Gene/longevity association studies at four autosomal loci (REN, THO, PARP, SOD2)

The possibility that four loci (REN, THO, PARP, SOD2) are associated with longevity was explored by comparing the genotypic pools of subjects older than 100 years with those of younger subjects matched for sex and geographic area (northern and southern Italy). The markers (all located within the respective gene) were HUMREN4; HUMTHO1; HUMPARP (gt)845nt; SOD2(C/T)401nt. In order to reduce the number of genotypes, multiallelic polymorphisms were recoded as diallelic according to allele size and frequency patterns (small: S, and large: L, alleles). A significant loss of LL homozygous genotypes was found at the THO locus in male but not in female centenarians with respect to matched controls. On the other hand no significant difference was found between case/control genotypic frequencies at REN, PARP, SOD2 loci. The latter loci therefore do not affect inter-individual variability in life expectancy (at least in terms of qualitative variants associated with the tested markers). However, the data is consistent with an association between the THO locus and longevity

Valutazione dell'espressione dell'RNA messaggero del fattore di trascrizione GATA-4 in cellule H9c2

Il GATA- 4 \ue8 un membro della famiglia dei fattori di trascrizione GATA; queste proteine leganti il DNA hanno ruoli importanti nella regolazione del differenziamento, proliferazione e morfogenesi degli organi durante l\u2019embriogenesi e la vita adulta. Sono stati identificati sei membri della famiglia GATA nei vertebrati e queste proteine nucleari regolano la trascrizione attraverso un legame alla sequenza consenso 5\u2019 \u2013WGATAR-3\u2019 presente nei promotori dei target genetici; tre membri di questa famiglia, GATA 4/5/6, sono espressi nell\u2019embriogenesi del cuore. Inoltre sia GATA- 4 che GATA- 6 sono espressi nel cuore adulto e presumibilmente regolano il fenotipo differenziato dei cardiomiociti. Il presente studio e' stato finalazzato alla ricerca dell\u2019RNA messaggero del GATA- 4 in cardiomioblasti H9c2, una linea di cellule muscolari cardiache di ventricolo di embrione di ratto. Una volta isolato l\u2019RNA, \ue8 stata eseguita la retrotrascrizione in cDNA usando il Transcriptor First Strand cDNA Sinthesis Kit, Roche Diagnostics. E\u2019stata poi condotta una reazione di polimerizzazione a catena (PCR) attraverso una Real Time PCR, Light Cycler, Roche Diagnostics, che ha permesso di amplificare un segmento di DNA che si trova tra due regioni di sequenza nota e usando come punto di partenza della DNA polimerasi due oligonucleotidi, detti primer, complementari a due sequenze che fiancheggiano il segmento da amplificare. La tecnologia utilizzata \ue8 quella delle sonde TaqMan che, degradate durante la fase di amplificazione, rilasciano un fluoroforo rilevato in \u201ctempo reale\u201d dallo strumento. Il numero di cicli, o crossing point (CP), in cui inizia l\u2019amplificazione dei campioni \ue8 l\u2019indice quantitativo a cui ci si riferisce. La fluorescenza emessa dai campioni nel corso della reazione di amplificazione e' stata rappresentata da due curve ottenute utilizzando lo stesso campione e che sono quindi indicative del grado di riproducibilit\ue0 della misura. Allo scopo di verificare se l\u2019amplificazione \ue8 avvenuta in modo corretto nei riguardi dell\u2019espressione del gene target del GATA \u2013 4, si \ue8 provveduto a verificare, mediante elettroforesi su gel di agarosio, la presenza del DNA amplificato. Il dosaggio dell\u2019RNA messaggero del GATA- 4 e dell\u2019espressione delle proteine, pu\uf2 quindi fornire utili informazioni sia nelle ricerche nelle quali sono studiati i processi di differenziamento delle cellule staminali in cardiomiociti, sia per verificare nei cardiomiociti adulti il ruolo di questo fattore di trascrizione nei processi di sopravvivenza cellulare

Stratospheric intrusion index (SI2) from baseline measurement data

This work introduces an index to identify deep stratospheric intrusions (SI) from measurement data alone, without requiring additional model-based information. This stratospheric intrusion index (SI2) provides a qualitative description of SI event behaviour by summarizing the information from different tracer variations. Moreover, being independent from any model constraint, the SI2 can also represent a valid tool to help in evaluating the capacity of chemistry-transport and chemistry-climate models in simulating deep stratosphere to troposphere transport. The in situ variations of ozone, beryllium-7 and relative humidity were used to calculate the index. The SI2 was applied on 8-year data recorded at the regional GAW station of Mt. Cimone (2165 m asl; 44.10N, 10.70E: Italy). The comparison of the SI2 behaviour with a pre-existing database obtained by also using model products, permitted us to tune a SI2-threshold value capable of identifying SI events efficiently. In good agreement with previous climatological studies across Europe, at Mt. Cimone, the averaged monthly SI frequency obtained by the SI2 analysis showed a clear seasonal cycle with a winter maximum and a spring-summer minimum. These results suggest that the presented methodology is efficient for both identifying SI events and evaluating their annual frequency at the considered baseline measurement site

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis.

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions

Opioid peptide response to spinal cord stimulation in chronic critical limb ischemia.

Twelve patients with chronic critical limb ischemia in whom a spinal cord stimulation (SCS) system had been implanted for at least one year had increased microvascular flow and achieved healing of trophic acral lesions. After switching off the system, the clinical improvement persisted for 10 days and the neurohormonal pattern showed high plasma values of beta-endorphin and Met-enkephalin, normal dynorphin B, endothelin-1 and catecholamines, and low nitric oxide. Met-enkephalin levels were further increased (P < 0.01) immediately after switching on the electrical stimulation again. The persistence of high plasma opioid levels after switching off the spinal cord stimulation explains the absence of subjective complaints and suggests an involvement of opioids in the regulation and improvement of the microcirculation

Comparison between stem cells harvested from wet and dry lipoaspirates

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p<0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 7 10(3) SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of ASC in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies

Epigenetic signature of early cardiac regulatory genes in human adipose-derived stem cells

Adipose-derived stem cells (ADSCs) are stromal mesenchymal stem cells isolated from lipoaspirates, and they display a broad potential to differentiate toward different lineages. The role of epigenetics in regulating the expression of their lineage-specific genes is under evaluation, however till date virtually nothing is known about the relative significance of cardiac-specific transcription factor genes in human ADSCs. The aim of this study was to investigate DNA promoter methylation and relevant histone modifications involving MEF-2C, GATA-4, and Nkx2.5 in native human ADSCs. CpG sites at the transcription start in their promoters were found unmethylated using methylation-specific PCR. Chromatin immunoprecipitation assay showed low levels of total acetylated H3 histone (acH3) and high levels of trimethylated lysine 27 in H3 histone (H3K27me3) which were associated with both GATA-4 and Nkx2.5 promoters, indicating their transcriptional repressive chromatin arrangement. On the other hand, the opposite was apparent for MEF-2C promoter. Accordingly, MEF-2C\u2014but not GATA-4 and Nkx2.5\u2014 transcripts were evidenced in native human ADSCs. These results suggest that the chromatin arrangement of these early cardiac regulatory genes could be explored as a level of intervention to address the differentiation of human ADSCs toward the cardiac lineage