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The extraordinary increase of the elderly in developed countries underscore the importance of studies on ageing and longevity and the need for the prompt spread of knowledge about ageing in order to satisfactorily decrease the medical, economic and social problems associated to advancing years, because of the increased number of individuals not autonomous and affected by invalidating pathologies. Centenarians are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases. Thus, they are the best example of extreme longevity, representing selected people in which the appearance of major age-related diseases, such as cancer, and cardiovascular diseases among others, has been consistently delayed or escaped. To discuss the relevance of genetics and life style in the attainment of longevity, five papers mostly focused on Italian centenarians have been assembled in this series. The aim is to realize, through a” positive biology” approach (rather than making diseases the central focus of research, “positive biology” seeks to understand the causes of positive phenotypes, trying to explain the biological mechanisms of health and well-being) how to prevent and/or reduce elderly frailty and disability.

The average life-span of the population of industrialized countries has improved enormously over the last decades. Despite evidence pointing to the role of food intake in modulating life-span, exceptional longevity is still considered primarily an inheritable trait, as pointed out by the description of families with centenarian clusters and by the elevated relative probability of siblings of centenarians to become centenarians themselves. However, rather than being two separate concepts, the genetic origin of exceptional longevity and the more recently observed environment-driven increase in the average age of the population could possibly be explained by the same genetic variants and environmentally modulated mechanisms (caloric restriction, specific nutrients). In support of this hypothesis, polymorphisms selected for in the centenarian population as a consequence of demographic pressure have been found to modulate cellular signals controlled also by caloric restriction. Here, we give an overview of the recent findings in the field of the genetics of human exceptional longevity, of how some of the identified polymorphisms modulate signals also influenced by food intake and caloric restriction, of what in our view have been the limitations of the approaches used over the past years to study genetics (sib-pair-, candidate gene association-, and genome-wide association-studies), and briefly of the limitations and the potential of the new, high-throughput, next-generation sequencing techniques applied to exceptional longevity.

Aging is the sum of the deleterious changes that occur as time goes by. It is the main risk factor for the development of cardiovascular disease, and aging of the vasculature is the event that most often impacts on the health of elderly people. The “free-radical theory of aging” was proposed to explain aging as a consequence of the accumulation of reactive oxygen species (ROS). However, recent findings contradict this theory, and it now seems that mechanisms mediating longevity act through induction of oxidative stress. In fact, calorie restriction − a powerful way of delaying aging − increases ROS accumulation due to stimulation of the basal metabolic rate; moreover, reports show that antioxidant therapy is detrimental to healthy aging. We also now know that genetic manipulation of the insulin-like-growth-factor-1/insulin signal (IIS) has a profound impact on the rate of aging and that the IIS is modulated by calorie restriction and physical exercise. The IIS regulates activation of nitric oxide synthase (eNOS), the activity of which is essential to improving lifespan through calorie restriction, as demonstrated by experiments on eNOS knockout mice. Indeed, eNOS has a key role in maintaining vascular integrity during aging by activating vasorelaxation and allowing migration and angiogenesis. In this review, we will overview current literature on these topics and we will try to convince the reader of the importance of vascular integrity and nitric oxide production in determining healthy aging.

Background The expression of calcium/calmodulin‐dependent kinase IV ( CaMKIV ) was hitherto thought to be confined to the nervous system. However, a recent genome‐wide analysis indicated an association between hypertension and a single‐nucleotide polymorphism ( rs10491334 ) of the human CaMKIV gene ( CaMK4 ) , which suggests a role for this kinase in the regulation of vascular tone. Methods and Results To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4 −/− mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild‐type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. Conclusions Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity.

1. Fisher EA, Ginsberg HN. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins. J Biol Chem 2002;277: 17377–80. 2. Karpe F, Lundahl B, Ehrenborg E, et al. A common functional polymorphism in the promoter region of the microsomal triglyceride transfer protein gene influences plasma LDL levels. Arterioscler Thromb Vasc Biol 1998; 18:756–61. 3. Geesaman BJ, Benson E, Brewster SJ, et al. Haplotypebased identification of a microsomal transfer protein marker associated with the human lifespan. Proc Natl Acad Sci USA 2003;100:14115–20. 5. Barzilai N, Atzmon G, Schechter C, et al. Unique lipoprotein phenotype and genotype associated with exceptional longevity. JAMA 2003;290:2030–40. 6. Agarwal KC. Therapeutic actions of garlic constituents. Med Res Rev 1996;16:111–24. 7. Berthold HK, Sudhop T. Garlic preparations for prevention of atherosclerosis. Curr Opin Lipidol 1998;9: 565–9. 8. Lin MC, Wang EJ, Lee C, et al. Garlic inhibits microsomal triglyceride transfer protein gene expression in human liver and intestinal cell lines and in rat intestine. J Nutr 2002;132:1165–8. 9. Glueck CJ, Fallat RW, Millett F, et al. Familial hyperalpha-lipoproteinemia: studies in eighteen kindreds. Metabolism 1975;24:1243–65. 10. Groenendijk M, Cantor RM, de Bruin TW, et al. The apoAI-CIII-AIV gene cluster. Atherosclerosis 2001;157: 1–11. 11. Wang CS, McConathy WJ, Kloer HU, et al. Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-III. J Clin Invest 1985;75: 384–90. 12. Chhabra S, Narang R, Lakshmy R, et al. Apolipoprotein C3 SstI polymorphism in the risk assessment of CAD. Mol Cell Biochem 2004;259:59–66. 13. Olivieri O, Bassi A, Stranieri C, et al. Apolipoprotein C-III, metabolic syndrome, and risk of coronary artery disease. J Lipid Res 2003;44:2374–81. 14. Atzmon G, Rincon M, Schechter CB, et al. Lipoprotein genotype and conserved pathway for exceptional longevity in humans. PLoS Biol 2006;4:e113. 15. Olivieri O, Martinelli N, Sandri M, et al. Apolipoprotein C-III, n-3 polyunsaturated fatty acids, and “insulinresistant” T-455C APOC3 gene polymorphism in heart disease patients: example of gene-diet interaction. Clin Chem 2005;51:360–7. 16. Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 1996;37:907–25. 17. Humphries SE, Talmud PJ, Cox C, et al. Genetic factors affecting the consistency and magnitude of changes in plasma cholesterol in response to dietary challenge. QJM 1996;89:671–80. 18. Lopez-Miranda J, Jansen S, Ordovas JM, et al. Influence of the SstI polymorphism at the apolipoprotein C-III gene locus on the plasma low-density-lipoproteincholesterol response to dietary monounsaturated fat. Am J Clin Nutr 1997;66:97–103. 19. Perez-Martinez P, Ordovas JM, Lopez-Miranda J, et al. Polymorphism exon 1 variant at the locus of the

Long-living individuals (LLIs) are used to study exceptional longevity. A number of genetic variants have been found associated in LLIs to date, but further identification of variants would improve knowledge on the mechanisms regulating the rate of aging. Therefore, we performed a genome-wide association study on 410 LLIs and 553 young control individuals with a 317K single-nucleotide polymorphism (SNP) chip to identify novel traits associated with aging. Among the top (p

Healthy aging is thought to reflect the combined influence of environmental factors (lifestyle choices) and genetic factors. To explore the genetic contribution, we undertook a genome-wide association study of exceptional longevity (EL) in 1055 centenarians and 1267 controls. Using these data, we built a genetic model that includes 150 single-nucleotide polymorphisms (SNPs) and found that it could predict EL with 77% accuracy in an independent set of centenarians and controls. Further in silico analysis revealed that 90% of centenarians can be grouped into 19 clusters characterized by different combinations of SNP genotypes—or genetic signatures—of varying predictive value. The different signatures, which attest to the genetic complexity of EL, correlated with differences in the prevalence and age of onset of age-associated diseases (e.g., dementia, hypertension, and cardiovascular disease) and may help dissect this complex phenotype into subphenotypes of healthy aging.

Paraoxonase 1 (PON1) has been suggested as a plausible candidate gene for human longevity due to its modulation of cardiovascular disease risk, by preventing oxidation of atherogenic low-density lipoprotein. The role of the PON1 192 QJR polymorphism has been analyzed for association with survival at old age in several populations, albeit with controversial results. To reconcile the conflicting evidence, we performed a large association study with two samples of 2357 Germans and 1025 French, respectively. We combined our results with those from seven previous studies in the largest and most comprehensive meta-analysis on PON1 192 Q/R and longevity to-date, to include a total of 9580 individuals. No significant association of PON1 192 Q/R with longevity was observed, for either R allele or carriership. This finding relied on very large sample sizes, is supported by different analysis methods and is therefore considered very robust. Moreover, we have investigated a potential interaction of PON1 192 Q/R with APOE epsilon 4 using data from four populations. Whereas a significant result was found in the German sample, this could not be confirmed in the other examined groups. Our large-scale meta-analysis provided no evidence that the PON1 192 Q/R polymorphism is associated with longevity, but this does not exclude the possibility of population-specific effects due to the influence of, and interaction between, different genetic and/or environmental factors (e.g. diet). (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Background The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered. Methodology/Principal Findings Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function. Conclusions/Significance Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan.

Background Bayesian networks are powerful instruments to learn genetic models from association studies data. They are able to derive the existing correlation between genetic markers and phenotypic traits and, at the same time, to find the relationships between the markers themselves. However, learning Bayesian networks is often non-trivial due to the high number of variables to be taken into account in the model with respect to the instances of the dataset. Therefore, it becomes very interesting to use an abstraction of the variable space that suitably reduces its dimensionality without losing information. In this paper we present a new strategy to achieve this goal by mapping the SNPs related to the same gene to one meta-variable. In order to assign states to the meta-variables we employ an approach based on classification trees. Results We applied our approach to data coming from a genome-wide scan on 288 individuals affected by arterial hypertension and 271 nonagenarians without history of hypertension. After pre-processing, we focused on a subset of 24 SNPs. We compared the performance of the proposed approach with the Bayesian network learned with SNPs as variables and with the network learned with haplotypes as meta-variables. The results were obtained by running a hold-out experiment five times. The mean accuracy of the new method was 64.28%, while the mean accuracy of the SNPs network was 58.99% and the mean accuracy of the haplotype network was 54.57%. Conclusion The new approach presented in this paper is able to derive a gene-based predictive model based on SNPs data. Such model is more parsimonious than the one based on single SNPs, while preserving the capability of highlighting predictive SNPs configurations. The prediction performance of this approach was consistently superior to the SNP-based and the haplotype-based one in all the test sets of the evaluation procedure. The method can be then considered as an alternative way to analyze the data coming from association studies.

There was an error in affiliation assignment for author Annibale Puca. This author should also be affiliated with: Istituto di Tecnologie Biomediche - Consiglio Nazionale delle Ricerche, Segrate, Italy.

A number of potential candidate genes in a variety of biological pathways have been associated with longevity in model organisms. Many of these genes have human homologs and thus have the potential to provide insights into human longevity. Recently, several studies suggested that FOXO3A functions as a key bridge for various signaling pathways that influence aging and longevity. Interestingly, Willcox and colleagues identified several variants that displayed significant genotype-gender interaction in male human longevity. In particular, a nested case-control study was performed in an ethnic Japanese population in Hawaii, and five candidate longevity genes were chosen based on links to the insulin-insulin-like growth factor-1 (IGF-1) signaling pathway. In the Willcox study, the investigated genetic variations (rs2802292, rs2764264, and rs13217795) within the FOXO3A gene were significantly associated with longevity in male centenarians. We validated the association of FOXO3A polymorphisms with extreme longevity in males from the Southern Italian Centenarian Study. Particularly, rs2802288, a proxy of rs2802292, showed the best allelic association-minor allele frequency (MAF) = 0.49; p = 0.003; odds ratio (OR) = 1.51; 95% confidence interval (CI), 1.15-1.98). Furthermore, we undertook a meta-analysis to explore the significance of rs2802292 association with longevity by combining the association results of the current study and the findings coming from the Willcox et al. investigation. Our data point to a key role of FOXO3A in human longevity and confirm the feasibility of the identification of such genes with centenarian-controls studies. Moreover, we hypothesize the susceptibility to the longevity phenotype may well be the result of complex interactions involving genes and environmental factors but also gender. © Mary Ann Liebert, Inc.

BACKGROUND: Mobilizing agents (MAs) have been suggested to improve the fungal degradation of polycyclic aromatic hydrocarbons (PAHs) in soil. Three different MAs (Tween 20, Tween 80 and soybean oil) were investigated for their ability to stimulate contaminant degradation by either Phlebia sp. DABAC 9 or Allescheriella sp. DABAC1 in a soil spiked with amixture of PAHs. RESULTS: Phlebia sp. and Allescheriella sp. markedly differed in their growth capabilities under non-sterile conditions and withoutMAs (3.0 versus 0.1 μg ergosterol g−1 soil, respectively). However, soybean oil led to a 35-fold increase of Allescheriella sp. growth. Contaminant degradations by Phlebia sp. DABAC 9 and Allescheriella sp. DABAC 1 were best supported by soybean oil and Tween 20, respectively. Enumeration of cultivable bacteria and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA showed that microbial density and biodiversity were positively affected by the mycoremediation especially with Allescheriella sp., the use of which led to an evident detoxification. CONCLUSIONS: Allescheriella sp. DABAC 1 appears to be a promising strain in the remediation of PAH-contaminated soils. The different response of the two fungi to MAs addition confirms the stringent need for a preliminary lab-scale assessment of fungus/MA combinations prior to application. L'articolo è disponibile sul sito dell'editore: http://www.onlinelibrary.wiley.com

Several epidemiological published data support the protective role of omega-3 consumption in coronary artery disease, sudden cardiac death and ventricular arrhythmias, but interestingly, this is not the case for atrial arrhythmias. The purpose of this study is to evaluate different fatty acid profile between AF/AFL subjects and healthy controls.Gas chromatography was employed to determine fatty acid percentage of erythrocyte membranes from 40 idiopathic AFL/AF patients and 53 healthy control subjects.AFL/AF erythrocyte membranes had significantly lower percentage of saturated fatty acid (43.1 +/- SD2.2 versus 47.8 +/- SD9.6, p0.001), monounsaturated fatty acid (18.2 +/- SD2.5 versus 22.6 +/- SD5.2, p0.001) and total trans fatty acid (0.2 +/- SD0.1 vs 1.3 +/- SD1.1, p0.001) than controls. Furthermore, fatty acid (FA) profiles of arrhythmic individuals showed an increased percent of total polyunsaturated fatty acid (PUFA) (36.7 +/- SD2.4 versus 26.4 +/- SD10.4, p0.001), PUFA n-3 (5.3 +/- SD1.1 versus 2.8 +/- SD1.8, p0.001) and n-6 (31.4 +/- SD2.2 versus 23.5 +/- SD9.9, p0.001).This study shows that the erythrocyte membranes FA composition of AF/AFL subjects differs from that of healthy controls.

The ability to survive to an extremely old age is a consequence of complex interactions among genes, environment, lifestyle and luck. In the last two centuries, life expectancy in western countries has doubled, increasing from 40 to 81 years (79 for males and 82 for females). The candidate factors to determine such mortality reduction are reduced exposure to infections and the subsequent reduction in inflammatory responses, and to some extent, improvement in diet and nutrition. Among the people born at the beginning of the previous century, a small portion of individuals (1 in 10,000 born) have reached 100 years, surviving approximately 20 years more than the general population. The successful longevity of these individuals shows a familial component, possibly genetic, as underlined by the centenarian sibling's increased chance of reaching 100 years of age compared to the general population. Genetic studies on long living individuals have led to the discovery of potential genetic causes of extreme longevity. These discoveries have highlighted the role of lipid metabolism as a potential key player in the ability to survive to extreme old age. Additional studies on the longevity phenotype have confirmed the role of lipids and lipid-associated cell activities in the predisposition to longevity, from lower eukaryotes to humans. The main focus of this review is the appreciation of demographic survival data and changes in recent diet with the above mentioned genetic and phenotypic biomarkers of longevity, in order to elucidate hypotheses on mechanisms of slow aging and disease resistance.

The exceptional longevity of centenarians is due in part to inherited genetic factors, as deduced from data that show that first degree relatives of centenarians live longer and have reduced overall mortality. In recent years, a number of groups have performed genetic association studies on long-living individuals (LLI) and young controls to identify alleles that are either positively or negatively selected in the centenarian population as consequence of a demographic pressure. Many of the reported studies have shown genetic loci associated with longevity. Of these, with the exception of APOE, none have been convincingly reproduced. We validated our populations by typing the APOE locus. In addition, we used 749 American Caucasian LLI, organized in two independent tiers and 355 American Caucasian controls in the attempt to replicate previously published findings. We tested Klotho (KL)-VS variant (rs952706), Cholesteryl Ester Transfer Protein (CETP) I405V (rs5882), Paraoxonase 1 (PON1) Q192R (rs662), Apolipoprotein C-III (APOC3) -641C/A (rs2542052), Microsomal Transfer Protein (MTP) -493G/T (rs2866164) and apolipoprotein E (APOE) epsilon2 and epsilon4 isoforms, (rs7412 and rs429358) haplotypes respectively. Our results show that, at present, except for APOE, none of the selected genes show association with longevity if carefully tested in a large cohort of LLI and their controls, pointing to the need of larger populations for case-control studies in extreme longevity.

Background: Atrial fibrillation (AF) and atrial flutter (AFL) are common cardiac conduction disorders affecting many people. Recent studies on sporadic cases of AF/AFL showed a significant association of the single nucleotide polymorphism rs2200733T with the disease, suggesting a genetic factor in the development of the disease. Objectives: To determine the association of rs2200733 with AF/AFL derived from an Italian population sample. Subjects: 78 patients with AF/AFL and 348 controls took part in the study. Design: Genetic case–control study. Results: The results indicate that there is a positive, significant association between the rs2200733 T allele and patients with AF/AFL of Italian origin (allelic p,0.001 with OR = 2.17). Conclusion: These results derived from a sample of the Italian population agree with previously reported findings from an Icelandic study, which also found that the minor allele rs2200733 was associated with AF/AFL disease. Cardiac conduction disorders may depend upon a variety of predisposing factors, including developmental and congenital defects, acquired injuries or ischaemia of the conduction system and rarely inherited genetic defects that alter the electrophysiological properties of the heart. Atrial fibrillation (AF) is the most common sustained arrhythmia (supraventricular tachyarrhythmia). 1 It is associated with increased cardiovascular morbidity and mortality and it is a leading cause of stroke. 2 Other predisposing disease states include diabetes mellitus, essential hypertension (EH), congestive heart failure, valvular disease and myocardial infarction. The electrophysiological mechanism underlying AF may include the following events: myocardial electrical conduction and structural remodelling, genetic changes, ectopic electrical activity within the pulmonary veins and oxidative stress of the cardiac tissue. 13 Thus, more than one mechanism may be involved in the onset of AF. AF and atrial flutter (AFL) are closely related. 4

Offspring of long-lived individuals are a useful model to discover biomarkers of longevity. The lipid composition of erythrocyte membranes from 41 nonagenarian offspring was compared with 30 matched controls. Genetic loci were also tested in 280 centenarians and 280 controls to verify a potential genetic predisposition in determining unique lipid profile. Gas chromatography was employed to determine fatty acid composition, and genotyping was performed using Taqman assays. Outcomes were measured for erythrocyte membrane percentage content of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids (omega-6 and omega-3), geometrical isomers of arachidonic and oleic acids, and total trans-fatty acids. Also, allele and genotyping frequencies at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci were considered. Erythrocyte membranes from nonagenarian offspring had significantly higher content of C16:1 n-7, trans C18:1 n-9, and total trans-fatty acids, and reduced content of C18:2 n-6 and C20:4 n-6. No association was detected at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci that could justify genetic predisposition for the increased trans C18:1 n-9, monounsaturated fatty acids and decreased omega-6 synthesis. We concluded that erythrocyte membranes derived from nonagenarian offspring have a different lipid composition (reduced lipid peroxidation and increased membrane integrity) to that of the general population.

Here we report the unexpected finding that specific human ANK2 variants represent a new example of balanced human variants. The prevalence of certain ANK2 (encodes ankyrin-B) variants range from 2 percent of European individuals to 8 percent in individuals from West Africa. Ankyrin-B variants associated with severe human arrhythmia phenotypes (eg E1425G, V1516D, R1788W) were rare in the general population. Variants associated with less severe clinical and in vitro phenotypes were unexpectedly common. Studies with the ankyrin-B(+/-) mouse reveal both benefits of enhanced cardiac contractility, as well as costs in earlier senescence and reduced lifespan. Together these findings suggest a constellation of traits that we term "ankyrin-B syndrome", which may contribute to both aging-related disorders and enhanced cardiac function.

The present study mainly investigated the ability of solid-state cultures of the non-pathogenic Fusarium oxysporum strain BAFC 738 to transform aromatic components to reduce the phytotoxicity in olive-mill dry residue (DOR), the waste from the two-phase manufacturing process. Lignin, hemicellulose, fats and water-soluble extractives contents of DOR colonized by the fungus for 20 weeks were reduced by 16%, 25%, 71% and 13%, respectively, while the cellulose content increased by 25%. In addition, the ethyl acetate-extractable phenolic fraction of the waste was reduced by 65%. However, mass-balance ultra-Wltration and size-exclusion chromatography experiments suggested that the apparent removal of that fraction, mainly including 2-(3,4-dihydroxyphenyl)ethyl alcohol and 2-(4-hydroxyphenyl) ethyl alcohol, was due to polymerization. Mn-peroxidase and Mn-independent peroxidase activities were found in F. oxysporum solid-state cultures, while laccase and aryl alcohol oxidase activities were not detected. Tests performed with seedlings of tomato (Lycopersicum esculentum L.), soybean (Glycine maximum Merr.), and alfalfa (Medicago sativa L.) grown on soils containing 6% (w/w) of bioconverted DOR (kg soil)¡1 showed that the waste’s phytotoxicity was removed by 20 weeks-old fungal cultures. By contrast, the same material exhibited a high residual toxicity towards lettuce (Lactuca sativa L.). L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com

Lifespan experiments of lower organisms and mammals along with recent studies of centenarians are making inroads into delineating genetic factors that determine the ability to achieve exceptional longevity. These models may be helpful for the discovery of both longevity-enabling genes as well as genes associated with increased propensity to develop specific diseases. Both academic and commercial laboratories are putting substantial resources into discovering such genes in order to better understand the genetic and environmental underpinnings of how some people age more slowly than others and markedly delay or even escape age-associated diseases.

Substantial evidence supports the familial aggregation of exceptional longevity. The existence of rare families demonstrating clustering for this phenotype suggests that a genetic etiology may be an important component. Previous attempts at localizing loci predisposing for exceptional longevity have been limited to association studies of candidate gene polymorphisms. In this study, a genome-wide scan for such predisposing loci was conducted by using 308 individuals belonging to 137 sibships demonstrating exceptional longevity. By using nonparametric analysis, significant evidence for linkage was noted for chromosome 4 at D4S1564 with a MLS of 3.65 ( P = 0.044). The analysis was corroborated by a parametric analysis ( P = 0.052). These linkage results indicate the likelihood that there exists a gene, or genes, that exerts a substantial influence on the ability to achieve exceptional old age. Identification of the genes in humans that allow certain individuals to live to extreme old age should lead to insights on cellular pathways that are important to the aging process.

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

Four families highly clustered for extreme longevity are described here, representing the first report of clustering for this phenotype. Families such as these may prove to be helpful in the further understanding of the genetic contribution to achieving exceptional longevity.

People have always been fascinated with extreme longevity. The ability to achieve extreme old age has been viewed as a miracle, as in the case of Methuselah, who lived 969 years, and as a curse in the case of Jonathan Swift’s strudleburgs, in his book Gulliver’s Travels. Claims of supercentenarians (age greater than 110 years) still capture the attention of the popular media and are accepted as valid without further investigation. Scientists and the lay public usually ask, “What’s their trick?”, expecting or perhaps hoping that there is a magic environmental factor that can be manipulated into a fountain of youth. Unsubstantiated and dangerous promises of eternal youth by antiaging practitioners who sell $20,000-per-year regimens of human growth hormone also profit from the belief held by many that immortality is a simple game of resetting the endocrinologic thermostat.

Objective: To conduct the genotype-phenotype correlation in a family in which several individuals share clinical and electrophysiologic features of paramyotonia congenita (PC). Background: PC, hyperkalemic periodic paralysis (HyperPP), and potassium-aggravated myotonias form the group of hereditary sodium channelopathies. Each of these disorders is associated with different point mutations in SCN4A , the gene encoding the α-subunit of the adult human skeletal muscle sodium channel. However, in HyperPP families, evidence of a causative gene different from SCN4A has been found. Methods: We conducted direct clinical examination, electrophysiologic (EMG/electroneurographic) and cardiologic studies, as well as laboratory screening in several affected and nonaffected members of the family. We performed the genotype-phenotype correlation by microsatellite linkage and cDNA-mutation analyses of the SCN4A gene. Results: Affected members in this family showed clinical and electrophysiologic features typical of PC. The disease phenotype segregated with the chromosomal region that includes the SCN4A gene. Analysis of the entire cDNA sequence of the SCN4A gene in the index case disclosed a G3826A transition, which results in the Val1276Ile substitution. However, PCR–single-stranded confirmation polymorphism and direct sequencing analysis of the segment coding for Val-1276 on genomic DNA confirmed the G3826A transition in the index case but was negative in 11 affected members of the family; however, neither mutations nor aberrant splicings causative of the PC phenotype in this family were found on SCN4A . Conclusion: The existence of a second gene different from SCN4A that can give rise to a clinical PC phenotype can be speculated upon.

Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins.

Through our transcriptional mapping effort in the Xp22 region, we have isolated by exon trapping a new transcript highly homologous to the Drosophila retinal degeneration C (rdgC) gene. rdgC encodes a serine/threonine phosphatase protein and is required in Drosophila to prevent light-induced retinal degeneration. This human gene is the first mammalian member of the serine-threonine phosphatase with EF hand motif gene family, and was thus named PPEF (Protein Phosphatase with EF calcium-binding domain). The expression pattern of the mouse Ppef gene was studied by RNA in situ hybridization on embryonic tissue sections. While rdgC is expressed in the visual system of the fly, as well as in the mushroom bodies of the central brain, we found that Ppef is highly expressed in sensory neurons of the dorsal root ganglia (DRG) and neural crest-derived cranial ganglia. The selective pattern of expression makes PPEF an important marker for sensory neuron differentiation and suggests a role for serine-threonine phosphatases in mammalian development.

Mutations in any of the genes encoding the alpha, beta or gamma-sarcoglycan components of dystrophin-associated glycoproteins result in both sporadic and familial cases of either limb-girdle muscular dystrophy or severe childhood autosomal recessive muscular dystrophy. The collective name 'sarcoglycanopathies' has been proposed for these forms. We report the identification of a fourth member of the human sarcoglycan family. We named this novel cDNA delta-sarcoglycan. Its mRNA expression is abundant in striated and smooth muscles, with a main 8 kb transcript, encoding a predicted basic transmembrane glycoprotein of 290 amino acids. Antibodies specifically raised against this protein recognized a single band at 35 kDa on western blots of human and mouse muscle. Immunohistochemical staining revealed a unique sarcolemmal localization. FISH, radiation hybrid and YAC mapping concordantly linked the delta-sarcoglycan gene to 5q33, close to D5S487 and D5S1439. The gene spans at least 100 kb and is composed of eight exons. The identification of a novel sarcoglycan component modifies the current model of the dystrophin-glycoprotein complex.