none of the other tested genetic variants was significantly different between centenarians and controls. Moreover, a particular haplotype combination (G -800/C -509/C 869/C 915) was notably lower in centenarians than in younger individuals (P=0.007). Finally, active TGF-beta1 plasma levels were significantly increased in the elderly group, but no relationship with TGF-beta1 genotypes was observed. These results suggest that, at least in this population, the variability of the TGF-beta1 gene influences longevity and that the age-related increase in plasma levels of active TGF-beta1 seems not to be genetically regulated.

A novel mitochondrial DNA-like sequence insertion polymorphism in Intron I of the FOXO1A gene.

The human forkhead box O1A (FOXO1A) gene belongs to the human forkhead gene family and acts downstream of the human insulin signalling pathway. In this study, polymorphisms of the Intron I of FOXO1A gene were studied in Italian healthy people and insulin resistant subjects. No significant association between the germ-line variability in the Intron I of FOXO1A and insulin resistance was observed. Interestingly, during the study, a new 39-bp sequence insertion polymorphism in Intron I of FOXO1A gene was described. The polymorphism was found to co-segregate in a co-dominant Mendelian fashion and to be present in an ethnically distinct population (Greeks). A BLAST search showed that the sequence shares 100% identity with a mtDNA (mitochondrial DNA) sequence coding for the ATP synthase 8 (ATPase8) and ATP synthase 6 (ATPase6) genes. Hence, FOXO1A Intron I is a polymorphic nuclear region involved in the exchange of DNA material between mitochondrial and genomic DNA, which is a well-established mechanism of evolutionary change in eukaryotes.

In vitro IL-6 production by EBV-immortalized B lymphocytes from young and elderly people genotyped for -174 C/G polymorphism in IL-6 gene: a model to study the genetic basis of inflamm-aging.

In the present investigation we analysed Interleukin 6 (IL-6) in vitro production by Epstein-Barr virus (EBV)-immortalized B lymphocytes established from 43 subjects, 15 young people and 28 elderly people, including 18 centenarians, after 3, 6, 9, 24, 48 and 72 h of culture. The subjects were genotypized for the C to G transition at nucleotide -174 of IL-6 gene promoter (-174 C/G) and were classified as C allele carriers (C+) and non-carriers (C-). We found that: (i) the interindividual difference in in vitro IL-6 production was wider in elderly individuals in respect to young individuals, leading to different coefficient of variation in the two groups; (ii) the -174 C/G polymorphism had an age-related effect on IL-6 in vitro production. Only among C- people, cells from elderly subjects produced significant higher level of IL-6 than cells from young subjects. These data are consistent with our previous results regarding the IL-6 serum levels in a large group of people of different age, including centenarians. Thus, the EBV-immortalized B lymphocytes can be considered a useful in vitro model for studying the genetic control of IL-6 production and its changes with age.

An allele of HRAS1 3'variable number of tandem repeats is a frailty allele: implication for an evolutionarily-conserved pathway involved in longevity.

The human HRAS1 belongs to an evolutionarily-conserved family of genes which enrolls among its members the yeast RAS2, a gene which regulates stress response and longevity in the Saccharomyces cerevisiae. In this paper we report that the frequency of the a3 allele of HRAS1 3'variable number tandem repeat (HRAS1 3'VNTR) decreases in centenarians in respect to young people, and we estimate that during aging a3 carriers have a relative mortality risk of 1.126 (95% CI=1.044-1.213). We propose that the germ-line variability at the HRAS1 locus impacts on the individual's capacity to reach the extreme limits of human life-span. Furthermore, we provide suggestive evidence that a3 HRAS1 3'VNTR allele and inherited variants of the mitochondrial genome (mtDNA haplogroups) do not affect independently human longevity, thus recalling the nucleus-mitochondrion interaction which regulates stress response and life-span in the yeast.