Sometime ago we obtained biomarker data suggesting that the earliest determining event in the expression of the extended longevity phenotype in our selected strains of Drosophila took place early in adult life at about 5–7 days of age. In a later series of experiments we documented that our La and Lb long lived strains underwent a specific up-regulation of the antioxidant defense system (ADS) genes and enzymes. This led to a reduction in oxidative damage and an extended longevity. In the current work, we assayed the activity of 17 metabolically important enzymes in 5–7 day old flies of 13 strains variously selected for different longevities. We conclude that the two sets of replicated long-lived strains have an altered metabolic pattern (relative to normal-lived animals) which is consistent with an increased flux through the pentose shunt and an enhanced NADP+ reducing system to support the increased activity of the ADS enzymes. This result can be interpreted as a shift of energy expenditure from reproduction to somatic maintenance. We conclude that theories based on differential energy allocations appear to empirically explain, at least in part, the mechanisms underlying the transformation of a normal longevity phenotype to an extended longevity phenotype.

To explain trends emerging from the study of longevity mutants, a modification of the reactive oxygen species (ROS) model of aging is suggested.

ROS do not appear to be produced in greater quantities during cellular activity unless specific factors are also present. These include raised cytosolic calcium and sodium ions, nitric oxide (NO) or dopamine. Metabolically active cells that are repeatedly exposed to these factors, especially in combination, show the most ROS damage and may contribute most to aging. This explains the importance of neurons, which is highlighted by genetic studies, and points to which cells are the most aging-sensitive.

Control pathways disrupted in long-lived mutants are those which control one or more of these ROS promoting factors. The daf-2/daf-16 pathway may interact with these factors in several ways. Changes in control networks may be propagated from a relatively small number of cells/junctions by neural connectivity and hormonal means.

Life span could be modified by genetic or environmental perturbations in Caenorhabditis elegans. Here we show that two extensions of life span are associated with oxidative stress resistance and upregulation of the gene expression of antioxidant enzymes. First, mutations in age-1 gene (PI3 kinase homologue) that confer life span extension, display oxidative stress resistance and increase in the gene expression of sod-3, one of two Mn-superoxide dismutases (SOD) and ctl-1, cytosolic catalase. In this study, these traits appear to be regulated by the following genetic pathway: daf-2 (insulin receptor family)-> daf-18 (PTEN homologue)-> age-1-> daf-16 (Fork head transcription factor family), similar to the genetic pathway for the life span extension. Second, we show that short-term exposure to hyperoxia extends life span slightly but significantly. This treatment increases oxidative stress resistance and the gene expression of three types of SOD isoforms. These results suggest that both of these two life span extensions are closely related with increase in the antioxidant defense function.

Oxidative stress is thought to be a causative factor for age-related damage in a wide variety of cellular constituents that can lead to dysfunction and various pathological conditions, including the inflammatory process. At the molecular level, the redox-sensitive transcription factor, NF-κB plays a key role in the regulation of the inflammatory process, along with cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). We studied the mechanism underlying the modulation of the inflammatory reaction with age by investigating NF-κB activation and the expression of COX-2, iNOS, and cytokines genes in hepatic tissues isolated from young and old rats. We expanded our investigation of these factors in rats injected with the inflammatory activator, lipopolysaccharide (LPS). Data showed that NF-κB activity was up-regulated with age and was further enhanced by LPS injection, indicating an increased susceptibility and sensitivity to the inflammatory stimulus with age. To explore further the molecular events leading to NF-κB activation, we investigated the inhibitory component of NF-κB complex, IκB. Cytosolic IκBα, but not IκBβ, was significantly decreased in both old and LPS-treated rats, signifying the enhanced migration of cytosolic NF-κB complex into the nucleus following dissociation from the inhibitor. The appearance of the polypeptide, p65, as determined in the nucleus, corresponded with the change in IκBα, providing further supporting evidence for the molecular process involved in NF-κB activation. Our additional investigation of two proinflammatory-related enzymes, COX-2 and iNOS, and three cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α, clearly showed aged-related increases, in corroboration with the NF-κB activation. Our results demonstrated that LPS injection caused the enhanced gene expression of inducible proinflammatory proteins, COX-2 and iNOS through NF-κB activation.

It has been proposed that uric acid is an important scavenger of deleterious oxygen species and peroxynitrite in biological systems. The cellular sources responsible for the generation of damage-causing reactive oxygen species (ROS) are widespread. Xanthine dehydrogenase (XDH) / oxidase (XOD) catalyzes the oxidation of xanthine to uric acid. The rosy (ry) gene encodes XDH/XOD in Drosophila melanogaster. XDH codes for uric acid which is a ROS scavenger. XOD however is an enzyme system implicated in ROS production. In this study, we investigated the roles of XDH in the fly’s immune defense response to infection and in the aging process. We first compared ROS generation and nitric oxide (NO) level in the whole body and the gut of XDH mutant with those of wild type. Our results suggested that XDH has a protective effect with respect to both ROS and NO generations, particularly in the gut. We also examined the effect of a XDH deletion mutant on the relative sensitivity of the organism against bacterial infection, on the immune inducibility of antimicrobial peptides and on the effect of aging in the defensive response to infection. Our results strongly suggest that XDH plays an important role in the innate immune response and that the age-associated deterioration of the innate immune response might be, at least in part, associated with the loss of XDH activity in the aging process.

Aging is associated with an accumulation of oxidative damage to proteins, lipids and DNA. Cellular mechanisms designed to prevent oxidative damage decline with aging and in diseases associated with aging. A long-lived mouse, the Ames dwarf, exhibits growth hormone deficiency and heightened antioxidative defenses. In contrast, animals that over express GH have suppressed antioxidative capacity and live half as long as wild type mice. In this study, we examined the generation of H2O2 from liver mitochondria of Ames dwarf and wild type mice and determined the level of oxidative damage to proteins, lipids and DNA in various tissues of these animals. Dwarf liver mitochondria (24 months) produced less H2O2 than normal liver in the presence of succinate (p<0.03) and ADP (p<0.003). Levels of oxidative DNA damage (8ÕHdG) were variable and dependent on tissue and age in dwarf and normal mice. Forty-seven percent fewer protein carbonyls were detected in 24-month old dwarf liver tissue compared to controls (p<0.04). Forty percent more (p<0.04) protein carbonyls were detected in liver tissue (3-month old) of GH transgenic mice compared to wild types while 12 month old brain tissue had 53% more protein carbonyls compared to controls (p<0.005). Levels of liver malonaldehyde (lipid peroxidation) were not different at 3 and 12 months of age but were greater in Ames dwarf mice at 24 months compared to normal mice. Previous studies indicate a strong negative correlation between plasma GH levels and antioxidative defense. Taken together, these studies show that altered GH-signaling may contribute to differences in the generation of reactive oxygen species, the ability to counter oxidative stress and life span.

The ultrastructural features of perikaryal mitochondria positive to the copper ferrocyanide cytochemical reaction due to SDH activity were investigated in Purkinje cells of adult rats fed a vitamin E (α-tocopherol) deficient diet (AVED) for 11 months. The mitochondrial volume fraction (volume density: Vv), the number of organelles/μm3 of tissue (numeric density: Nv) and their average volume (V) were estimated by computer-assisted morphometry. The data obtained were compared with our previous results on 3, 12 and 24 month-old normally fed rats. In a comparison with age-matched controls, AVED animals showed significant decreases of the three morphometric parameters taken into account. These reductions were also observed in old, normally fed rats vs. the young and adult groups, but in AVED rats Vv and V decreased to a higher extent. In adult control animals, the percent of larger organelles (0.32 μm3 >) decreases to less than 1%. Vitamin E deficiency resulted in a steeper reduction of this fraction of organelles, i.e. only 0.5% in the 0.24–0.32 μm3 size range accounted for the largest mitochondria in the AVED group. Taken together, these data document a significant impairment of mitochondrial efficiency in old and AVED rats. We interpret these findings to support that the underlying processes of aging and vitamin E deficiency may share common mechanisms. Considering the antioxidant action of α-tocopherol and the SDH role in cellular bioenergetics, inadequate protection from free radical attacks appears to represent an important determinant in the age-related decline of the mitochondrial metabolic competence.

The aim of the present study was to determine whether oxidative stress contributes to aging of the liver in a mouse model. Liver was obtained from young (3–5 months old) and aged (18–24 months old) mice. No age-induced gross changes in liver morphology were detected by light microscopy. Apoptosis was measured using the fragment end labeling of DNA for the immunohistochemical identification of the apoptotic nuclei. The total apoptotic cells represented 1% of the total cells in livers of young mice and 8% in those of aged mice. Among the total apoptotic cells in livers of aged animals, 15% were hepatocytes, 40% sinusoidal endothelial cells, and 45% bile duct cells. Hepatic lipid peroxidation, expressed as malonaldehyde levels, protein oxidation, measured by protein carbonyl content, and DNA oxidation, measured as 8-hydroxy-2′-deoxyguanosine (oxo(8)dG), were significantly increased in the livers of aged animals as compared to younger mice. The apoptotic cells presented elevated levels of oxidized DNA, detected by immunohistochemistry using an antibody directed against oxo(8)dG in serial sections. These results suggest that livers of aged animals presents evidence of increased oxidative injury and apoptosis. Because the apoptotic cells in the aged livers are mostly bile duct cells and sinusoidal endothelial cells, the cells most sensitive to oxidative stress injury, it can be hypothesized that reactive oxygen species-induced apoptosis in these cells contributes to the aging of the liver.

The oxygen radical-induced DNA lesion 8-oxo,7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most commonly measured marker of oxidative DNA damage, which is currently considered a main cause of aging. However, a detailed study of the age-related variations of this marker in both mitochondrial (mtDNA) and nuclear (nDNA) DNA of post-mitotic organs throughout the life span has not been previously performed. In this investigation 8-oxodG steady-state levels were simultaneously measured in mtDNA and nDNA in the heart and brain of Sprague-Dawley rats at up to five different ages covering most of the adult life span, 4, 8, 12, 17 and 24 months of age, using exactly the same digestion of DNA to deoxynucleosides and chromatographic procedures for mtDNA and nDNA. 8-oxodG levels were maintained without changes during young and middle age in all cases, but showed statistically significant increases at the older ages studied in the majority of the kinds of DNA investigated. These age-related increases in oxidative damage occurred in brain nDNA at 17 and 24 months of age, in heart nDNA at 24 months of age, and in brain mtDNA at 24 months of age, whereas no significant age-related changes were detected in heart mtDNA. Besides, 8-oxodG levels were various fold higher in mtDNA than in nDNA, both in brain and heart, at all the ages studied. The results show that oxidative damage to DNA is higher in the mtDNA than in the nDNA of post-mitotic tissues throughout the whole life span of the rat and that and increase in mtDNA and nDNA oxidative stress occurs in most cases in old animals.

During the last years, the hypothesis that aging and diseases are two distinct phenomena, and that successful aging is possible for most humans, has been put forward.

We studied the TCR Vβ repertoire of T lymphocytes of healthy longevals and centenarians as crossing point of genetic predisposition and environmental effects to longevity, using the Spectra-typing method.

TCR Vβ1, Vβ8, and Vβ20 were found to be expanded in the longeval population, compared with the younger control population.

This repertoire can have been shaped by the selective action of particular HLA alleles, or by the clonal expansion of specific T cell clones, able to modulate the immune response to endogenous and exogenous antigens. Moreover, the skewed Vβ usage and the clonal expansion seem to be the effects of physiological changes occurring with aging and not pathological signs of malignity.

Background. Elevated serum total cholesterol (TC) and triglycerides (TG) are risk factors for atherosclerosis and ischemic heart disease. Adult growth hormone deficiency (AGHD) is associated with elevated TC and TG. Many treatment protocols for AGHD use relatively high doses of growth hormone (GH) given at low frequency, which is associated with increased incidences of edema, joint pains, and carpal tunnel syndrome. We have treated > 2200 patients using a low-dose high frequency (LDHF) dosing regimen of GH which results in similar beneficial subjective responses, and fewer of the side-effects associated with the higher-dosage treatment at a substantial cost savings. Clinically, in addition to increased insulin-like growth factor I (IGF-I), we observed lower TG and TC levels and no elevation of prostate specific antigen levels in treated patients.

Methods. A retrospective analysis of IGF-I, TG, TC, and PSA data from our patient population was performed to test our hypothesis that positive objective responses of IGF-I, TG, and TC occur and that elevation of PSA does not occur in response to LDHF dosing regimen of GH. The mean duration of treatment of the analyzed data ranged from 181 to 259 days.

Results. The mean plasma IGF-I level rose significantly (p<.00001) to a level 37% greater than baseline with treatment. TC and TG decreased significantly (p<.001) in those patients with elevated baseline values, and did not change significantly in those with normal baseline values. PSA concentrations decreased non-significantly during treatment, and few cases of edema, joint pain, or carpal tunnel were reported.

Conclusions. Treatment of AGHD using the LDHF dosing regimen of GH resulted in significant increases in IGF-I, significant reductions in TC and TG levels in patients with elevated baseline values, no increase in PSA concentrations, and fewer side effects than other dosing regimens.

Peroxynitrite produced from nitric oxide and superoxide has been proposed to cause neuronal dysfunction and cell death in aging and age-related degenerative diseases. 3-Nitrotyosine, an oxidation product of tyrosine by peroxynitrite, was reported to increase in degenerating brains. In this paper, involvement of peroxynitrite in neuronal cell death was studied by analyses of human brains and in vitro experiments on cell death induced by a peroxynitrite-generating agent, SIN-1. 3-Nitrotyrosine-containing proteins were detected in lipofuscin, a typical aging-related pigment in human brains. The cytotoxicity of peroxynitrite was examined in human dopaminergic SH-SY5Y cells by use of SIN-1. SIN-1 induced apoptotic cell death in the cells, and increased the level of 3-nitrotyrosine-containing proteins. The intracellular transduction of death signal was studied in apoptosis induced by peroxynitrite. Apoptosis was induced by sequential death cascade, collapse of mitochondrial membrane potential, activation of caspases and fragmentation of nuclear DNA. In addition, phosphorylation of p38 mitogen activated phosphokinase (MAPK) was found to be associated with apoptosis by SIN-1, as shown by inhibition of apoptotic process by SB202190, a p38 inhibitor. Involvement of peroxynitrite in the cell death is discussed in relation to neuronal degeneration in aging and age-associated diseases.

Brain infarction is one of the most important age-associated diseases. We have developed aged animal models for brain ischemia, and found the age-related neuronal vulnerability to brain ischemia. Investigation of that mechanism would lead to the effective treatment of brain infarction in the elder population. Recent advancement of gene transfer technique has provided strong tools for the neuronal and vascular biology. We described our recent approaches of gene transfer to blood vessels, including cerebral circulation, using adenoviral vectors. Cerebral blood vessels, atherosclerotic endothelium, and ischemic brain tissue are good targets of gene transfer. Development of these techniques would offer new therapeutic strategies for the age-related neuronal vulnerability and other age-associated diseases.

In this series of experiments, we found that Sanguisorbae Radix extract possesses strong free radical-scavenging activity in vitro and in vivo. This crude drug protected against renal disease, which is closely associated with excessive generation of reactive oxygen species. We also showed that Sanguisorbae Radix extract can suppress lipid peroxidation and stimulate an antioxidant defense ability in SAM, suggesting that this crude drug may be an effective agent for ameliorating the pathological conditions related to excessive generation of free radicals and oxidant damage, particularly in the aging process.

The importance of apolipoprotein E (apoE) in the central nervous system (CNS) became increasingly clear since the descovery that apoE ε4 allele is a major risk factor for Alzheimer’s disease. ApoE is one of the major apolipoproteins that acts as a ligand for the cellular uptake of lipoproteins via apoE receptors, members of low-density lipoprotein receptor (LDLR) family, in the CNS. Recently, LDLR family has been shown to have new functions that modulate intracellular signalling and affect neuronal and glial functions, survival and regeneration. However, the pattern of expression of apoE receptors in the CNS has not been fully clarified yet. The LDLR, very low density lipoprotein receptor (VLDLR), LDLR-related protein (LRP), and apolipoprotein E receptor 2 (apoER2) are known to bind to and internalize apoE-containing lipoproteins. Here we summarize the expression of apoE receptors in the CNS and demonstrate additional our original data on cell type specific expression and regulation of those receptors in the CNS, using in situ hybridization and RT-PCR. The cells used in our study were highly enriched cultures of neurons, astrocytes, microglia and oligodendrocytes isolated from rat brain and neuroblastoma cell line, Neuro2a. All of these four types of receptors were shown to be expressed in neurons, astrocytes, microglia and oligodendrocytes, while LDLR and LRP were expressed in Neuro2a cells. We further examined the regulation of the expression of these receptors by altering the cholesterol content of the cells, and found that only the LDLR expression was downregulated following internalization of lipoprotein cholesterol and upregulated by cholesterol deprivation, in neuronal and astroglial cells. These data together with previous studies suggest that LDLR, VLDL, LRP, and apoER2 may be involved in apoE-mediated lipid uptake and/or intracellualr signalling in the cells of the CNS cells, i.e., neurons, astrocytes, microglia, and oligodendrocytes.