Chromosome studies of in vitro senescent lymphocytes: nonrandom trisomy 2

Chromosome studies were carried out in long-term (142 and 184 d) human lymphocyte in vitro cultures in order to investigate the cytogenetic status of aging lymphocytes. The female donors were subdivided into three subgroups according to their age: 20-40 year-old (three individuals), 70-90 year-old (five persons), and centenarians (three persons). Besides some aneuploidy and structural abnormalities, telomere fusions were detected in all donor cells, and associations of acrocentric chromosomes were found in six persons in the three age-groups. Clonal trisomy 2 was present in three individuals (two from the 70-90 year-group and one centenarian with a clone +2, +8). While telomeric fusions and acrocentric associations seem to be more related to in vitro aging, trisomy 2 also appears dependent on the age of the cell donors.     

Variation of Telomerase Activity and Morphology in Porcine Mesenchymal Stem Cells and Fibroblasts during Prolonged in vitro Culture

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-ß-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P < 0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P < 0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of ß-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0 / G1 phase was significantly (P < 0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.     

Triphala extract negates arecoline-induced senescence in oral mucosal epithelial cells in vitro

BackgroundArecoline found in areca nut causes oral submucous fibrosis. Triphala is an Ayurvedic medicinal preparation used to improve overall physical wellness that has also been shown to improve oral health.ObjectivesTo assess the activity of Triphala extract on arecoline-induced senescence in oral mucosal epithelial cells in vitro.Materials and methodsOral mucosal epithelial cells were isolated and cultured in vitro. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess the viability of treated cells, while senescence was assessed by senescence-associated-β-galactosidase staining. Cell surface marker expression was analyzed by flow cytometry. Finally, real-time quantitative polymerase chain reaction was performed to examine gene expression levels.ResultsTriphala extract (5 µg/mL) reversed the cell senescence activity of arecoline, as evidenced by reduced β-galactosidase activity, increased Ki-67 marker expression, and reduced expression of senescence-related genes p16 and p21.ConclusionTriphala extract helped to reduce the pathological effects of arecoline-induced pathogenesis.Clinical relevance.Arecoline found in the areca nut causes oral pathological conditions including oral submucous fibrosis. Our results showed that Triphala counteracted the adverse effects of arecoline, in particular, negating senescence in oral mucosal epithelial cells. As a translational effect, Triphala treatment could restore normal epithelial thickness in oral submucous fibrosis, thus reducing the clinical severity of the disease. This reestablishment of oral homeostasis would help to improve oral health-related quality of life in patients with oral submucous fibrosis.     

2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence

Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48 kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process.     

Plant D-2-hydroxyglutarate dehydrogenase participates in the catabolism of lysine especially during senescence

D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH (d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.     

Donepezil attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells through SIRT1 activation

Cellular senescence of endothelial cells is a damage and stress response which induces pro-inflammatory, pro-atherosclerotic, and pro-thrombotic phenotypes. Donepezil is a drug used for the treatment of mild to moderate dementia of the Alzheimer’s disease (AD). The aim of the present study was to investigate the attenuation of endothelial cell senescence by donepezil and to explore the mechanisms underlying the anti-aging effects of donepezil. Our results indicated that high glucose (HG) markedly decreased cell viability of human umbilical vein endothelial cells (HUVECs), and this phenomenon was reversed by treatment with donepezil. Importantly, our results displayed that the frequency of senescent (SA-ß-gal-positive) cells and the expression level of senescence genes (PAI-1 and p21) were significantly higher in the HG group compared with the normal glucose (NG) group, and these changes were blocked by treatment with donepezil. Also, our results showed that donepezil inhibits the generation of reactive oxygen species (ROS), which promotes cellular senescence. Pretreatment with nicotinamide (NAM), a sirtuin 1 (SIRT1) inhibitor, inhibited the reduction in senescence associated with donepezil. Indeed, our results indicated that donepezil increased the SIRT1 enzyme activity. Therefore, these results show that donepezil delays cellular senescence that is promoted under HG condition via activation of SIRT1.     

Telomere dynamics in human cells

Human telomeres are intrinsically dynamic structures, with multiple biological processes operating to generate substantial length heterogeneity. Processes that operate specifically at the terminus, that include the end-replication problem coupled with C-strand resection, result in gradual telomere erosion with ongoing cell division. Rates of telomere erosion can be modulated by cell culture conditions and pleiotropic effects. Other processes, that are not consistent with the end replication problem, generate sporadic large-scale changes in telomere length. These events are detected in normal human cells and tissues; the severely truncated telomeres that result are potentially fusogenic and may lead to the types of genetic rearrangements that typify early-stage neoplasia. The processes that underlie sporadic telomeric deletion are unclear, but may include intra-allelic recombination within the T-loop structure, unequal sister chromatid exchange and replication fork stalling. The relative contributions of these processes in the generation of the heterogeneous telomere length profiles observed in human cells are discussed.     

Evidence for a graft-transmissible substance which delays apical senescence in Pisum sativum L.

Apical senescence in an early flowering line of pea, G2, is greatly delayed by short days. This behavior is controlled by two dominant genes. Apical senescence of ungrafted, insensitive (I) lines is unaffected by photoperiod. When I-type scions with one of the two required genes were grafted onto G2, apical senescence of the I-type was delayed in short days, but not in long days. Flowering of the I-type was unaffected. The apex of the G2 stock was unaffected as well. Apical senescence of an I-type line lacking both photoperiod genes was not delayed when grafted on G2 in short days. It is concluded that G2 plants grown in short days produce a graft-transmissible factor which delays apical senescence of photoperiodically insensitive lines.