Identification of novel genes associated with longevity in Drosophila melanogaster - a computational approach.

Despite a growing number of studies on longevity in Drosophila, genetic factors influencing lifespan are still poorly understood. In this paper we propose a conceptually new approach for the identification of novel longevity-associated genes and potential target genes for SNPs in non-coding regions by utilizing the knowledge of co-location of various loci, governed by the three-dimensional architecture of the Drosophila genome. Firstly, we created networks between genes/genomic regions harboring SNPs deemed to be significant in two longevity GWAS summary statistics datasets using intra- and inter-chromosomal interaction frequencies (Hi-C data) as a measure of co-location. These networks were further extended to include regions strongly interacting with previously selected regions. Using various network measures, literature search and additional bioinformatics resources, we investigated the plausibility of genes found to have genuine association with longevity. Several of the newly identified genes were common between the two GWAS datasets and these possessed human orthologs. We also found that the proportion of non-coding SNPs in borders between topologically associated domains is significantly higher than expected by chance. Assuming co-location, we investigated potential target genes for non-coding SNPs. This approach therefore offers a stepping stone to identification of novel genes and SNP targets linked to human longevity.

The effects of inspiratory muscle training in older adults.

PURPOSE: Declining inspiratory muscle function and structure and systemic low-level inflammation and oxidative stress may contribute to morbidity and mortality during normal ageing. Therefore, we examined the effects of inspiratory muscle training (IMT) in older adults on inspiratory muscle function and structure and systemic inflammation and oxidative stress, and reexamined the reported positive effects of IMT on respiratory muscle strength, inspiratory muscle endurance, spirometry, exercise performance, physical activity levels (PAL), and quality of life (QoL). METHODS: Thirty-four healthy older adults (68 ± 3 yr) with normal spirometry, respiratory muscle strength, and physical fitness were divided equally into a pressure-threshold IMT or sham-hypoxic placebo group. Before and after an 8-wk intervention, measurements were taken for dynamic inspiratory muscle function and inspiratory muscle endurance using a weighted plunger pressure-threshold loading device; diaphragm thickness by using B-mode ultrasonography; plasma cytokine concentrations by using immunoassays; DNA damage levels in peripheral blood mononuclear cells by using comet assays; spirometry, maximal mouth pressures, and exercise performance by using a 6-min walk test; PAL by using a questionnaire and accelerometry; and QoL using a questionnaire. RESULTS: Compared with placebo, IMT increased maximal inspiratory pressure (+34% ± 43%, P = 0.008), diaphragm thickness at residual volume (+38% ± 39%, P = 0.03), and peak inspiratory flow (+35% ± 42%, P = 0.049) but did not change other spirometry measures, plasma cytokine concentrations, DNA damage levels in peripheral blood mononuclear cells, dynamic inspiratory muscle function, inspiratory muscle endurance, exercise performance, PAL, or QoL. CONCLUSION: These novel data indicate that in healthy older adults, IMT elicits some positive changes in inspiratory muscle function and structure but neither attenuates systemic inflammation and oxidative stress nor improves exercise performance, PAL, or QoL.

Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma cytokine response to maximum sustainable voluntary ventilation.

The influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training (IMT) on the cytokine response to maximum sustainable voluntary ventilation (MSVV) is unknown. Twelve healthy males were divided equally into an IMT or placebo (PLA) group, and before and after a 6-wk intervention they undertook, on separate days, 1 h of (1) passive rest and (2) MSVV, whereby participants undertook volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with heavy cycling exercise. Plasma cytokines remained unchanged during passive rest. There was a main effect of time (P < 0.01) for plasma interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) concentrations and a strong trend (P = 0.067) for plasma interleukin-1 receptor antagonist concentration during MSVV. Plasma IL-6 concentration was reduced after IMT by 27 ± 18% (main effect of intervention, P = 0.029), whereas there was no change after PLA (P = 0.753). There was no increase in a systemic marker of oxidative stress [DNA damage in peripheral blood mononuclear cells (PBMC)], and diaphragm fatigue was not related to the increases in plasma IL-1ß and IL-6 concentrations. A dose-response relationship was observed between respiratory muscle work and minute ventilation and increases in plasma IL-6 concentration. In conclusion, increases in plasma IL-1ß and IL-6 concentrations during MSVV were not due to diaphragm fatigue or DNA damage in PBMC. Increases in plasma IL-6 concentration during MSVV are attenuated following IMT, and the plasma IL-6 response is dependent upon the level of respiratory muscle work and minute ventilation.

The effects of inspiratory muscle training on plasma interleukin-6 concentration during cycling exercise and a volitional mimic of the exercise hyperpnea.

It is unknown whether the respiratory muscles contribute to exercise-induced increases in plasma interleukin-6 (IL-6) concentration, if this is related to diaphragm fatigue, and whether inspiratory muscle training (IMT) attenuates the plasma IL-6 response to whole body exercise and/or a volitional mimic of the exercise hyperpnea. Twelve healthy males were divided equally into an IMT or placebo (PLA) group, and before and after a 6-wk intervention they undertook, on separate days, 1 h of 1) passive rest, 2) cycling exercise at estimated maximal lactate steady state power (EX), and 3) volitional hyperpnea at rest, which mimicked the breathing and respiratory muscle recruitment patterns achieved during EX (HYPEX). Plasma IL-6 concentration remained unchanged during passive rest. The plasma IL-6 response to EX was reduced following IMT (main effect of intervention, P = 0.039) but not PLA (P = 0.272). Plasma IL-6 concentration increased during HYPEX (main effect of time, P < 0.01) and was unchanged postintervention. There was no evidence of diaphragm fatigue (measured by phrenic nerve stimulation) following each trial. In conclusion, plasma IL-6 concentration is increased during EX and HYPEX and this occurred in the absence of diaphragm fatigue. Furthermore, IMT reduced the plasma IL-6 response to EX but not HYPEX. These findings suggest that the respiratory muscles contribute to exercise-induced increases in plasma IL-6 concentration in the absence of diaphragm fatigue and that IMT can reduce the magnitude of the response to exercise but not a volitional mimic of the exercise hyperpnea.

DNA damage and cytotoxicity in pancreatic beta-cells expressing human CYP2E1.

Epidemiological studies have identified nitrosamines as a risk factor for Type I (insulin dependent) diabetes mellitus. These compounds require bioactivation by cytochrome P450 2E1 (CYP2E1) for exertion of their toxic effects. Two mammalian insulin secreting pancreatic beta-cell lines BRIN BD11h2E1 and INS-1h2E1, which express human full length CYP2E1 cDNA, were used to elucidate the role of CYP2E1-mediated nitrosamine bioactivation in pancreatic beta-cell dysfunction and destruction. These cell lines were shown to metabolise dimethylnitrosamine to produce formaldehyde at rates of 3.41 +/- 0.24 and 3.65 +/- 0.26 nmol/minmg microsomal protein, respectively. Following incubation with various concentrations of the nitrosamines dimethylnitrosamine, N-nitrosopyrrolidine and 1-nitrospiperidine, all of which are bioactivated by CYP2E1, cytotoxicity and DNA damage were assessed using either the neutral red assay or comet assay respectively. Exposure of CYP2E1 expressing cells to nitrosamines resulted in significant dose-dependent decreases in cell viability, which were not seen in cells which did not express CYP2E1. Following culture with nitrosamine concentrations as low as 2.5mM 1-nitrosopiperidine, cell viability was significantly lower in BRIN BD11h2E1 and INS-1h2E1 cell lines in comparison to the BRIN BD11 and INS-1 parental cell lines (72.5 +/- 4.96 and 66.4 +/- 3.09% in BRIN BD11h2E1 and INS-1h2E1 versus 109.0 +/- 3.40 and 100.0 +/- 3.25% in BRIN BD11 and INS-1 respectively, P < 0.001). The highest dose of any of the nitrosamines tested failed to significantly reduce cell viability in the cells which lacked CYP2E1. Expression of CYP2E1 did not cause any change in the basal level of DNA damage in any of the cell lines. However, 16 h exposure to various nitrosamines resulted in significant dose-dependent DNA damage in the BRIN BD11h2E1 and INS-1h2E1 cells compared to their respective non CYP2E1-expressing parental controls, e.g. DNA damage increased from 34.38 +/- 1.25 to 44.01 +/- 1.56% DNA in comet tail in BRIN BD11h2E1 cells incubated with 10 or 40 mM N-nitrosopyrrolidine, respectively (P < 0.001). Similar treatment of the BRIN BD11 and INS-1 cell lines did not result in a significant increase in DNA damage (20.33 +/- 1.0 and 22.4 +/- 0.98% DNA in comet tail). The pancreatic beta-cell is richly vascularised and expresses CYP2E1. This study suggests that expression of human CYP2E1 in pancreatic beta-cells make them highly susceptible to cytotoxicity and DNA damage by nitrosamines and other agents bioactivated by CYP2E1.

Investigation of killer cell immunoglobulin-like receptor gene diversity: IV. KIR3DL1/S1.

The polymorphic nature of the KIR3DL1/S1 gene complex has been investigated through the development of a polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP) allele typing system. The KIR3DL1/S1 system was applied to a healthy group of Northern Irish individuals to establish allele frequencies within this Caucasian population. Additionally, cell line DNA and CEPH families, both from the 13th International Histocompatibility Workshop, and local families have been investigated. The generated data emphasize the complexity and highly polymorphic nature of this KIR gene complex; 11 allelic variations were identified, 2 of which are novel to this study. Use of the PCR-SSOP system has confirmed the presence of multiple copies of KIR3DL1/S1 alleles in a number of individuals.

Role of CYP2E1 in ketone-stimulated insulin release in pancreatic B-cells.

The role of CYP2E1 in ketone-stimulated insulin release was investigated using isolated pancreatic islets of Langerhans and two mammalian insulin secreting pancreatic beta-cell lines engineered to stably express human CYP2E1 (designated BRIN BD11h2E1 and INS-1h2E1). Isolated rat pancreatic islets were shown to express the CYP2E1 isoform which was inducible by pretreatment of animals with acetone. The cDNA encoded CYP2E1 was expressed and inducible in the engineered cells as shown by Western blotting. The transfected protein was enzymatically active in the heterologous cells as determined by p-nitrophenol hydroxylation rates (0.176 +/- 0.08 vs. 0.341 +/- 0.08 nmol/min/mg microsomal protein in BRIN BD11 control cells and BRIN BD11h2E1 cells respectively, P < 0.001; 0.204 +/- 0.03 vs. 0.633 +/- 0.102 nmol/min/mg microsomal protein in INS-1 and INS-1h2E1, respectively, P < 0.001). Cultivation of CYP2E1 expressing BRIN BD11h2E1 and INS-1h2E1 cells in 40 mM ethanol increased the rate of p-nitrophenol hydroxylation (0.968 +/- 0.09 nmol/min/mg microsomal protein, P < 0.001 and 0.846 +/- 0.103 nmol/min/mg microsomal protein, P < 0.001, respectively) providing further evidence that the heterologous protein is inducible. Cultivation of control cells with ethanol had no observable effect (0.186 +/- 0.05 and 0.195 +/- 0.03 in BRIN BD11 and INS-1, respectively). These cell lines also express NADPH-cytochrome P450 reductase protein which was enzymatically active (0.632 +/- 0.023 in parental BRIN BD11 vs. 0.657 +/- 0.066 without ethanol and 0.824 +/- 0.014 nmol/min/mg microsomal protein with ethanol in BRIN BD11h2E1, P < 0.05; and 1.568 +/- 0.118 in parental INS-1 vs. 1.607 +/- 0.093 without ethanol and 1.805 +/- 0.066 nmol/min/mg microsomal protein with ethanol in INS-1h2E1, P < 0.05) thereby providing a functional cytochrome P450 system. The insulin secretory response of control cell lines and islets was similar to cell lines and islets which had been chemically pretreated, to induce CYP2E1 expression, in response to known nutrient secretagogues. However, insulin output was significantly higher in pretreated islets (1.3-fold, P < 0.05) and CYP2E1 expressing cell lines (BRIN BD11h2E1 2.3-fold, P < 0.001; INS1-1h2E1 1.6-fold, P < 0.001) when stimulated with the ketone 3-hydroxybutyrate than control islets and parental cell lines respectively. Similar acute exposure to acetoacetate enhanced insulin secretion by 1.3-fold (P < 0.05) in pretreated islets, 2.6-fold (P < 0.001) in ethanol pretreated BRIN BD11h2E1 and 1.4-fold (P < 0.001) in ethanol pretreated INS-1h2E1 cells compared to the respective control islets or ethanol pretreated control parental cells. Therefore, these studies highlight a possible role for CYP2E1 in pancreatic cell dysfunction.

Increased frequency of the 2437T allele of the heat shock protein 70-Hom gene in an aged Irish population.

The frequency of the functional polymorphism, T2437C transversion (Met-->Thr), in the HSP 70-Hom gene was investigated within a healthy aged Irish population using oligonucleotide probes. The 2437T polymorphic nucleotide was observed to increase in the elderly, although not attaining statistical significance. The TT genotype was observed to be significantly increased within the Irish aged population (p=0.03), while conversely the TC genotype was significantly decreased in the aged subjects (p=0.01). These findings would support the theory that the change from a Met (non-polar and hydrophobic) residue to a Thr (polar and neutral) residue may disrupt the peptide-binding specificity of HSP 70-Hom and have an effect on its functional efficiency. One postulates that the highly significant p-value obtained for the TC genotype may infer that the presence of both the T and the C allele (heterozygosity) resulting in the generation of two different HSP 70-Hom protein species may negatively influence longevity.

mt4216C variant in linkage with the mtDNA TJ cluster may confer a susceptibility to mitochondrial dysfunction resulting in an increased risk of Parkinson's disease in the Irish.

Polymorphism of the mtDNA genome has been implicated as playing a role in the development and pathogenesis of Parkinson's disease (PD). A PCR-RFLP methodology was employed to generate genetic haplotypes for a cohort of 90 PD sufferers. No association was observed between the various mtDNA haplotypes observed and PD in comparison to healthy aged controls. The longevity-associated European J haplogroup and T haplogroup were identified and were both found to be in tight linkage with the mt4216C polymorphism. The mt4216C variant was observed at a significantly increased frequency in the PD cases (28%) in comparison to the healthy aged controls (15%; p=0.014). However, when the frequency of the mt4216C variant was examined in a cohort of 200 young controls (18-45 years) a similar frequency to the PD cases (25%) was observed. The frequencies obtained for the two branches of the J haplogroup (J1 and J2) and the T haplogroup in the cohort of PD subjects also reflected those observed for the young controls used in the previous longevity study. These findings lead one to postulate that the mt4216C variant, in linkage with the mtDNA TJ cluster, may influence mitochondrial dysfunction, resulting in an increased risk of PD.

HLA haplotypes and TNF polymorphism do not associate with longevity in the Irish.

Polymorphism of the human leukocyte antigen has been implicated in a number of autoimmune disorders including ageing. In the course of the present study, no association of the human leukocyte antigen (HLA)-A1, B8, DR3 haplotype with a male Irish aged population, as previously reported, was observed. Two polymorphic nucleotides in the TNF cluster (G-308A TNF-alpha and G+252A TNF-beta), associated with increased TNF-alpha production, were shown to be in tight linkage disequilibrium with the class I and II HLA loci, generating HLA haplotypes with extended linkage disequilibrium. However, no age-related allele or genotype frequencies were observed for either polymorphic nucleotide.

Study of age-association with cytokine gene polymorphisms in an aged Irish population.

The release of cytokines is of crucial importance in the regulation of the type and magnitude of the immune response in the elderly. A number of studies have shown different levels of cytokine production in the elderly. In the present study, a range of polymorphisms were chosen within the genes of cytokines (IL-2, IL-6, IL-8, IL-10, IL-12 and IFN-gamma) that have been observed at different levels within the elderly and analysed for age-association. No association was observed for the polymorphic cytokine markers and the healthy aged Irish population (or with respect to gender) examined in this study. These findings would suggest that polymorphism of cytokine genes may not play as crucial a role in healthy ageing as previously believed.

A novel polymorphic triplet repeat in intron five of the alpha-synuclein gene: no evidence of expansion or allelic association with idiopathic Parkinson's disease in the Irish.

The recent discovery of two mutations associated with autosomal dominant Parkinson's disease (PD) has led to the hypothesis that the alpha-synuclein gene plays a role in the pathogenesis of PD. Here we report a novel triplet CAA repeat within the unusually large intron 5 sequence of the alpha-synuclein gene. Microsatellite analysis revealed a high degree of polymorphism within the Irish population with seven alleles detected, ranging from eight to 17 CAA repeats. Analysis of the allele/genotype frequency differences observed between an Irish idiopathic PD cohort (eta = 98) and a healthy aged control group ( eta= 92) revealed no strong association with either group. All PD subjects displaying homozygous profiles were examined for expansion of the trinucleotide repeat, but no expansion was observed. These results would suggest that polymorphism of the alpha-synuclein gene may not play as significant a role in the pathogenesis of idiopathic PD as previously hypothesised.

Mitochondrial DNA damage in lymphocytes: a role in immunosenescence?

An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA(4977) and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA(4977) and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA(4977) was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.