Mechanisms of Ageing and Development

Published by Elsevier
Online ISSN: 0047-6374
Publications
Article
The effects of treatment with the dihydropyridine Ca+2 antagonist darodipine (PY 108-068) on age-related changes in the cerebral capillary network was studied using alkaline phosphatase histochemistry with quantitative image analysis. The investigation was performed on male Wistar rats of 12 months (adult reference group) and 27 months. The 27-month-old rats consisted of two groups, the first of control untreated animals and the second of rats receiving an oral dose of 5 mg/kg/day of darodipine from the 21st to the 27th month. The cerebral areas examined included the frontal cortex, the occipital cortex, Ammon's horn of the hippocampus, and the dentate gyrus. The number and the average length of alkaline phosphatase-positive capillaries were decreased in old rats, when compared with adult rats. The intercapillary distance, which is considered as a sensitive parameter for capillary density was increased in aged rats in comparison to adult rats. The capillary diameter was increased slightly or unchanged in old rats. The Ammon's horn and the frontal cortex were the cerebral areas most affected by age-dependent changes of the capillary network. Treatment with darodipine increased the number and the average length of alkaline phosphatase-reactive capillaries and reduced the intercapillary distance and the diameter of cerebral capillaries in old rats. The pericapillary microenvironment of the Ammon's horn was the most sensitive to treatment with darodipine. The above results showed that treatment with darodipine is capable of counteracting some microvascular changes occurring in the brain of aged rats. This suggests that the blockade of dihydropyridine-type Ca2+ channels has a positive effect on the brain microvascular system and may counteract the impairment of pericapillary microenvironment occurring with aging.
 
Article
The effect of long-term treatment with the dihydropyridine-type Ca2+ antagonist darodipine (PY 108-068) on the expression of neurofilament (NF) protein (200 kDa-NF subunit) immunoreactivity in the cerebellar cortex of aged male Wistar rats was assessed using immunohistochemical techniques associated with image analysis. In 12-month-old rats (adult) used as reference animals, 200 kDa-NF subunit immunoreactivity was observed primarily in axons of basket neurons localized in the molecular layer and surrounding the cell body of Purkinje neurons. A specific immunoreactivity was also found in the initial segment of Purkinje neuron axons, and in axons of the white matter of the cerebellar cortex. In 24-month-old rats (aged) a significant decrease in the area occupied by immunoreactive structures was noticeable in comparison with adult animals. A 6-month treatment (from the 18th to the 24th month of life) with an oral daily dose of 10 mg/kg of darodipine restored in part the expression of 200 kDa-NF subunit immunoreactivity in the cerebellar cortex. These data indicate that treatment with the dihydropyridine-type Ca2+ channel blocker darodipine is able to counter in part the age-related loss in the expression of NF protein in the rat cerebellar cortex. This suggests that darodipine may reduce neuronal cytoskeletal changes occurring in aging and in neurodegenerative disorders.
 
Article
The influence of long term treatment with the dihydropyridine-type Ca2+ antagonist darodipine (PY 108-068) on age-dependent changes in calbindin D-28K immunoreactivity in the cerebellar cortex of male Wistar rats was assessed. In 12-month-old rats used as an adult reference group, specific calbindin D-28K immunoreactivity was found within the cytoplasm of Purkinje neurons and their dendritic processes. The number of Purkinje neurons displaying calbindin D-28K immunoreactivity was decreased in the cerebellar cortex of aged in comparison with adult rats. The pattern of calbindin D-28K immunoreactivity was similar in the cerebellar cortex of 24-month-old rats (aged), although a significant decrease in the intensity of immunoreactivity was noticeable. Treatment of aged rats with darodipine for 6 months increased the percentage of immunoreactive Purkinje neurons and the intensity of calbindin D-28K immunoreactivity in the cytoplasm of Purkinje neurons. Calbindin D-28K is a Ca2+ binding protein probably involved in the modulation of Ca2+ homeostasis. The observation of a positive effect of darodipine treatment on calbindin D-28K immunoreactivity in the cerebellar cortex suggests that manipulation of dihydropyridine-type Ca2+ channels may contribute to counter age-dependent changes of Ca2+ homeostasis.
 
Expression of CD11a and CD18 on CD4 T-cells from young and old mice. The shaded contour relates to expression on control cells, and the dotted line shows expression on cells from infected mice.  
Expression of CD54 and CD62L on CD4 T-cells from young and old mice. The shaded contour relates to expression on control cells, and the dotted line shows expression on cells from infected mice.  
Contour mapping of CD4 + NK + cells in young mice (left) and old mice (right) during infection (top) as compared to uninfected controls (bottom).  
Expression of CD49e on CD4 T-cells from young and old mice. The shaded contour relates to expression on control cells, and the dotted line shows expression on cells from infected mice.
T-cell receptor gene element usage by CD4 T-cells in control and infected young and old mice. Analysis of variance showed no significant differences before or after infection with tuberculosis.
Article
The results of this study present data in support of the hypothesis that T lymphocytes in both young and old mice infected with virulent Mycobacterium tuberculosis undergo changes in expression of cell surface integrin/adhesion molecules as determined by flow cytometric analysis. These data thus further support the hypothesis that a reduced ability of T-cells in old mice to adequately and promptly accumulate at sites of inflammation induced by bacterial implantation is a central parameter underlying the increased susceptibility of these mice to this intracellular bacterial infection. In addition, however, no changes were observed in terms of the T-cell receptor expression (repertoire) of these animals, indicating that this facet of immunity is preserved in aging.
 
5-AzaC treatment suppresses the 1,25-D3-induced PCD in C6.9 cells. (A) Cells were treated for 24 h with vehicle alone and then cultured in serum-free medium for 4 days. (B) Cells were treated with 10 − 7 M 1,25-D3 for 24 h, then rinsed and cultured in a serum-free medium without 1,25-D3. (C, D) Cells pretreated with 5-AzaC (see Section 2) were treated for 24 h with vehicle alone (C) or 10 − 7 M 1,25-D3 (D) and then cultured in a serum-free medium without 1,25-D3. (E, F) Naive C6.9 cells were treated with vehicle alone (E) or 10 − 7 M 1,25-D3 (F) during 24 h in serum-free medium. Then they were cultured in serum-free medium with 5-AzaC (3 mM) from day 2 to day 4. Photographs were taken on day 5. 
Inhibition of internucleosomal DNA fragmentation by 5-AzaC in C6.9 cells treated with 10 − 7 M 1,25-D3. DNA was extracted at day 7 from C6.9 cells and the formation of oligonucleosomal fragments was determined by agarose gel electrophoresis. Lane 1, molecular weight markers; lane 2, DNA from control cells; lane 3, DNA from cells treated for 24 h with 10 − 7 M 1,25-D3 and then cultured for 6 days in serum-free medium; lane 4, DNA from cells treated from day 2 to day 4 with 3 vM 5-AzaC; lane 5, DNA from cells treated for 24 h with 10 − 7 M 1,25-D3 and then exposed to 3 vM 5-AzaC from day 2 to day 4.
Expression of c-myc gene in C6.9 cells treated for 24 h with 10 − 7 M 1,25-D3 and then exposed to 3 vM 5-AzaC from day 2 to day 4. Total RNA was extracted from cells 3 days (lanes 1-4) after a pretreatment for 24 h with vehicle alone (lane 1), or with 10 − 7 M 1,25-D3 (lane 2), or after treatment with 3 vM 5-AzaC (lane 3), or after a pretreatment for 24 h with 10 − 7 M 1,25D3 and then treatment with 3 vM 5-AzaC (lane 4). Blots were serially hybridized with radiolabelled c-myc, VDR and GAPDH probes.
Effects of a coculture of 5-AzaC-treated and naive C6.9 cells on 1,25-D3-induced cell death. 1,25-D3 was added at day 0 for 24 h. Then, serum-free culture medium was replaced every 2 days. MTT assay was performed at day 7.
Article
In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well established that DNA methylation is an important mechanism involved in gene expression, DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD) after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to study whether DNA methylation was involved in the control of PCD. We found that 1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2'-deoxycytidine. This effect remains detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene remains unaffected. These results suggest that a change in DNA methylation pattern could suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even genes inducing change in the differentiated state of these cells.
 
Article
The response of IMR-90 human fetal lung fibroblasts at high population doubling level (PDL > 42) to 1,25-dihydroxyvitamin D3[1,25(OH)2D3] was investigated to clarify whether some metabolic and molecular parameters of senescent cells are affected by the hormone treatment. Pyruvate kinase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity significantly increased after treatment of confluent-phase cells with 10 nM 1,25(OH)2D3 for 24 h. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3 to affect the enzyme activities, while estradiol-17 beta and progesterone produced a slight increase in glucose-6-phosphate dehydrogenase and lactate dehydrogenase levels, respectively. 1,25(OH)2D3 also affected fibroblast proliferation, protein content/cell and DNA synthesis. The cell number significantly decreased after a 48 h incubation with 1,25(OH)2D3 at various concentrations (0.01-1 nM) when compared with control fibroblasts, while an increase in the protein content/cell was demonstrated. The same experiment, carried out by protracting the incubation with the hormone for 72 h, showed a similar trend, but 10 nM 1,25(OH)2D3 was also able to inhibit cell proliferation and to stimulate protein synthesis. The incorporation of [3H]thymidine into DNA increased after the treatment of high PDL fibroblasts with 0.01-1 nM of hormone for 48 h in comparison with controls.
 
Article
A significant positive correlation between bone mineral density (BMD) and serum dehydroepiandrosterone sulfate (DHEA-S) was found in 120 postmenopausal women (51-99 years old) but no correlation was seen between BMD and serum estradiol. In subset analysis, strong positive correlation of serum DHEA-S and estrone with BMD was observed in postmenopausal women aged less than 69 years old. To study a possible role of DHEA-S in preventing osteoporosis, we characterized aromatase activity converting androgens to estrogens in human osteoblasts, because postmenopausal women maintain considerable levels of adrenal androgens. Glucocorticoids at 10(-9) to 10(-7) M induced transiently the expression of and the enzymatic activity of aromatase cytochrome P450 (P450AROM) in primary cultured osteoblasts. 1,25-Dihydroxyvitamin D3 (1,25-(OH)(2)D(3)) alone did not induce the aromatase activity, but enhanced and maintained the glucocorticoid-induced P450AROM gene expression. Analysis of the activity of P450AROM gene 1b (I.4) promoter, which is used dominantly in human osteoblasts, indicated that the region from -888 bp to -500 bp, which does not contain a typical vitamin D responsive element, is responsible for the enhancing effect of 1,25-(OH)(2)D(3). These results may suggest that adrenal androgen, DHEA, is converted to estrone in osteoblast by P450AROM, which is positively regulated by glucocorticoid and 1,25-(OH)(2)D(3), and is important in maintaining BMD in the sixth to the seventh decade, after menopause.
 
Article
The level of inositol 1,4,5-trisphosphate (Ins1,4,5P3) was determined in human and rabbit red blood cells of different ages. In human erythrocytes, fractionated by discontinuous density gradient centrifugation, Ins1,4,5P3 was 290 nM in the 0.3% low density (youngest) cells compared to values of 107 nM in the whole red blood cell population. A progressive increase in Ins1,4,5P3 was then observed during erythrocyte aging from values of 63 nM in mature erythrocytes to 128 nM in the oldest cells. Determinations of Ins1,4,5P3 in rabbit erythrocytes provided values of 180 nM. Phenylhydrazine was administered to three animals to induce reticulocytosis. Ins1,4,5P3 in rabbit reticulocytes was significantly lower than in the whole red cell population, remained lower in young red blood cells and then increased to normal values during cell maturation. These results provide evidence for an increase of Ins1,4,5P3 during red blood cell aging and could contribute to explain the age-dependent loss of deformability and of Ca2+ homeostasis of these cells.
 
Article
Fructose-1,6-diphosphate aldolase has been purified to homogeneity 62.0 and 58.3 fold from young and old nematodes respectively. The aldolase preparations from young (7 days) and old (35 days) animals are indistinguishable in their electrophoretic mobility, molecular weight of the tetramer (158,000) and monomer (40,000), and Km, although the "old" enzyme is more heat stable than the "young" enzyme. Enzyme from old animals has only about 55% specific activity per mg purified protein of the "young" enzyme and its catalytic activity per unit of enzyme antigen is about 50% of that of the enzyme from young animals. Immunological identity of purified enzyme from old and young animals was established by the Ouchterlony technique by antiserum produced against purified "young" enzyme and antiserum against purified "old" enzyme. Thus, this work shows for the first time that the altered form of an enzyme which appears in senescent animals apparently does not possess extra antigenic sites which are acquired as a function of age.
 
Article
In human red blood cells phosphoglucomutase exists in multiple molecular forms with different isoelectric points determined by two distinct loci called PGM1 and PGM2. With regard to the phosphoglucomutase PGM1 and PGM2 isoenzymes, the latter appear to be more important in erythrocyte metabolism owing to their ability to mutate ribose monophosphates and synthetize glucose-1,6-bisphosphate. In this paper we show that, beside undergoing age-related postranslational modifications, both phosphoglucomutase PGM1 and PGM2 forms decrease their activities as the mean cell age increases. Under the experimental conditions used to separate erythrocytes by age the comparison of the younger erythrocytes with the older shows that total phosphoglucomutase, phosphoribomutase and glucose-1,6-bisphosphate synthetic activities decay by 55%, 26% and 28%, respectively. We consider that these results substantiate the multifunctionality of PGM2 isoenzymes. Furthermore we discuss the role of these forms in the age-related decay of erythrocyte metabolism.
 
Article
Postsynthetic changes in the enzyme and structural proteins play an important role in the mechanisms of ageing of the lens, when development of protein aggregates and -S-S- linking occurs. Protection of the free -SH groups through the glutathione peroxidase and glutathione reductase system is a prerequisite to avoid these phenomena. Investigations on these two enzymes in young and old bovine lens tissues showed that they are themselves subjected to age-dependent modifications. With increasing age, the specific activity decreases while a simultaneous increase in heat lability occurs. This means that in the course of postsynthetic processes the stability of the conformation decreases and that finally the catalytic properties are lost.
 
Article
The diet known as calorie restriction (CR) is the most reproducible way to extend the lifespan of mammals. Many of the early hypotheses to explain this effect were based on it being a passive alteration in metabolism. Yet, recent data from yeast, worms, flies, and mammals support the idea that CR is not simply a passive effect but an active, highly conserved stress response that evolved early in life's history to increase an organism's chance of surviving adversity. This perspective updates the evidence for and against the various hypotheses of CR, and concludes that many of them can be synthesized into a single, unifying hypothesis. This has important implications for how we might develop novel medicines that can harness these newly discovered innate mechanisms of disease resistance and survival.
 
Article
The paper of Kitani cited in the title has raised an apparent contradiction regarding the validity of certain aspects of the membrane hypothesis of aging (MHA). He collected data showing that a number of detoxifying liver enzyme activities, although decline with age in male Fischer 344 rats, remain at an unchanged level in females of the same strain. He concluded that the main assumption of the MHA, according to which intracellular enzyme activities generally decline with age, cannot be maintained, and invoked me (p. 312) 'ellipsis to provide in the future ample (and convincing) evidence' in this respect. The present paper answers this criticism by showing that the apparent contradiction mentioned above is based on a misunderstanding on behalf of Kitani. Namely, MHA speaks about the general, density-dependent decline of the catalytic rate constant of any enzyme (k(cat)), i.e., activity per mole of enzyme, being the true specific activity of the enzymes. This parameter inevitably decreases at the increased physical density of the intracellular colloids during aging. This statement derives from the molecular enzyme kinetic models, and has extensively been proven experimentally, too. On the other hand, Kitani speaks about enzyme activities per mg total protein content of certain tissue extracts, which is a very illdefined parameter, since the concentration of the measured enzyme remains unknown. Therefore, this latter parameter is irrelevant from the point of view of MHA in any aspect.
 
Article
A substantial number of human tumors utilize a telomerase-independent telomere length maintenance mechanism referred to as alternative lengthening of telomeres (ALT). Although it is known that ALT is a telomere-specific, loss of function phenotype, which involves lengthening of telomeres by homologous recombination-mediated replication of telomeric DNA, many of the details of these processes require elucidation. Here we discuss the current literature on ALT and telomere capping, specifically focusing on how alterations in telomere capping functions may permit activation of ALT and explain the phenotypic characteristics of cells in which this occurs.
 
Article
The IgM antibody response to sheep red blood cells (SRBC) appeared significantly earlier in A/J strain mice (already in 10-day-old animals), but after 21 days responses were higher in B10 mice. These differences disappeared after reaching adulthood and IgM responses after either primary or secondary immunization were thereafter comparable in these strains. High-responder A/J mice made significantly more IgG antibodies than low-responder B10 mice from 21 days of age and strong differences lasted until the age of 19 months, when IgG antibody production was again similar. Potentiation of IgM formation by simultaneous application of 10 micrograms of LPS was higher in B10 mice until 19 months of age. On the other hand, potentiation of the IgG response was markedly high in B10 mice only in adult animals (3 months). Thereafter the potentiation was higher in A/J mice. The onset of Ig secretion in A/J mice was at 15 days and markedly increased at the age of 30 days. Levels of immunoglobulin synthesis remained extremely low in B10 mice. Age-related changes in IgG antibody production generally correlated with the decline of MHC class II antigen expression on peritoneal macrophages in these strains.
 
Article
We investigated miRNA expression changes associated with stress-induced premature senescence (SIPS) in primary cultures of human diploid fibroblast (HDF) and human trabecular meshwork (HTM) cells. Twenty-five miRNAs were identified by miRNA microarray analysis and their changes in expression were validated by TaqMan real-time RT-PCR in three independent cell lines of HTM and HDF. SIPS in both HTM and HDF cell types was associated with significant down-regulation of four members of the miR-15 family and five miRNAs of the miR-106b family located in the oncogenic clusters miR-17-92, miR-106a-363, and miR-106b-25. SIPS was also associated with up-regulation of two miRNAs (182 and 183) from the miR-183-96-182 cluster. Transfection with miR-106a agomir inhibited the up-regulation of p21(CDKN1A) associated with SIPS while transfection with miR-106a antagomir led to increased p21(CDKN1A) expression in senescent cells. In addition, we identified retinoic acid receptor gamma (RARG) as a target of miR-182 and showed that this protein was down-regulated during SIPS in HDF and HTM cells. These results suggest that changes in miRNA expression might contribute to phenotypic alterations of senescent cells by modulating the expression of key regulatory proteins such as p21(CDKN1A) as well as by targeting genes that are down-regulated in senescent cells such as RARG.
 
Article
Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) which converts active glucocorticoids to inactive metabolites. As 11 beta HSD is also expressed in the intestine, this enzyme may be an important regulator of intestinal maturation. To investigate this, we have performed the systematic study of the development of intestinal 11 beta HSD activity and its cofactor preference as well as of the effect of 11 beta HSD inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and Na,K-ATPase in the intestine. The activity of 11 beta HSD was low in ileum of suckling rats and significantly increased during the weaning period. In colon, the activity was already high in suckling rats and gradually rose during the postnatal development. 11 beta HSD activity was undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic 11 beta HSD utilized predominantly NAD as a cofactor, but displayed significant sensitivity also to NADP. Ileal 11 beta HSD had similar sensitivity to both cofactors. With NAD as a cofactor, ileal 11 beta HSD had a Km (59 +/- 10 nM) compatible with the colonic enzyme (81 +/- 14 nM). Carbenoxolone administration to suckling and weanling rats in vivo did not result in any changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone significantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-affinity type of 11 beta HSD is expressed not only in colon but also in ileum and that 11 beta HSD is an important factor in the regulation of tissue levels of active glucocorticoids in developing colon but not in the small intestine.
 
Article
Age-related decline in thymic T-cell development in 22-month-old C57BL/6J X DBA/2J (BXD) recombinant inbred strains of mice was functionally and phenotypically analyzed and genetically mapped. There was a positive correlation of the concanavalin A (Con A)-induced thymocyte proliferative response with the capability of thymocytes to mature to the CD4(+)CD8(+) stage. The accumulation of CD4(-)CD8(-) stage of thymocytes in 22-month-old BXD mice was further identified to be associated with a developmental block between the CD25(-)CD44(+) and the CD25(+)CD44(+) stages. The quantitative trait loci regulating the Con A-induced thymocyte proliferative response were mapped to mouse chromosome 1, 3, and 11, nearest to 32.1 centimorgan (cM), 5.6 cM, and 18.0 cM, respectively. Our results suggest that several genetic loci regulate the intra-thymic T-cell maturation process and play an important role in determining age-related decline in thymic T-cell development.
 
Article
A significant increase in the utilization of the VH gene families VH11 and Q52 was observed in LPS-stimulated splenic B lymphocytes from aged mice compared to young mice. VH gene usage was assayed by in situ DNA/RNA hybridization using VH family-specific and kappa chain probes. The observed age-dependent differences appear to reflect the preferential use of VH11 and Q52 VH gene use by the CD5 + B lymphocyte subset whose numbers in the spleen increase with age. The increased use of VH11 by splenic cells from old mice is associated with clonal expansions of splenic CD5 + B lymphocytes.
 
Article
We investigated the effects of age and (+/-)-methyl-3-ethyl-2,3,3a,4-tetrahydro-1 H-in-dolo[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (vinconate), an indolonaphthyridine derivative, on calcium channels, neurotransmitter receptor systems and immunophilin in Fischer rat brain using quantitative receptor autoradiography. [3H]MK-801, [3H]glycine, sodium-dependent D-[3H]aspartate, [3H]FK-506 and [3H]PN200-110 were used to label N-methyl-D-aspartate (NMDA) receptors, glycine receptors, excitatory amino acid transport sites, FK-506 binding proteins (FKBP) and voltage-dependent L-type calcium channels, respectively. [3H]Glycine and sodium-dependent D-[3H]aspartate binding significantly decreased in the frontal cortex, parietal cortex, striatum, nucleus accumbens, hippocampus, thalamus, substantia nigra and cerebellum of 24 month old rats in comparison with 6 month old animals. In contrast, [3H]MK-801, [3H]FK-506 and [3H]PN200-110 binding showed no significant changes in the brain of 24 month old rats. Intraperitoneal chronic treatment with vinconate (10 and 30 mg/kg, once a day for 4 weeks) dose-dependently ameliorated the significant reduction in [3H]glycine and sodium-dependent D-[3H]aspartate binding in the brain of 24 month old rats. These results demonstrate that glycine receptors and excitatory amino acid transport sites are more susceptible to aging processes than NMDA receptors, immunophilin and voltage-dependent L-type calcium channels. Furthermore, our findings suggest that vinconate may have a beneficial effect on age-related changes in glycine receptors and excitatory amino acid transport sites.
 
Article
Dopamine neurons in the substantia nigra of human brain are selectively vulnerable and the number decline by aging at 5-10% per decade. Enzymatic and non-enzymatic oxidation of dopamine generates reactive oxygen species, which induces apoptotic cell death in dopamine neurons. Parkinson's disease (PD) is also caused by selective cell death of dopamine neurons in this brain region. The pathogenesis of Parkinson's disease remains to be an enigma, but it was found that an endogenous MPTP-like neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl(R)salsolinol, NM(R)Sal], may be one of the pathogenic agents of PD. NM(R)Sal increases in cerebrospinal fluid from untreated parkinsonian patients, and two enzymes, a (R)salsolinol synthase and a neutral N-methyltransferase, synthesize this neurotoxin in the nigro-striatum. The activity of a neutral N-methyltransferase is significantly higher in lymphocytes from parkinsonian patients than in control. The mechanism of cell death by this toxin was proved to be by the induction of apoptosis, by use of dopaminergic SH-SY5Y cells. The apoptosis was suppressed by anti-oxidants, suggesting that the generation of reactive oxygen species may initiate cellular death process. These results indicate that in aging and PD oxidative stress induces degeneration of dopamine neurons, and the antioxidant therapy may delay the decline of dopamine neurons in the brain.
 
Article
Oxidative stress may contribute to the cellular alterations, which occur as the result of aging, and the nervous system is particularly vulnerable to aging associated oxidative injury. The multicatalytic proteasome (MCP) is responsible for the majority of protein degradation and is sensitive to oxidative stress. To determine if MCP activity is altered during aging, studies were conducted in multiple tissues from aged Fisher 344 rats. Analysis of heart, lung, kidney, and liver revealed decreased MCP activity in 12, 24, and 28 month old rats, compared with 3 week or 3 month old animals. The spinal cord, hippocampus, and cerebral cortex demonstrated age dependent decreases in MCP activity, but at no timepoint was MCP activity decreased in either the brain stem or cerebellum. Oxidative injury and the lipid oxidation product 4-hydroxynonenal caused decreased MCP activity in neural PC6 cells, while application of MCP inhibitors was sufficient to induce cell death in neural PC6 cells. Together, these data indicate a role for MCP inhibition in cellular dysfunction associated with aging and oxidative injury.
 
Article
The human premature aging protein Werner (WRN), deficient in Werner syndrome (WS), is localized mainly to the nucleolus in many cell types. DNA damage or replication arrest causes WRN to redistribute from the nucleolus to the nucleoplasm into discrete foci. In this study, we have investigated DNA damage specific cellular redistribution of WRN. In response to agents causing DNA double strand breaks or DNA base damage, WRN is re-distributed from the nucleolus to the nucleoplasm in a reversible manner. However, after ultraviolet (UV) irradiation such redistribution of WRN is largely absent. We also show that WRN is associated with the insoluble protein fraction of cells after exposure to various kinds of DNA damage but not after UV irradiation. Further, we have studied the DNA damage specific post-translational modulation of WRN. Our results show that WRN is acetylated after mytomycin C or methyl methane-sulfonate treatment, but not after UV irradiation. Also, DNA damage specific phosphorylation of WRN is absent in UV irradiated cells. Inhibition of phosphorylation fails to restore WRN localization. Thus, our results suggest that the dynamics of WRN protein trafficking is DNA damage specific and is related to its post-translational modulation. The results also indicate a preferred role of WRN in recombination and base excision repair rather than nucleotide excision repair.
 
Article
The effects of aging on glutamate neurotransmission in the brain is reviewed and evaluated. Glutamate is the neurotransmitter in most of the excitatory synapses and appears to be involved in functions such as motor behaviour, cognition and emotion, which alter with age. However, relatively few studies have been conducted to study the relationship between glutamate and aging of the brain. The studies presented here indicate the existence of a number of changes in the glutamatergic system during the normal process of aging. First, an age-related decrease of glutamate content in tissue from cerebral cortex and hippocampus has been reported, although it may be mainly a consequence of changes in metabolic activity rather than glutamatergic neurotransmission. On the other hand, studies in vitro and in vivo have shown no changes in glutamate release during aging. Since glutamate sampled in most of these studies is the result of a balance between release and uptake processes, the lack of changes in glutamate release may be due to compensatory changes in glutamate uptake. In fact, a reduced glutamate uptake capacity, as well as a loss in the number of high affinity glutamate transporters in glutamatergic terminals of aged rats, have been described. However, the most significant and consistent finding is the decrease in the density of glutamatergic NMDA receptors with age. A new perspective, in which glutamate interacts with other neurotransmitters to conform the substrates of specific circuits of the brain and its relevance to aging, is included in this review. In particular, studies from our laboratory suggest the existence of age-related changes in the interaction between glutamate and other neurotransmitters, e.g. dopamine and GABA, which are regionally specific.
 
Article
We have examined the telomere length in NHOK explanted from 28 donors between the ages of 21 and 84 years. Genomic DNA was isolated from exponentially replicating NHOK and digested with HinFI to yield terminal restriction fragments (TRF). The TRF length ranged from 4.1 to 7.0 kbp with a mean of 5.3 +/- 0.8 kbp, which was significantly shorter than that (8.9 +/- 1.0 kbp) of normal human oral fibroblasts (NHOF). The TRF length was inversely correlated to the increase of donor age in NHOK (m=-23 bp per year; r=-0.60; P<0.001). Also, the heterogeneity of TRF length in cultured NHOK decreased with increased donor age (r=-0.38, P<0.05). These data indicated that clonogenic NHOK cells had replicated in situ and showed a progressive shortening of TRF length. The short telomere length and decreased telomeric length heterogeneity in immortalized cells suggested that there is a critical minimum for cell survival.
 
Article
Unlabelled: Unlike other tissues such as breast, colon and renal cell carcinoma, it is not an easy task to single out any representative oncogene or tumor suppressor genes in the development of hepatocellular carcinoma (HCC), which play a pivotal role. To investigate putatively altered main pathways in HCC, F344 male rats were treated with a single injection of N-nitrosodiethylamine (DEN), followed by either twice/week injections of nodularin for 10 weeks or thioacetamide (TAA) in drinking water for 39 weeks. p53 expression was dramatic in both hepatocytes and mesenchymal cells after a single injection of DEN, however, PCR-SSCP assay could not detect any p53 mutation during the development of hepatocellular adenoma (HCA). The data indicate that wtp53 response was mostly for removal of damaged cells during the initiation of carcinogenesis. When treated with DEN-TAA, induction of gankyrin expression during hepatic fibrosis preceded the loss of pRB protein, accompanied with significant expressions of G1phase cyclins and CDKs. Moreover, p16(INK4A) exon 1 was hypermethylated during the development of poorly differentiated HCCs. These changes would result in complete inactivation of the pRB regulatory pathway during hepatocarcinogenesis. Induction of TGF-beta1 expression with loss of its receptor expression occurred rapidly in the altered hepatocytes by DEN-nodularin treatment. Conclusion: Therefore, escape from TGF-beta1 induced apoptosis and severe degradation of pRB protein during the early stage of carcinogenesis can perform a symphony to proliferate and to transform the altered hepatocytes to tumor cells. Inactivation of p16(INK4A) and p53 genes at the later stage of carcinogenesis would endow HCC with malignancy, which is highly resistant to any therapeutic trials.
 
Article
Hypermethylation of CpG islands, resulting in the inactivation of tumor suppressor genes, is an early event in the development of some malignancies. Recent studies suggest that this abnormal methylation may be a function of aging. The number of CpG islands that methylate with age is unknown. We used restriction landmark genome scanning (RLGS) to approximate the extent to which CpG islands change methylation status during aging. Comparison of more than 2000 loci in T lymphocytes isolated from newborn, middle age, and elderly people revealed that 29 loci ( approximately 1%) changed methylation status during aging, with 23 increasing methylation, and six decreasing. The same subset also changed methylation status with age in the esophagus, lung, and pancreas, but in variable directions. Virtual genome scanning identified one of these loci as a member of the forkhead family, recently implicated in aging, and another as an EST fragment. The methylation status of both correlated with level of expression. Confirming studies in multiple tissues from normal and DNMT1(+/-) mice demonstrated only one age dependent change in the methylation of more than 2000 loci, occurring in liver and kidney. These results indicate that the methylation status of the majority of CpG islands in both mice and humans is tightly controlled during aging, and that changes are infrequent and in humans confined to a specific subset of genes.
 
Article
Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R123-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.
 
Article
Using 125-I-labelled red kidney bean phytohemagglutinin (125-I-PHA), we have found that spleen cells from old BC3F1 mice bind this plant mitogen equally well, if not better, than spleen cells from young BC3F1 mice, although PHA-induced blastogenesis of spleen cells from old mice is sharply reduced. Analyes demonstrated that there is neither significant alteration of binding affinity nor decreased total number of membrane receptor sites for PHA in senescing mouse spleen cells. The amount of PHA which was initially bound to spleen cells in serum-free medium appeared to be insufficient for a subsequent full stimulation of blastogenesis ([3-H]thymidine incorporation) in either young or old mouse spleen cells; when washed free of unbound extracellular PHA and upon clutivation in serum-containing culture medium, spleen cells rapidly released more than 90% of the bound PHA. Also, temperatures which change cell membrane morphology played a significant role in the binding and retention of PHA. However, no difference was observed between young and old mouse spleen cells in all these phenomena of PHA-cell membrane interaction.
 
Article
Angiogenesis, the formation of new vessels from pre-existing vasculature, is impaired in aging. This is due, in part, to a lack of regulatory molecules such as nitric oxide (NO). We wished to test the hypothesis that there are deficits in the pathways that mediate NO production during angiogenesis (as defined by fibrovascular invasion into a polyvinyl alcohol (PVA) sponge implant), in aged mice in comparison to young mice. Sponges were implanted subcutaneously in young (6-8 months old, n=11) and aged (23-25 months old, n=13) mice and sampled at 14 and 19 days. Sections from the implants were stained with antibodies against vascular endothelial growth factor receptor 2 (VEGFR-2), Akt, phosphorylated Akt (p-Akt), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), inducible NOS (iNOS), and 3-nitrotyrosine (3-NT, a marker for nitrosylated proteins). Expression of VEGFR-2 was similar in the sponges of young and aged mice. Moreover, there were no significant differences in levels of Akt or its phosphorylated form in sponges from young and aged mice at 14 and 19 d. In marked contrast, levels of eNOS, p-eNOS and iNOS were significantly decreased in sponges from aged mice relative to young mice (p<0.02 for eNOS, p-eNOS and <0.01 for iNOS between young and aged mice). Concomitantly, there was diminished expression of 3-NT in the sponges from aged mice (p<0.05). Our data indicate that defects in the activation of nitric oxide synthases result in decreased NO production in aged tissues relative to young tissues. We propose that the subsequent lack of NO contributes to impaired angiogenesis in aging.
 
Article
It has been known for some 70 years that restricting the food intake of laboratory rats extends their mean and maximum life span. In addition, such life extension has been observed over the years in many other species, including mice, hamsters, dogs, fish, invertebrate animals, and yeast. Since this life-extending action appears to be due to a restricted intake of energy, this dietary manipulation is referred to as caloric restriction (CR). CR extends life by slowing and/or delaying the ageing processes. The underlying biological mechanism responsible for the life extension is still not known, although many hypotheses have been proposed. The Growth Retardation Hypothesis, the first proposed, has been tested and found wanting. Although there is strong evidence against the Reduction of Body Fat Hypothesis, efforts have recently been made to resurrect it. While the Reduction of Metabolic Rate Hypothesis is not supported by experimental findings, it nevertheless still has advocates. Currently, the most popular concept is the Oxidative Damage Attenuation Hypothesis; the results of several studies provide support for this hypothesis, while those of other studies do not. The Altered Glucose-Insulin System Hypothesis and the Alteration of the Growth Hormone-IGF-1 Axis Hypothesis have been gaining favor, and data have emerged that link these two hypotheses as one. Thus, it may now be more appropriate to refer to them as the Attenuation of Insulin-Like Signaling Hypothesis. Finally, the Hormesis Hypothesis may provide an overarching concept that embraces several of the other hypotheses as merely specific examples of hormetic processes. For example, the Oxidative Damage Attenuation Hypothesis probably addresses only one of likely many damaging processes that underlie aging. It is proposed that low-intensity stressors, such as CR, activate ancient hormetic defense mechanisms in organisms ranging from yeast to mammals, defending them against a variety of adversities and, when long-term, retarding senescent processes.
 
Article
Classical evolutionary theory predicts the existence of genes with antagonistic effects on longevity and various components of early-life fitness. Quantitative genetic studies have provided convincing evidence that such genes exist. However, antagonistic pleiotropic effects have rarely been attributed to individual loci. We examine several classes of longevity-assurance genes: those involved in regulation of the gonad; the insulin-like growth factor pathway; free-radical scavenging; heat shock proteins and apoptosis. We find initial evidence that antagonistic pleiotropic effects are pervasive in each of these classes of genes and in various model systems--although most studies lack explicit studies of fitness components. This is particularly true of human studies. Very little is known about the early-life fitness effects of longevity loci. Given the possible medical importance of such effects we urge their future study.
 
Article
We investigated the age-related changes in neuronal cell death and synaptophysin of the hippocampal CA1 sector in mice using immunohistochemistry. Microtubule-associated protein 2a, b (MAP2) and synaptophysin immunoreactivity was measured in 2-, 8-, 18-, 42- and 59-week-old mice. MAP2 immunoreactivity was unchanged in the hippocampal CA1 sector up to 42 weeks after birth. In 59-week-old mice, however, a significant decrease in MAP2 immunoreactivity was observed in the hippocampal CA1 sector. Total number of synaptophysin-positive boutons was also unchanged in the hippocampal CA1 sector up to 42 weeks of birth. In 59-week-old mice, however, a significant increase in synaptophysin-positive boutons was observed in the hippocampal CA1 sector. These results demonstrate that dendrites and axons in the hippocampal CA1 neurons are particularly susceptible to ageing processes. In contrast, a marked increase in synaptophysin-positive boutons was found in the hippocampal CA1 sector of aged mice. These findings suggest that increase in synaptophysin-positive boutons may play a role in the maintenance of the structural components in the hippocampal CA1 sector of aged mice although most postsynaptic CA1 pyramidal neurons are generated. Thus, our findings provide further valuable information on age-related neurodegeneration and deficits in hippocampus-dependent memory and synaptic plasticity.
 
Article
Patients with type 2 diabetes mellitus (NIDDM) are at risk for macrovascular disease complications, such as myocardial infarction (MI) or stroke from plaque rupture. Cytokines play a key role in plaque vulnerability. IFN-γ inhibits collagen synthesis thereby affecting plaque stability. High IL-6, TNF-α, and dyslipidemia are risk factors for thrombosis. Abnormal increments of HSP70 in atherosclerotic plaques might lead to plaque instability and rupture caused by chronic inflammation, which up-regulates the expression of pro-inflammatory cytokines (IL-6 and TNF-α) in human monocytes. Studies of a polymorphic PstI site lying in the coding region at position 1267 of the HSP70-2 gene have shown that the BB genotype is associated with NIDDM. We screened 60 old NIDDM patients with carotid stenosis and 107 old healthy controls for 1267 HSP70-2 polymorphism in order to establish if an association with plaque frailty exists. Different genotypic distributions were observed between patients and healthy controls. An increased relative risk was associated with the B allele (p = 0.0107; odds ratio = 1.861). HSP70-2, IL-6, IFN-γ, TNF-α gene expressions within the plaques and serum levels of triglyceride, total cholesterol and LDL cholesterol were tested from patients stratified according to their B+ (AB and BB) and B- (AA) genotypes. Plaque morphology (soft or fibrous-calcified) and the incidence of cerebral ischaemia were also assessed. B+ patients showed increased HSP70-2, IL-6, IFN-γ, TNF-α and dyslipidemia as compared to B- carriers. The frequency of soft plaques increased in B+ in comparison to B- patients (67% versus 13%; odds ratio 13.0, p = 0.0006). A higher frequency of cerebral ischaemia (ictus or transient ischaemic attack (TIA)) was present in B+ than in B- genotype (53% versus 20%; odds ratio 4.57, p < 0.05) Hence, 1267 HSP70-2 polymorphism may be of use in identifying B+ NIDDM patients at risk for carotid plaque rupture and cerebral ischaemia.
 
Article
The mammalian immune system defends the organism against pathogens, and possibly cancer, but is known to become dysregulated with increasing age. This results in greater morbidity and mortality due to infectious disease in old people. The most important changes occur in T cell immunity, manifested sometimes dramatically as altered clonal expansions of cells of limited antigen specificity and a marked shrinkage of the T cell antigen receptor repertoire. At the same time, it was independently reported that CMV seropositivity was associated with many of the same T cell changes that were being identified as biomarkers of immune ageing. It has now become clear that CMV is commonly the driving force behind the oligoclonal expansions and altered phenotypes and functions of CD8 cells seen in most old people. These changes are much less obvious in centenarians and most extreme in people whom longitudinal studies have shown to possess an "immune risk profile". This is a cluster of immunological parameters of which CMV seropositivity is one component and which predicts incipient mortality in an elderly population. Taken together, these findings suggest the hypothesis that persistence of CMV as a chronic antigenic stressor is a major contributor to immunosenescence and associated mortality.
 
Article
Survival of Drosophila melanogaster was estimated in 128 successive generations for 5 years. The resultant 116 samples (58 for females and 58 for males) containing series of individual values of life span (LS; 50-90 values in each series) were analyzed. Each of 58 pairs of samples belonged to a definite generation in a continuous succession where every next generation was an offspring of the preceding one. In total, 10180 Drosophila flies (5100 females and 5080 males) were studied. The mean life span (MLS) was found to be considerably heterogeneous in the series of generations: in many pairs of consecutive generations, MLSs significantly differed within two errors of the mean (P<0.05), and the minimum and maximum MLSs in the series differed from each other almost twofold. The use of the nonparametric Kruskal-Wallis test made it possible to conclusively demonstrate highly significant (P<0.001) differences between LS distributions and their medians in the series of successive generations of D. melanogaster. Highly significant (P<0.0001) positive correlations (from 0.61 to 0.91) between the parameters characterizing the minimum (t(10)), mean, and maximum (t(90)) LSs were found. This indicates considerable consistency of the LS variations in successive generations of the same population, with the proportions of individuals with low and high LSs remaining about the same irrespective of the MLS. It was demonstrated with the use of mathematical simulation that the MLS changes in successive generations may be regarded as an oscillatory process and described as a sum of several (three to five) harmonic components. The calculated determination coefficients were high (93.51 and 88.93% for females and males, respectively). This indicates that the mathematical model used for simulation adequately described the observed variations in MLS. The results are discussed in terms of population gerontology and the problem of geroprotector effectiveness.
 
Article
The gene p53 has been fashioned as the guardian of the genome and as prototype of the tumour suppressor gene (TSG) whose function must be inactivated in order for tumours to develop. The ubiquitous expression of truncated p53 protein isoforms, results in "premature ageing" of laboratory mouse strains engineered for expressing such isoforms. These facts have been construed in the argument that p53 evolved in order to protect organisms with renewable tissues from developing cancer yet, because p53 is also an inducer of cellular senescence or apoptosis after extensive DNA damage, it becomes a limiting factor for tissue renewal by depleting tissues from stem/precursor cells thus leading to whole-organism ageing. From that point of view p53 displays antagonist pleiotropy contributing to the establishment of degenerative diseases and ageing. Therefore, tumour suppression becomes a balancing act between cancer prevention and ageing. Nevertheless, here we present current evidence showing that the aforementioned argument is rather inconsistent and unwarranted on evolutionary grounds. The evolutionary perspective indicates that p53 evolved so as to play a subtle but very important role during development while its role as a TSG is only important in animals that are protected from most sources of extrinsic mortality, thus suggesting that p53 was primarily selected for its developmental role and not as a TSG. Therefore no real antagonist pleiotropy can be attached to p53 functions and their relationship with whole-organism ageing might be a laboratory artefact.
 
Article
It was recently reported that the plant polyphenol resveratrol, found, e.g., in grape berry skins, extended lifespan in the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. This lifespan extension was dependent on an NAD(+)-dependent histone deacetylase, Sir2 in Drosophila and SIR-2.1 in C. elegans. The extension of lifespan appeared to occur through a mechanism related to dietary restriction (DR), the reduction of available nutrients without causing malnutrition, an intervention that extends lifespan in diverse organisms from yeast to mammals. In Drosophila, lifespan extension by DR is associated with a reduction in fecundity. However, a slight increase in fecundity was reported upon treatment with resveratrol, suggesting a mode of action at least partially distinct from that of DR. To probe this mechanism further, we initiated a new study of the effects of resveratrol on Drosophila. We saw no significant effects on lifespan in seven independent trials. We analysed our resveratrol and found that its structure was normal, with no oxidative modifications. We therefore re-tested the effects of resveratrol in C. elegans, in both wild-type and sir-2.1 mutant worms. The results were variable, with resveratrol treatment resulting in slight increases in lifespan in some trials but not others, in both wild type and sir-2.1 mutant animals. We postulate that the effect of resveratrol upon lifespan in C. elegans could reflect induction of phase 2 drug detoxification or activation of AMP kinase.
 
Article
We investigated mainly immunohistochemical changes of nestin (a marker of neuroepithelial stem cells) and Ki-67 (a marker of proliferating cells) proteins related to ageing in the mouse hippocampus and subventricular zone (SVZ) using young adult (8 weeks old) and middle-aged (40 weeks old) mice. In the present study, no significant changes in neurons and astrocytes of the hippocampal CA1 sector were found in a middle-aged male ICR mice without severe senile weakness, as compared with young adult animals. In contrast, a significant change in the number of microglia was found in the hippocampal CA1 sector of the middle-aged mice. Furthermore, no significant changes in the number of nestin- and Ki-67-positive cells were observed in the hippocampal CA1 sector of the middle-aged mice. On the other hand, decreases in the number of nestin- and Ki-67-immunopositive cells were observed in the SVZ of the middle-aged mice. Furthermore, a migration of nestin- and Ki-67-immunoreactive cells in the corpus callosum was not observed in the SVZ of the middle-aged mice. In the dentate gyrus, significant decreases in the number of Ki-67-immunopositive cells were observed in the middle-aged mice. Our study also showed that nestin immunoreactivity was observed in both Ki-67-postive cells and astrocytes in the SVZ of young adult mice. These findings emphasize the need to recognize ageing as important factors in studies of microglia, which may help to clarify the role of glial cell structure and function during ageing processes. Furthermore, the present findings suggest that ageing processes may decrease neurogenesis in the corpus callosum, SVZ and dentate gyrus. Thus our present findings provide valuable information for the neurogenesis during ageing processes.
 
Article
Cellular senescence, a stress induced growth arrest of somatic cells, was first documented in cell cultures over 40 years ago, however its physiological significance has only recently been demonstrated. Using novel biomarkers of cellular senescence we examined whether senescent cells accumulate in tissues from baboons of ages encompassing the entire lifespan of this species. We show that dermal fibroblasts, displaying markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16, accumulate in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reaches a value of over 15% of total fibroblasts, whereas 80% of cells contain high levels of the heterochromatin protein HIRA. In skeletal muscle, a postmitotic tissue, only a small percentage of myonuclei containing damaged telomeres were detected regardless of animal age. The presence of senescent cells in mitotic tissues might therefore be a contributing factor to aging and age related pathology and provides further evidence that cellular senescence is a physiological event.
 
Article
Human mesenchymal stem cells (hMSC) represent a promising cell-based therapy for a number of degenerative conditions. Understanding the effect of aging on hMSCs is crucial for autologous therapy development in older subject whom degenerative diseases typically afflict. Previous investigations into the effects of aging on hMSC have proved contradictory due to the relative narrow age ranges of subjects assessed and the exclusive reliance of in vitro assays. This study seeks to address this controversy by using a wider range of donor ages and by measuring indices of cellular aging as well as hMSC numbers ex vivo and proliferation rates. CFU-f analysis and flow cytometry analysis using a CD45(low)/D7fib(+ve)/LNGF(+ve) gating strategy were employed. In addition a variety of markers of cellular aging, oxidative damage and senescence measured. A reduction in CFU-f and CD45(low)/D7fib(+ve)/LNGF(+ve) cell numbers were noted in adulthood relative to childhood. Indices of aging including oxidative damage, ROS levels and p21 and p53 all increased suggesting a loss of MSC fitness with age. These data suggest that hMSC numbers obtained by marrow aspiration decline with age. Furthermore, there is an age-related decline in overall BM MSC "fitness" which might lead to problems when using autologous aged MSC for cell-based therapies.
 
Article
The secondary and tertiary immune responses to TNP-KLH and the autoanti-immunoglobulin response were studied in 1-, 3- and 6-month-old 129/J and 129/Sv mice. A profound inability of aging 129/Sv mice (lifespan: 9 months) to produce gamma 2a anti-TNP antibodies relative to their normal counterparts was observed. However, this markedly low response could not be related to clearance of antibody by RFs since all 129 mice studied produced significant amounts of RFs after immunization with this T-dependent antigen. Furthermore, the results of the plaque forming cell assays indicated that young (1 month old) 129/Sv mice have an accelerated immune response while older mice produce significantly low direct and indirect responses compared to the control groups. The data suggest a fundamental dysfunction of B and/or T cells in 129/Sv mice and that spontaneous production of RFs in older mice may affect the ability of B and T cells to cooperate in a secondary response.
 
Article
The regulation of cell growth and differentiation and also expression of a number of genes by retinoids are mediated by nuclear retinoid receptors (RARs and/or RXRs). In this study we investigated age-related alteration in both RAR and RXR receptor subtypes gene expression and tissue transglutaminase (tTG) activity before and after supplementation with 13-cis retinoic acid (13cRA) in human peripheral blood mononuclear cells (PBMCs). Healthy men (40) were divided in two groups according to their age (young group: 26.1+/-4.1 years and old group: 65.4+/-3.8 years). Each volunteer received 13cRA (Curacné), 0.5mg/(kgday)) during a period of 4 weeks. We have shown that RXRbeta expression was decreased significantly (p=0.0108) in PBMCs of elderly men when compared to that of young volunteers. Distribution of retinoic acid receptor subtype expression in PBMCs was found in the order: RXRbeta>RARgamma>RXRalpha>RARalpha. The tTG activity in PBMCs reflected a trend to be enhanced after 13-cis retinoic acid supplementation. In conclusion, we demonstrate a significant decrease in the expression of RXRbeta subtype of rexinoid receptors in PBMCs of healthy elderly men. Our data suggest that in healthy elderly men reduction of RXRbeta expression in PBMCs might be a common feature of physiological senescence.
 
Article
The leading causes of death for individuals with Werner syndrome (WS) are myocardial infarction (MI) and stroke. The WS gene encodes a nuclear protein with both helicase and exonuclease activities. While individuals with WS have mutations that result in truncated, inactive proteins, several sequence variants have been described in apparently unaffected individuals. Some of these gene polymorphisms encode non-conservative amino acid substitutions, and it is expected that the changes would affect enzyme activity, although this has not been determined. Two research groups have studied the Cys/Arg 1367 polymorphism (located near the nuclear localization signal) in healthy and MI patients. Their results suggest that the Arg allele is protective against MI. We have characterized the Cys (C) and Arg (R) forms of the protein and find no notable difference in helicase and nuclease activities, or in nuclear/cytoplasmic distribution. The frequency of the C/R alleles in healthy individuals and subjects with coronary artery disease (CAD) drawn from the Baltimore Longitudinal Study of Aging (BLSA) was also examined. There was no indication that the R allele was protective against CAD. We conclude that the C/R polymorphism does not affect enzyme function or localization and does not influence CAD incidence in the BLSA cohort.
 
SIR-2.1, DAF-16 and 14-3-3 proteins interact in vivo . (A) SIR-2.1 is co- immunoprecipitated with DAF-16 and 14-3-3 proteins. Lysates from geIn3 ; DAF-16 < GFP worms were immunoprecipitated with anti-SIR-2.1 antibody and immunoblotted with anti-GFP antibody (to detect the presence of DAF-16) and anti-14-3-3 antibody. The immunoblotting of SIR-2.1 served as a control. (B) DAF-16 is co-precipitated with 14-3-3 proteins. Lysates from geIn3 ; DAF- 16 < GFP worms were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-14-3-3 antibody. 
Life span extension by overexpression of sir-2.1 , but not daf-2 mutation, is suppressed by par-5 RNAi. (A) par-5 RNAi slightly shortens the life span of wild-type and completely suppresses the life span extension by sir-2.1 overexpression (N2 + pRF4 on L4440 vector RNAi: 18.2 Æ 0.5 days; on par-5 RNAi: 15.6 Æ 0.3 days; geIn3 ( sir-2.1 overexpression strain) on L4440 vector RNAi: 21.2 Æ 0.6 days; on par-5 RNAi: 16.4 Æ 0.5 days). (B) Life span of daf- 2 ( e1370 ) is not affected by par-5 RNAi ( daf-2 ( e1370 ) on L4440 vector RNAi: 42.3 Æ 2.1 days; on par-5 RNAi: 44.1 Æ 1.5 days). Life span data shown were from a single representative experiment. Life span analysis was performed several times with similar results. 
Overexpression of par-5 and ftt-2 extends life span in a DAF-16 dependent manner. (A) Overexpression of par-5 and ftt-2 extends life span. Life spans were as follows (mean Æ S.E.): wild type with pRF4 17.8 Æ 0.4 days was extended to 23.1 Æ 0.9 days in PAR-5 < GFP ( lpEx21 ) and 24.7 Æ 0.6 days in FTT-2 < GFP ( lpEx22 ). (B) daf-16 RNAi suppresses the life span extension by the overexpression of par-5 and ftt-2 . Life span extension by par-5 overexpression is completely suppressed by daf-16 RNAi (PAR-5 < GFP ( lpEx21 ) on L4440 vector RNAi: 22.2 Æ 0.7 days; on daf-16 RNAi: 13.5 Æ 0.3 days). Similarly life span extension by overexpression of ftt-2 is significantly suppressed by daf-16 RNAi (FTT-2 < GFP ( lpEx22 ) on L4440 vector RNAi: 24.4 Æ 0.5 days; on daf-16 RNAi: 17.7 Æ 0.4 days). N2 + pRF4 served as a control. Life span of N2 + pRF4 on L4440 vector RNAi and daf-16 RNAi are 18.9 Æ 0.4 days and 14.3 Æ 0.3 days, respectively. Life span data shown were from a single representative experiment. Life span analysis was performed several times with similar results. 
Expression of PAR-5 < GFP and FTT-2 < GFP under normal growing conditions. (A) PAR-5 < GFP is broadly expressed in the nervous system and the intestine. (B) The detailed view of PAR-5 < GFP in the head region. GFP expression is observed in the head neurons and is absent in the pharynx. (C) There is strong PAR-5 < GFP expression in the intestine and the ventral nerve cord. (D) FTT-2 < GFP is mainly expressed in the pharynx and the nervous system. There is weak GFP expression in the intestine. (E) Strong GFP expression can be seen in the pharynx and head neurons of FTT-2 < GFP worms. (F) Weak GFP expression is observed in the intestine of FTT-2 < GFP worms. 
Article
14-3-3 proteins are evolutionarily conserved and ubiquitous proteins that function in a wide variety of biological processes. Here we define a new role for C. elegans 14-3-3 proteins in life span regulation. We identify two C. elegans 14-3-3 proteins as interacting proteins of a major life span regulator, the C. elegans SIR2 ortholog, SIR-2.1. Similar to sir-2.1, we find that overexpression of either 14-3-3 protein (PAR-5 or FTT-2) extends life span and that this is dependent on DAF-16, a forkhead transcription factor (FOXO), another important life span regulator in the insulin/IGF-1 signaling pathway. Furthermore, we show that both 14-3-3 proteins are co-expressed with DAF-16 and SIR-2.1 in the tissues critical for life span regulation. Finally, we show that DAF-16/FOXO also physically interacts with the 14-3-3 proteins. These results suggest that C. elegans 14-3-3 proteins can regulate longevity by cooperating with both SIR-2.1 and DAF-16/FOXO.
 
Article
Skin aging is a complicated process associated with the passage of time and environmental exposure, especially to UV light. This aging phenomenon is related to alterations in various cellular mechanisms, such as changes in apoptosis, perturbations to cellular signaling, and an increased genetic instability. In this study, we investigated changes of proteins involved in intrinsic aging by the proteomic analysis of human sun-protected (upper inner arm) young and aged dermis. One of the proteins upregulated in aged dermis was identified as 14-3-3epsilon. This protein is an isoform of 14-3-3 protein, which is involved in cellular processes like signal transduction, cell cycle arrest, and apoptosis. 14-3-3epsilon is consistently found to be upregulated in the sun-protected dermis of aged skin, by Western blotting and immunohistochemical staining. In addition, we demonstrate that the expression of 14-3-3epsilon is further upregulated in the sun-exposed (photodamaged) dermis, and that the UV irradiation of young skin significantly upregulates 14-3-3epsilon in vivo. Our results suggest the possibility that the cellular processes related to 14-3-3epsilon protein play an important role in the photoaging and intrinsic aging of human skin.
 
Article
Cartilage of chick embryos, 7 to 16 days of age, was incubated with [14C]-proline, [14C]lysine, or [14C]glutamic acid. The biosynthesis of [14C]hydroxyproline, total [14C]hydroxylysine, unglycosylated [14C]hydroxylysine and glycosylated [14C]-hydroxylysine was assayed. Increased hydroxylation of [14C]proline was observed up to day 14; and the total [14C]hydroxylysine reached a plateau from day 10 to day 16. Glycosylated [14C]hydroxylysine decreased after day 10.Although in the samples from 8- to 11-day-old tibiae, the values of glutamic acid expressed as dpm/bone per μCi per h increased, the participation in the process of hydroxyproline biosynthesis appeared constant if the values were expressed as percentage of total14C.Differences in glycosylation of hydroxylysine during the embryogenesis of chicken indicates a considerable heterogeneity of collagen.
 
Article
Uptake from the circulation and subsequent intracellular degradation of foreign and potentially harmful substances are key functions of hepatic Kupffer cells. While ageing is generally associated with decreased clearance by the reticulo-endothelial system, the effect of ageing on specific Kupffer cell functions is poorly understood. This study measured the ability of Kupffer cells of isolated perfused rat livers from young and old rats to both phagocytose and subsequently degrade exogenous radiolabelled mitochondria. Using electron microscopy and stereological techniques it was determined that there was no change in the volume density of Kupffer cells between 2 and 24 months, implying that the size of the Kupffer cell population increased (along with the total liver size) with age. However, despite this increase size there was no parallel increase in the capacity of the liver to take up or degrade radiolabelled mitochondria, implying that, in aged rats, Kupffer cell uptake and intracellular degradation was less efficient.
 
Article
The polyphenol quercetin has recently been found to extend lifespan and increase stress resistance in the nematode Caenorhabditis elegans. The forkhead transcription factor DAF-16 has previously been linked to these effects. However, by using a daf-16(mgDf50) mutant strain, we show that quercetin exposure leads to increased mean lifespans up to 15%. Furthermore, quercetin-treated daf-16(mgDf50) worms show an enhanced resistance to thermal and oxidative stress. Our data reveal that DAF-16 is not obligatorily required for quercetin-mediated longevity and stress resistance.
 
Top-cited authors
Claudio Franceschi
  • University of Bologna
Rajindar S Sohal
  • University of Southern California
Daniela Monti
  • University of Florence
Fabiola Olivieri
  • Università Politecnica delle Marche
Miriam Capri
  • University of Bologna