Abstract: Physical inactivity leads to increased incidence of a variety of diseases and it can be regarded as one of the end points of the exercise-associated hormesis curve. On the other hand, regular exercise, with moderate intensity and duration, has a wide range of beneficial effects on the body including the fact that it improves cardio-vascular function, partly by a nitric oxide-mediated adaptation, and may reduce the incidence of Alzheimer's disease by enhanced concentration of neurotrophins and by the modulation of redox homeostasis. Mechanical damage-mediated adaptation results in increased muscle mass and increased resistance to stressors. Physical inactivity or strenuous exercise bouts increase the risk of infection, while moderate exercise up-regulates the immune system. Single bouts of exercise increases, and regular exercise decreases the oxidative challenge to the body, whereas excessive exercise and overtraining lead to damaging oxidative stress and thus are an indication of the other end point of the hormetic response. Based upon the genetic setup, regular moderate physical exercise/activity provides systemic beneficial effects, including improved physiological function, decreased incidence of disease and a higher quality of life.

Abstract: Exercise is associated with increased ATP need and an enhanced aerobic and/or anaerobic metabolism, which results in an increased formation of reactive oxygen species (ROS). Regular exercise seems to decrease the incidence of a wide range of ROS-associated diseases, including heart disease, type II diabetes, rheumatic arthritis, Alzheimer and Parkinson diseases, and certain cancers. The preventive effect of regular exercise, at least in part, is due to oxidative stress-induced adaptation. The oxidative challenge-related adaptive process of exercise is probably not just dependent upon the generated level of ROS but primarily on the increase in antioxidant and housekeeping enzyme activities, which involves the oxidative damage repair enzymes. Therefore, the effects of exercise resemble the characteristics of hormesis. In addition, it seems that the oxidative challenge-related effects of exercise are systemic. Skeletal muscle, liver, and brain have very different metabolic rates and functions during exercise, but the adaptive response is very similar: increased antioxidant/damage repair enzyme activity, lower oxidative damage, and increased resistance to oxidative stress, due to the changes in redox homeostasis. Hence, it is highly possible that the well-known beneficial effects of exercise are due to the capability of exercise to produce increased levels of ROS. Or in other words, it seems that the vulnerability of the body to oxidative stress and diseases is significantly enhanced in a sedentary compared to a physically active lifestyle.

Abstract: Diesel exhaust (DE) is a major airborne pollutant of urban areas. It contains various polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs. In this study, gpt delta mice were treated with inhalation of 1 or 3 mg m(-3) DE, or a single intratracheal instillation of diesel exhaust particles (DEP) or DEP extract. In the lungs of mice treated with inhalation of 3 mg m(-3) DE for 12 weeks, the mutant frequency (MF) was 3.2-fold higher than that of the control group (1.90 x 10(-5) and 0.59 x 10(-5), respectively). An instillation of DEP and DEP extract resulted in a significant dose-dependent linear increase in MF. In mice treated with 0.5 mg DEP and 0.2 mg DEP extract, the MFs were 3.0- and 2.7-fold higher than that of the control group, respectively. The mutagenic potency (MF mg(-1)) of DEP extract (5.6 x 10(-5)) was double that of DEP (2.7 x 10(-5)), suggesting that the mutagenicity of the latter is derived primarily from compounds in the extract, which itself is responsible for ca. 50% of the weight of DEP. G:C-->A:T transitions were the predominant gpt mutation induced by all three treatments and G:C-->T:A transversions were induced by DEP and DEP extract. Guanine bases centered in nucleotide sequences such as GGA, TGA, CGG, and CGT were the major mutation targets of all three treatments. Thus, our results suggest that the mutagens contained in DEP such as PAH and nitrated PAHs induce mutations and may be responsible for carcinogenesis caused by inhalation of DE.

Abstract: MER5 (also called peroxiredoxin III, PrxIII) is a member of peroxiredoxin family that has antioxidant activity. The present study was performed to investigate its in vivo function using MER5 knockout mice. MER5 knockout mice were born in normal frequency and could grow to maturity, but we found that intracellular ROS levels are significantly higher in the macrophages of the knockout mice. We examined roles of MER5 function for the oxidative stress responses by intratracheal inoculation of lipopolysaccharide (LPS) to the mice. Lung inflammation such as inflammatory cell infiltration and airway wall thickening was more severely detected in the knockout mice. At the same time, oxidative damage on DNA and proteins was more strongly detected in lung tissues of the knockout mice, including 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and protein carbonylation. The degrees of lung inflammation and oxidative damage were positively related with LPS doses. Our results indicate that MER5 knockout mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that MER5 acts as an important scavenger of reactive oxygen species (ROS) under oxidative stress.

Abstract: Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.

Abstract: Exercise is often said to increase the generation of reactive oxygen species that are potentially harmful. On the other hand, regular exercise has various health benefits even late in life. The specific aim of this study was to explore effects of regular exercise on oxidative status of DNA in aged animals. We report that 2 months of regular treadmill running of aged rats (21 month old) significantly reduced 8-oxodG content to the level of young adult animals (11 month old) in both nuclear and mitochondrial DNA of the liver. The mitochondrial DNA showed 10-fold higher content of the oxidative lesion than the nuclear DNA. The levels in old animals were 2- and 1.5-fold higher than that in young adults for the nucleus and mitochondria, respectively. The activity of the repair enzyme OGG1 was upregulated significantly in the nucleus but not in mitochondria by the exercise. To our knowledge, this is the first report demonstrating that regular exercise can reduce significantly oxidative damage to both the nuclear and mitochondrial DNA. We suggest that the apparent beneficial outcomes in reducing the DNA damage by regular exercise can be interpreted in terms of hormetic effect by moderate oxidative stress and potential adaptation to stronger stresses.

Abstract: Megalin and cubilin are the major endocytic receptors responsible for resorption of glomerular filtrate proteins, particularly albumin, in the renal proximal tubule. In order to better understand the mechanism of the development of albuminuria with age in rats, we investigated age-related change of the amount and cellular localization of both receptors in the kidney. Immunoblot analysis of the kidney extracts showed that the amount of megalin significantly decreased with age. Although there was no age-related change in the amount of intact cubilin, the amount of cubilin fragments increased with age. Immunohistochemical study revealed that megalin and cubilin were predominantly localized in brush border membrane of proximal tubular cells in young rats, but the receptors tended to diffuse into the cytoplasm in the old rats. Interestingly, low but significant amounts of megalin and cubilin were present in the glomerular cells in addition to the proximal tubular cells. The quantity of receptors progressively increased in the glomerulus with age. This age-related increase might be to compensate for the age-related defect of the uptake of albumin by the proximal tubules. Thus, although it is unclear whether megalin and cubilin in the glomerulus contribute to the uptake of albumin in primary urine, the age-related increase in the amount of albumin in urine might at least partly be due to quantitative and qualitative alterations of both receptors in the proximal tubule.

Abstract: To explore mechanisms of the beneficial consequences of regular exercise, we studied the effects of regular swimming and treadmill exercise on oxidative stress in the brain and liver of rats. Protein carbonyl was significantly reduced and the activity of proteasome was upregulated in the brain extracts of young and middle-aged animals after 9 weeks of swimming training. Furthermore, their cognitive functions were significantly improved. In separate experiments, the activation of transcription nuclear factor kappaB was attenuated in the liver of old rats after 8 weeks of regular treadmill exercise and the DNA binding activity of glucocorticoid receptor reduced with age was restored, suggesting that inflammatory reactions are alleviated by the regimen. This was accompanied by upregulation of the glutathione level and reduced reactive oxygen species generation. Similar training reduced the 8-oxodeoxyguanosine content in the nuclear and mitochondrial DNA of the liver of old rats. Thus, these findings, together with reports of other investigators, suggest that moderate regular exercise attenuates oxidative stress. The mild oxidative stress possibly elicited by regular exercise appears to manifest a hormesis-like effect in nonmuscular tissues, constituting beneficial mechanisms of exercise by adaptively upregulating various antioxidant mechanisms, including antioxidative and repair-degradation enzymes for damaged molecules. Importantly, the adaptation induced by regular exercise was effective even if initiated late in life.

Abstract: Reactive oxygen species (ROS) are continuously generated during aerobic metabolism. Certain levels of ROS, which could be dependent on the type of cell, cell age, history of ROS exposure, etc., could facilitate specific cell functions. Indeed, ROS stimulate a number of stress responses and activate gene expression for a wide range of proteins. It is well known that increased levels of ROS are involved in the aging process and the pathogenesis of a number of neurodegenerative diseases. Because of the enhanced sensitivity of the central nervous system to ROS, it is especially important to maintain the normal redox state in different types of neuro cells. In the last decade it became clear that regular exercise beneficially affects brain function as well, and can play an important preventive and therapeutic role in stroke and in Alzheimer's and Parkinson's diseases. The effects of exercise appear to be very complex and could include neurogenesis via neurotrophic factors, increased capillarization, decreased oxidative damage, and increased proteolytic degradation by proteasome and neprilysin. Data from our and other laboratories indicate that exercise-induced modulation of ROS levels plays a role in the protein content and expression of brain-derived neurotrophic factor, tyrosine recepetor kinase B, and cAMP response element binding protein, resulting in better function and increased neurogenesis. The enhanced activities of proteasome and neprilysin result in decreased accumulation of carbonyls and amyloid beta-proteins, as well as improved memory. It appears that exercise-induced modulation of the redox state is an important means by which exercise benefits brain function, increases the resistance against oxidative stress, and facilitates recovery from oxidative stress.

Abstract: Dietary restriction (DR) or caloric restriction (CR) is the well-established means to retard aging, leading to prolongation of mean and maximum life span in many animal models. We have been interested in the possibility of extending the span of health of elderly people rather than increasing longevity, and therefore studied the effects of DR/CR initiated late in life in rodent models. We restricted food for 2-3.5 months in mice or rats of middle or old ages, which would perhaps be equivalent to 50-70 years of age in humans. We found that: (1) Potentially harmful altered proteins were reduced in the animals' tissues. (2) Extended half-life of protein in aged animals was shortened in mouse hepatocytes, suggesting improved protein turnover. (3) Reduced proteasome activity was upregulated in rat liver and skeletal muscle. (4) Protein carbonyls were decreased in rat liver mitochondria and skeletal muscle cytoplasm, and also oxidative DNA damage was reduced in rat liver nucleus, suggesting amelioration of oxidative stress. (5) Reduced apo A-IV and C-III metabolism in aged mouse was restored, suggesting increase in reduced fatty acid mobilization. (6) The carbonyl modification in histones that was paradoxically reduced in aged rat was increased to the level of a young animal, suggesting restoration of reduced transcription. These findings in rodents suggest a possibility that DR/CR is beneficial if applied in middle-aged or early senescent obese people. We argue, however, that application of late life DR/CR can be harmful if practiced in people who are already eating modestly.

Abstract: In the current investigation we tested how swimming training (T) (8 week, 5 times/week, 2 h/day), and detraining (DT) affects brain functions and oxidative stress markers in rat brain. The free radical concentration, measured by electron paramagnetic resonance, decreased in brain of T and DT rats compared to controls (C). The level of brain-derived neurotrophic factor (BDNF) increased as a result of training, but decreased below the control level after 6 weeks of detraining. In addition, the concentration of nerve growth factor (NGF) also declined with DT. The passive avoidance test was used to assess the memory of rats, and training-induced improvement was observed but the enhancement disappeared with detraining. When the content of mitochondrial electron transport complexes, as a potent free radical generator, was evaluated by the blue native gel method, no significant alterations were observed. The repair of nuclear and mitochondrial 8-oxodeoxyguanosine, as measured by the activity of OGG1, showed no significant difference. Therefore, the results suggest that regular exercise training improves memory, decreases the level of reactive oxygen species, and increase the production of BDNF and NGF. On the other hand, it appears that the beneficial effects of training are reversible in the brain, since detraining down-regulates the neurotrophin level, and memory. It is suggested that exercise training is more likely to beneficially effect the production of reactive oxygen species and the related oxidative damage.

Abstract: We studied carbonylation, a form of oxidative modification of proteins, of histones in rat livers. Histones H1, H2B/H2A, and H3 were significantly carbonylated but the modification was almost undetectable in H4. Contrary to the generally accepted view of increased protein carbonylation with age, the modification of histones was significantly lower in old (30-month-old) than in young (5-month-old) animals. Dietary restriction of older animals for 2 months resulted in increase in carbonylation comparable to that at the young level. These findings may have physiological implications in chromatin structure/function in aging and beneficial effects of DR by influencing transcription, replication, and/or repair activities.

Abstract: BACKGROUND: Japanese monkey, Macaca fuscata, is recognized as the monkey species inhabiting the northernmost area in the world, and thus likely to possess unique fat-depositing mechanisms to resist cold weather in winter. We report that obese females are present in the Wakasa group of Japanese monkey reared in an open enclosure of the Primate Research Institute, Kyoto University. METHODS AND RESULTS: Eight of 12 females were categorized as obese, showing percentage body fat of over 22%. The levels of serum leptin (mean +/- SD, 4.9 +/- 2.3 ng/ml) measured in these obese monkeys were significantly higher than those of non-obese peers of the same group (n = 4; 1.2 +/- 0.5 ng/ml) and another Japanese monkey group (Takahama, n = 14; 0.8 +/- 0.25 ng/ml); however, serum levels of adiponectin, insulin, glucose, hemoglobin A1c, and fructosamine did not differ between obese and non-obese monkeys. Few serum lipid parameters such as triglyceride and cholesterol showed lower levels in obese monkeys than their non-obese peers. CONCLUSIONS: These results show that these obese monkeys in the Wakasa group have not developed obesity-related diseases/disorders such as diabetes. In the Wakasa group, the frequency of obese individuals was high in some maternal lineages, suggesting that genetic factors responsible for obesity may have been inherited in these lineages.

Abstract: We originally identified senescence marker protein 30 (SMP30) as a distinctive protein whose expression decreases in an androgen-independent manner with aging. Here, we report its sequence homology found in two kinds of bacterial gluconolactonases (GNLs) by using the blast search. Then, through a biochemical study, we identify SMP30 as the lactone-hydrolyzing enzyme GNL of animal species. SMP30 purified from the rat liver had lactonase activity toward various aldonolactones, such as d- and l-glucono-delta-lactone, d- and l-gulono-gamma-lactone, and d- and l-galactono-gamma-lactone, with a requirement for Zn(2+) or Mn(2+) as a cofactor. Furthermore, in SMP30 knockout mice, no GNL activity was detectable in the liver. Thus, we conclude that SMP30 is a unique GNL in the liver. The lactonase reaction with l-gulono-gamma-lactone is the penultimate step in l-ascorbic acid (AA) biosynthesis, and the essential role of SMP30 in this synthetic process was verified here by a nutritional study using SMP30 knockout mice. These knockout mice (n = 6), fed a vitamin C-deficient diet, did not thrive; i.e., they displayed symptoms of scurvy such as bone fracture and rachitic rosary and then died by 135 days after the start of receiving the deficient diet. The AA levels in their livers and kidneys at the time of death were <1.6% of those in WT control mice. In addition, by using the SMP30 knockout mouse, we demonstrate that the alternative pathway of AA synthesis involving d-glucurono-gamma-lactone operates in vivo, although its flux is fairly small.

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Abstract: Blue-native and colorless-native gel electrophoresis combined with subsequent 2D-SDS-PAGE and MALDI mass spectrometry are successfully applied for understanding the role of mitochondria in cellular dysfunction, aging, and cellular death. The partial mitochondrial proteome maps of various tissues (liver, brain, kidney, heart, and skeletal muscle) obtained from rat serve now as a database for the elucidation of age-dependent changes, including alterations in protein-protein interactions as well as in posttranslational modifications.

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Abstract: A number of observations have been made to examine the role that mitochrondrial energetics and superoxide anion production play in the aging of wild-type Caenorhabditis elegans. Ultrastructural analyses reveal the presence of swollen mitochondria, presumably produced by fusion events. Two key mitochondrial functions - the activity of two electron transport chain complexes and oxygen consumption - decreased as animals aged. Carbonylated proteins, one byproduct of oxidative stress, accumulated in mitochondria much more than in the cytoplasm. This is consistent with the notion that mitochondria are the primary source of endogenous reactive oxygen species. However, the level of mitochondrially generated superoxide anion did not change significantly during aging, suggesting that the accumulation of oxidative damage is not due to excessive production of superoxide anion in geriatric animals. In concert, these data support the notion that the mitochondrial function is an important aging determinant in wild-type C. elegans.

Abstract: In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.

Abstract: The age-associated spontaneous spongy degeneration in the brain stem of senescence-accelerated mouse (SAM) P8 strain has been suggested to be closely associated with the ability to learn and memorize. In this study, we investigated the effects of dietary restriction (DR) initiated from weaning on learning and memory and histologic changes of the brain stem in P8 and control R1 mice. Although no effect of DR was observed in the retention of the passive-avoidance response in both the P8 and R1 mice, the acquisition of the task was significantly improved by DR in P8 mice. On the other hand, the total area and number of vacuoles in the brain stem was significantly higher in ad libitum-fed (AD)-P8 mice than in AD-R1 mice. However, no significant effect was observed on the vacuole formation in the brain stem of P8 mice by DR. These observations suggest that the improvement of the acquisition of the task by DR in P8 mice is possibly due to changes in neuronal function rather than histologic alteration in brain stem.

Abstract: RATIONALE: Senescence marker protein-30 (SMP30) is a multifunctional protein providing protection to cellular functions from age-associated deterioration. We previously reported that SMP30 knockout (SMP30Y/-) mice are capable of being novel models for senile lung with age-related airspace enlargement and enhanced susceptibility to harmful stimuli. OBJECTIVES: Aging and smoking are considered as major contributing factors for the development of pulmonary emphysema. We evaluated whether SMP30Y/- mice are susceptible to oxidative stress associated with aging and smoking. METHODS: Age-related changes of protein carbonyls in lung tissues from the wild-type (SMP30Y/+) and SMP30Y/- mice were evaluated. Both strains were exposed to cigarette smoke for 8 wk. Histopathologic and morphologic evaluations of the lungs, protein carbonyls and malondialdehyde in the lung tissues, total glutathione content in the bronchoalveolar lavage fluid, and degree of apoptosis of lung cells were determined. MEASUREMENTS AND MAIN RESULTS: In the lungs of SMP30Y/- mice, protein carbonyls tended to increase with aging and were significantly higher than the age-matched SMP30Y/+ mice. Cigarette smoke exposure generated marked airspace enlargement (23.3% increase of the mean linear intercepts) with significant parenchymal destruction in the SMP30Y/- mice but not in the SMP30Y/+ mice (5.4%). The protein carbonyls, malondialdehyde, total glutathione, and apoptosis of lung cells were significantly increased after 8-wk exposure to cigarette smoke in the SMP30Y/- mice. Conclusions: Our results suggest that SMP30 protects mice lungs from oxidative stress associated with aging and smoking. The SMP30Y/- mice could be useful animal models for investigating age-related lung diseases, including cigarette smoke-induced pulmonary emphysema.

Abstract: In spite of the potential benefit of lifelong food restriction to retard aging and extend life span, it is unrealistic in human. The restriction late in life may be more practical. There are, however, only limited studies on the effect of late onset caloric or dietary restriction. We and other investigators have shown that the late life restriction rejuvenates some parameters that decline with age in rats and mice. Although such studies may provide a basis for human application of late-life caloric or dietary restriction, the prolongation of maximum life span would not be expected in view of the current status of the long-lived population in which maximum life span potential appears to have already been achieved. The late life caloric restriction, however, could extend the health span if the extent were appropriate.

Abstract: N-Glycosylases excise the damaged adducts from DNA. 7,8-Dihydro-8-oxoguanine in human cells is repaired by OGG1 and hNTH1. The activities of hOGG1 and hNTH1 were measured, using modified and 32P labelled oligonucleotides, in bronchial biopsy samples of smoking patients with non-small cell lung carcinoma. The activity of hOOG1 was significantly higher in biopsies from tumour tissues compared with intra-individual control samples. On the contrary, the activity of endonuclease III homologue, hNTH1, was lower in tumours compared to controls. These opposing alterations in DNA repair enzymes may affect cancer growth due to the increased formation of AP sites.

Abstract: The hormesis theory purports that biological systems respond with a bell-shaped curve to exposure to chemicals, toxins, and radiation. Here we extend the hormesis theory to include reactive oxygen species (ROS). We further suggest that the beneficial effects of regular exercise are partly based on the ROS generating capability of exercise, which is in the stimulation range of ROS production. Therefore, we suggest that exercise-induced ROS production plays a role in the induction of antioxidants, DNA repair and protein degrading enzymes, resulting in decreases in the incidence of oxidative stress-related diseases and retardation of the aging process.

Abstract: We have tested the hypothesis that training with moderate- (MT), strenuous- (ST), or over- (OT) load can cause alterations in memory, lipid peroxidation, protein oxidation, DNA damage, activity of 8-oxoG-DNA glycosylase (OGG1) and brain-derived neurotrophic factor (BDNF), in rat brain. Rat memory was assessed by a passive avoidance test and the ST and OT group demonstrated improved memory. The content of BDNF was increased only in the OT group. The oxidative damage of lipids and DNA, as measured by thiobarbituric acid reactive substances (TBARS), and 8-hydroxydeoxyguanosine (8-OHdG), did not change significantly with exercise. Similarly, the activity of DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was not altered with exercise training. On the other hand, the content of reactive carbonyl derivatives (RCDs) decreased in all groups and the decrease reached significance levels in the ST and OT groups. The activity of the proteasome complex increased in the brain of OT. The findings of this study imply that over-training does not induce oxidative stress in the brain and does not cause loss of memory. The improved memory was associated with enhanced BDNF content.

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Abstract: Physical exercise above a certain load has been suggested as being a cause of oxidative stress. We have tested whether training with moderate (MT), strenuous (ST), or over (OT) load can cause alterations in the activities of antioxidant enzymes, lipid peroxidation, protein oxidation, DNA damage, or activity of 8-oxoG-DNA glycosylase (OGG1) in rat liver. The levels of corticosterone decreased in all exercising groups but the differences were not significant. Adrenocorticotrophin hormone (ACTH) levels decreased, not significantly, in MT and OT compared to C. Activity levels of antioxidant enzymes did not change significantly in the liver. The levels of reactive carbonyl derivative (RCD) content decreased in the liver of exercising animals, and the differences reached significance between control and moderately trained groups. The changes in the levels of lipid peroxidation (LIPOX) were not significant, but were lower in the exercised groups. The 8-hydroxydeoxyguanosine (8-OHdG) levels increased in the OT group, and the activity of OGG1 measured from crude cell extracts tended to increase in MT and ST. The findings of this study imply that overtraining induces oxidative damage to nuclear DNA, but not to liver lipids and proteins.

Abstract: Dietary restriction (DR) is only one well-established non-genetic experimental means to retard aging in various species of animals. Here we demonstrated that DR in mice for 3 months initiated late in life, at the age of 22 months, partially restores age-related decline of serum apolipoprotein A-IV (apo A-IV) level and the gene expression found in the liver of ad libitum fed young animals as revealed by fasting. In contrast, increase in gene expression of apo C-III by fasting was higher in the aged than that in the young, but it was reduced to the level of young animals in DR counterparts of the aged. In view of the implication that apo A-IV and C-III are involved in the activation and inhibition of lipoprotein lipase, respectively, the adult onset DR can conceivably upregulate the activity of this enzyme that is likely reduced in aged animals and thus improve the lipid metabolism. The present findings suggest that DR initiated even relatively late in life may reduce risk of age-related diseases associated with impaired lipid metabolism.

Abstract: A healthy diet and regular exercise are among the major factors that influence quality of life (QOL) in old age. Exercise is believed to be beneficial to improve QOL, retarding age-related decline of physiological functions and preventing age-related diseases. Regular physical exercise can possibly improve age-related functional decline and delay onset of age-related diseases by attenuating potentially harmful oxidative damage and suppressing inflammatory processes even in older age.

Abstract: Senescence marker protein-30 (SMP-30) has been proposed as an important aging marker and is now functionally identified as a Ca2+ binding protein. SMP-30 has been shown to blunt cell death caused by intracellular Ca2+ accumulation by enhancing plasma membrane Ca(2+)-pumping activity. Although SMP-30 is reported decrease during aging, at present, neither has the mechanism underlying this decrease been fully defined, nor have the mechanisms related to the modulation of SMP-30 been extensively explored. In the current study, we used the well-known anti-aging action of the calorie restriction (CR) paradigm to explore age-related changes in SMP-30 gene expression. The thrust of our investigation was based on CR's ability to defend against age-related oxidative stress and the inflammatory process. The kidney and liver from Fischer 344 rats at 6, 12, 18 and 24 months of age were utilized for this study. The rats were divided into two groups, ad libitum (AL)-fed and 40% restricted CR. Results showed that SMP-30 expression declined with age and that this decline was clearly blunted by CR. To correlate changes between SMP-30 gene expression and the oxidative status, SMP-30 expression and the production of reactive oxygen species (ROS) during aging and free-radical generating lipopolysaccharide (LPS) were monitored. Our data showed that the down-regulation of SMP-30 was accompanied by increased ROS generation and LPS-induced ROS. The potent anti-aging and anti-oxidative action of CR effectively suppressed the age-related down-regulation of SMP-30 by ROS reduction.

Abstract: BACKGROUND: Changes in apolipoprotein (Apo) metabolism can cause an increased incidence of diseases such as cardiovascular disorders and diabetes with advancing age. Limited reports are available on this topic, however. OBJECTIVE: To investigate age-related changes in mobilization of stored lipid, we studied the effects of fasting on the gene expression of Apos in the liver as well as serum triglyceride (TG) and cholesterol levels in the serum. METHODS: Using young (6- to 8-month-old) and old (24- to 28-month-old) fasted and re-fed mice, Northern blots of hepatic mRNAs for Apos A-I, A-IV, C-II, C-III, and liver-type fatty acid-binding protein and HPLC analyses of serum lipids were conducted. RESULTS: Fasting induced 4- and 20-fold increases in the mRNA of Apo C-II and A-IV, respectively, in young mice while only 1.1- and 7-fold increases, respectively, were detected in old mice. In contrast, the Apo C-III gene expression was significantly reduced by fasting in the young mice but the reduction was small in the old. In view of the stimulating effect of Apo C-II and A-IV and the inhibiting effect of C-III on lipoprotein lipase (LPL), these findings suggest that the fasting-induced activation of LPL may be considerably decreased in old mice. The amount of TG in very low-density lipoprotein (VLDL), a major form of the transport of TG to peripheral tissues, was significantly greater in the young than in the old mice. Despite possible activation of LPL by fasting, the amount of TG in VLDL, a major form of the transport of TG to peripheral tissues, was significantly greater in the young mice than in the old. It is indicated that the synthesis of VLDL in the liver is high in the young but low in the old mice, which also may be true for the rate of transport of TG. CONCLUSION: The present findings suggest that mobilization of lipids is impaired in old animals due to decreased gene expression of Apos, possibly leading in the long run to excessive lipid accumulation in tissues such as the liver, adipose tissues and blood vessels even in normal feeding, and resulting in an increased incidence of age-related diseases.

Abstract: The combined effects of aging and regular physical exercise was investigated on the production of reactive oxygen species (ROS), lipid peroxidation, glutathione status, and the activity of nuclear factor-kappaB (NF-kappaB) in rat liver. A group of 24 male F344 rats was divided into the following categories: adult control (18 months), adult exercised (18 months), and aged control (28 months) and aged exercised (28 months). The ROS formation increased as a function of age and exercise training decreased the rate of ROS formation in the two age groups. Significant positive correlation was found between ROS production and lipid peroxidation (LIPOX). The reduced glutathione (GSH) level was higher and the oxidized glutathione (GSSG) level lower in exercised groups compared with the sedentary controls (P<0.05). An age-associated increase in NF-kappaB activity was attenuated by the regular exercise. The content of p50 and p65 subunits of NF-kappaB increased with age and decreased with exercise training. The content of inhibitory factor-kappaB was inversely related to NF-kappaB activation. Regular exercise-induced adaptive responses, including attenuation of an increase in ROS production, LIPOX level, NF-kappaB activation, and reduced GSH/GSSG ratio, appear to be capable, even in old age, of reducing increases in inflammatory and other detrimental consequences that are often associated with advancing age.

Abstract:

Abstract: Alterations of serum apolipoproteins A-I (apo A-I) and A-IV and their mRNAs in young and old mice by fasting and refeeding were investigated by polyacrylamide gel elecrophoresis and Northern blot, respectively. After fasting for three days, serum apo A-I concentration in young mice (6-9 month-old) was increased about 1.5 fold while that of old animals (25-34 month-old) did not change significantly. Apo A-I mRNA was increased about 3-fold and 1.7-fold in the liver and small intestine of the young mice, respectively. The increase in old animals was not more than 1.5-fold in both tissues. The serum apo A-IV was elevated 2-fold and its mRNA was markedly (ca. 50-fold) induced in the liver of fasted young mice, whereas the increase of the mRNA was less than 2-fold in the small intestine. In contrast, induced levels of the protein in serum and its mRNA in both tissues were much less in old mice.In view of the roles of apo A-I and A-IV in triglyceride mobilization and reverse cholesterol transport, the present findings suggest that the reduced induction of the mRNAs for these apolipoproteins in the liver by prolonged fasting and possibly under normal feeding conditions can be an important factor in the impaired immobilization of lipid in old animals, and may, in turn, have implication in age-related diseases such as coronary, cerebral and other vascular disorders.

Abstract: Reactive oxygen and nitrogen species generated either as products of aerobic metabolism or as a consequence of environmental mutagens, oxidatively modify DNA. Formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (endo III) or their functional mammalian homologues repair 7,8-dihydro-8-oxoguanine (8-oxoG) and damaged pyrimidines, respectively, to curb the deleterious effects of oxidative DNA alterations. A single bout of physical exercise can induce oxidative DNA damage. However, its effect on the activity of repair enzymes is not known. Here we report that the activity of a functional homolog of Fpg, human 8-oxoG DNA glycosylase (hOGG1), is increased significantly, as measured by the excision of 32P labeled damaged oligonucleotide, in human skeletal muscle after a marathon race. The AP site repair enzyme did not change significantly. Despite the large individual differences among the six subjects measured, data suggest that a single-bout of aerobic exercise increases the activity of hOGG1 which is responsible for the excision of 8-oxoG. The up-regulation of DNA repair enzymes might be an important part of the regular exercise induced adaptation process.

Abstract: The active involvement of physical exercise in the evolution of a variety of cancers is well documented. However, its role in solid leukemia tumor development is essentially unknown. Solid leukemia tumor cells were transplanted into 21 hybrid BDF1 control mice, exercise-trained mice that did not exercise during leukemia and exercise-trained mice that exercised during leukemia. The tumor size of the continuously exercising group was ~50% of that of control and exercise-terminated animals 18 days after the transplantation. The activity of antioxidant enzymes and the levels of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine were not different in the tumors of the three groups. The level of carbonylated proteins was smaller in tumors of continuously exercising animals. The mutant form of cell regulatory protein p53 and vascular endothelial growth factor were present in similar amounts in the tumor cells of each group. On the other hand, the protooncogene Ras and I-kappaB proteins were present in higher concentrations in tumors of continuously exercising rats. The present data suggest that exercise during leukemia attenuates the development of tumors in mice. The selective alteration of regulatory proteins might play a role in the beneficial effects of exercise during leukemia.

Abstract: Many reports have been published on the effects of lifelong dietary restriction (DR) on a variety of parameters such as life span, carcinogenesis, immunosenescence, memory function, and oxidative stress. There is, however, limited available information on the effect of late onset DR that might have potential application to intervene in human aging. We have investigated the effect of DR initiated late in life on protein and protein degradation. Two months of DR in 23.5-month-old mice significantly reduced heat-labile altered proteins in the liver, kidney, and brain. DR reversed the age-associated increase in the half-life of proteins, suggesting that the dwelling time of the proteins is reduced in DR animals. In accordance with this observation, the activity of proteasome, which is suggested to be responsible for degradation of altered proteins, was found increased in the liver of rats 30 months of age subjected to 3.5 months of DR. Thus, DR can increase turnover of proteins, thereby possibly attenuating potentially harmful consequences by altered proteins. Likewise, DR in old rats reduced carbonylated proteins in liver mitochondria, although the effect was not observed in cytosolic proteins. Fasting induced apoA-IV synthesis in the liver of young mice for efficient mobilization of stored tissue fats, while it occurred only marginally in the old. DR for 2 months from 23 months of age partially restored inducibility of this protein, suggesting the beneficial effect of DR. Taking all these findings together, it is conceivable that DR conducted in old age can be beneficial not only to retard age-related functional decline but also to restore functional activity in young rodents. Interestingly, recent evidence that involves DNA array gene expression analysis supports the findings on the age-related decrease in protein turnover and its reversion by late-onset DR.

Abstract: Dietary restriction (DR) from weaning or young adult stages in rodents throughout their usual life span has been shown to prolong longevity and lower or delay (or both) the occurrence of many late-onset diseases, but the effects of DR when performed at middle age or later have not been well investigated. We have studied the effect of DR initiated late in life on altered proteins and protein degradation. DR initiated in 23.5-month-old mice and continued for 2 months reduced the amount of altered proteins and shortened the half-life of proteins, including oxidatively modified ones. Furthermore, our more recent studies in rats demonstrated that DR initiated later in life (26.5 months old) restored the activities of proteasomes that have been implicated in removal of altered proteins. Thus, DR initiated even relatively late in life appears to have beneficial effects, restoring an animal's youthful condition in terms of the age-related accumulation of altered proteins.

Abstract: The nematode Caenorhabditis elegans has proven a robust genetic model for studies of aging, including the roles of oxidative stress and protein damage. In this review, we focus on the genetics of select long-lived (e.g., age-1, daf-2, daf-16) and short-lived (e.g., mev-1) mutants that have proven useful in revealing the relationships that exist among oxidative stress, life span, and protein oxidation. The former are known to control an insulin/IGF-1-like pathway in C. elegans, while the latter affect mitochondrial function.

Abstract: Regular physical exercise retards a number of age-associated disorders, in spite of the paradox that free radical generation is significantly enhanced with exercise. Eight weeks of treadmill running resulted in nearly a 40% increase in maximal oxygen uptake in both middle-aged (20-month-old) and aged (30-month-old) rats. The age-associated increase in 8-hydroxy-2'-deoxyguanosine (8-OHdG) content was significantly attenuated in gastrocnemius muscle by exercise. The 8-OHdG repair, as measured by the excision of 32P-labeled damaged oligonucleotide, increased in muscle of exercising animals. The reactive carbonyl derivatives (RCD) of proteins did not increase with aging. However, when the muscle homogenate was exposed to a mixture of 1 mM iron sulfate and 50 mM ascorbic acid, the muscle of old control animals accumulated more RCD than that of the trained or adult groups. The chymotrypsin-like activity of proteasome complex increased in muscle of old trained rats. We suggest that regular exercise-induced adaptation attenuates the age-associated increase in 8-OHdG levels, and increases the activity of DNA repair and resistance against oxidative stress in proteins.

Abstract: The ability of hepatocytes isolated from young (7-10 months) and old (31 months) male F344/Jcl and F344/DuCrj rats to express heat shock protein (hsp) 27, hsp70 and hsp90 was determined after a mild heat shock (42.5 degrees C for 30 min). The induction of these three mRNA levels by the heat shock was 50-80% lower in hepatocytes isolated from old F344/Jcl rats than in those from young rats. However, the hepatocytes from old F344/DuCrj showed a marked increase (200-250%) in the induction of hsp mRNAs by heat shock when compared to cells from young rats. Because heat shock transcription factor (HSF) plays a critical role in regulating the transcription of hsp genes, the effect of age on the binding activity HSF to heat shock element (HSE) was also studied. Again, the induction of binding activity of HSF to HSE was significantly increased with age in hepatocytes from F344/DuCrj rats while the reverse was true for the cells from F344/Jcl. The induced levels of hsp mRNAs were positively correlated with the binding activity of HSF to HSE in hepatocyte extracts from both F344 substrains, suggesting that the diverse age-related changes of heat-shock response in F344 substrains occurs in HSF activity. The contradictory age-related change in the heat-shock response is discussed with the differences in biochemical and genetic properties of substrains of F344 rats.

Abstract: Many studies have shown that lifelong dietary restriction (DR) can retard aging processes. Very few reports, however, are found that examined the effect of late onset DR on biochemical parameters in aging animals [Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002b. Dietary restriction initiated in late adulthood can reverse age-related alterations of protein and protein metabolism. Ann. NY Acad. Sci. 959, 50-56]. We studied the effect of every-other-day feeding, initiated at the age of 26.5 months and continued for 3.5 months, on antioxidant enzymes, protein carbonyls, and proteasomes of the gastrocnemius muscle and tendon in rats. Age-related increase in the activity and content of Cu, Zn-SOD and the content of Mn-SOD was attenuated by the DR in both tissues. The same was true for glutathione peroxidase and catalase activities. Significant increase with age in protein reactive carbonyl derivatives (RCD) in the tendon was noted that was partially reversed by the DR. No significant change of RCD, however, was observed in the skeletal muscle. The age-related and DR-induced changes of the RCD in the tendon appeared to be associated with proteasome activity that decreases with age and increases by the DR. It is suggested that the late onset DR can have beneficial effects on the locomotive functions by reducing age-associated potentially detrimental oxidative protein damage in the tendon.

Abstract: The biochemical mechanisms by which regular exercise significantly benefits health and well being, including improved cognitive function, are not well understood. Four-week-old (young) and 14-month-old (middle aged) Wistar rats were randomly assigned to young control and young exercised, middle-aged control and middle-aged exercised groups. Exercise groups were exposed to a swimming regime of 1 h a day, 5 days a week for 9 weeks. The passive avoidance test showed that middle-aged exercised rats had significantly (P<0.05) better short- (24 h) and long-term (72 h) memory than aged-matched control rats. Conditioned pole-jumping avoidance learning was improved markedly in both age groups by exercise. Brain thiobarbituric acid-reactive substances and 8-hydroxy-2'deoxyguanosine content in the DNA did not change significantly, while the protein carbonyl levels decreased significantly (P<0.05) in both exercised groups. This decrease was accompanied by an increase in the chymotrypsin-like activity of proteasome complex in the exercised groups, whereas trypsin-like activity did not differ significantly between all groups. The DT-diaphorase activity increased significantly (P<0.05) in the brain of young exercised animals. These data show that swimming training improves some cognitive functions in rats, with parallel attenuation of the accumulation of oxidatively damaged proteins.

Abstract: Exercise increases the generation of reactive oxygen and nitrogen species (RONS) and by causing adaptation, could decrease the incidence of RONS-associated diseases. A single bout of exercise, depending upon intensity and duration, can cause an increase in antioxidant enzyme activity, decrease levels of thiols and antioxidant vitamins, and result in oxidative damage as a sign of incomplete adaptation. Increased levels of RONS and oxidative damage are initiators of a specific adaptive response, such as the stimulation of the activation of antioxidant enzymes, thiols, and enhanced oxidative damage repair. Regular exercise has the capability to develop compensation to oxidative stress, resulting in overcompensation against the increased level of RONS production and oxidative damage. Regular exercise causes adaptation of the antioxidant and repair systems, which could result in a decreased base level of oxidative damage and increased resistance to oxidative stress.

Abstract: Aging is characterized by accumulation of potentially harmful altered proteins that could lead to gradual deterioration of cellular functions and eventually result in increased probability of death. Metabolic turnover of proteins thus plays an essential role in maintaining the life of an organism. In this article we summarize our current knowledge on age-related changes in protein turnover with special reference to degradation. Increase in half-life of proteins with advancing age is well documented. Qualitative rather than quantitative changes of proteasomes appear to be responsible for this change. Dietary restriction and moderate long-term exercise seem to restore higher proteasome activity and turnover rate of proteins in aged animals.

Abstract:

Abstract: We were interested in the effects of immobilization (IM), a single bout of exercise (E) and immobilization followed by exercise (EIM) on memory and oxidative damage of macromolecules in hippocampus of rat brain. Eight hours of IM resulted in impairment of passive avoidance test (memory retrieval deficit) and increased latency to start locomotion in an open-field test. Two hours of swimming did not significantly alter the memory retrieval deficit and latency, while the EIM group had longer latency and similar memory than control and E groups. The oxidative damage of lipids, proteins and nuclear DNA increased significantly in IM group and no increase was observed in E and EIM animals. The activity of proteasome was not altered in any groups. The activity of glutamine synthetase (GS) was decreased in IM group (P < 0.05), this down regulation was not observed in E and EIM groups. These data suggest that oxidative damage of macromolecules is associated with impaired cognitive function. Single bout of exercise after immobilization eliminates the oxidative damage of macromolecules and normalizes memory function, probably by its ability to restore the activity level of GS and eliminate the consequences of immobilization-induced prolonged efflux of glutamate.

Abstract: Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.

Abstract: Life long dietary restriction has a variety of beneficial effects such as retarding the age-related decline of physiological functions as well as the onset of diseases, thus prolonging the life span of experimental animals. In the present work we investigated the effect of DR initiated late in life on rats regarding oxidative modifications of liver mitochondrial proteins in terms of generation of carbonyl moieties by western blot using antibodies against dinitrophenylhydrazones after the reaction of the proteins with dinitrophenylhydrazine. Protein carbonyls in mitochondria of 30-month-old animals increased by about 55% as compared with their 10-month-old counterparts. Every-other-day feeding initiated at the age of 26.5 months and continued for 3.5 months resulted in the decrease of the carbonyls to the level of the ad libitum fed young animals. Our findings suggest that DR initiated late in life can have a beneficial effect by reducing the accumulated oxidative damage in mitochondrial proteins.

Abstract: Both regular physical exercise and low levels of H(2)O(2) administration result in increased resistance to oxidative stress. We measured the accumulation of reactive carbonyl derivatives and the activities of proteasome complex and DT-diaphorase in cardiac muscle of trained and untrained rats after chronic i.p. administration of 1 ml t-butyl H(2)O(2) (1 mmol/kg for 3 weeks every second day). Twenty-four rats were randomly assigned to a control group administered with saline, control administered with H(2)O(2), and exercised administered either saline or H(2)O(2). The activity of DT-diaphorase significantly increased in H(2)O(2) administered and exercised groups, indicating that an increase in H(2)O(2) levels stimulate the activity of this enzyme. The cardiac muscle of H(2)O(2) administered nonexercised animals accumulated significantly more carbonyl than control group (P < 0.05). The exercise and H(2)O(2) administration resulted in less oxidatively modified protein than found in nonexercised groups (P < 0.05). The peptide-like activity of proteasome complex was induced by the treatment of H(2)O(2) and exercise and exercise potentiate the effect of H(2)O(2). On the other hand, the chymotrypsin-like and trypsin-like activities were stimulated only by physical training and H(2)O(2) administration. The data suggest that chronic administration of H(2)O(2) after exercise training decreases the accumulation of carbonyl groups below the steady-state level and induces the activity of proteasome and DT-diaphorase. Hence, the stimulating effect of physical exercise on free radical generation is an important phenomenon of the exercise-induced adaptation process since it increases resistance to oxidative stress. Regular exercise training is a valuable physiological means of preconditioning the myocardium to prolonged oxidative stress.

Abstract: The oxygen flux into the mitochondria of skeletal muscle increases with exercise. However, the extent of oxidative damage to mitochondrial proteins of skeletal muscle has only been estimated. We studied the alteration of reactive carbonyl derivatives (RCD) in mitochondrial and cytosolic fractions of skeletal muscle following 9 weeks of swimming training in rats. The RCD content of mitochondria was significantly elevated compared with the cytosolic fraction of both control and exercised animals. Accumulation of RCD in muscle mitochondria of the exercised group was also significantly elevated (P < 0.05). On the other hand, alteration of the accumulation of RCD was not apparent in the cytosolic fraction of skeletal muscle. The activity of proteasome complex, however, was increased in the cytosolic fraction of exercised muscle (P < 0.05). The data suggest that mitochondria of skeletal muscle accumulate significantly larger amounts of RCD than the cytosolic fraction and the tendency of the accumulation varies in cell fractions. Exercise training increases the accumulation of protein damage in mitochondria of skeletal muscle but cytosolic proteins are protected by increased activity of proteasome complex and possibly by other antioxidant enzymes.

Abstract: Moderate daily exercise is known to be beneficial to health, reducing risks of a number of age-related disorders. Molecular mechanisms that bring about these effects are not clear. In contrast, it has been claimed that some types of prolonged physical exertion are detrimental to health because active oxygen species are generated excessively by enhanced oxygen consumption. Using two age groups of rats, young (4 week) and middle aged (14 months), we investigated the effects of long-term swimming training on the oxidative status of phospholipids, proteins, and DNA. The concentration of thiobarbituric acid reactive substances and 4-hydroxynonenal protein adducts did not differ in the gastrocnemius muscle between exercised and nonexercised animals in the two age groups. The extent of carbonylation in a protein of molecular weight around 29 KDa and the amount of 8-hydroxydeoxyguanosine in nuclear DNA were smaller (p<.05) in the exercised rats than in the sedentary animals. Activities of DT-diaphorase (C1: 29.3+/-1.9; C2: 36.1+/-2.6; E1: 27.2+/-1.3; C2: 33.4+/-2.9 nmol/mg protein) and proteasome, a major proteolytic enzyme for oxidatively modified proteins were significantly higher in the exercised animals of both age groups (p<.05). The adaptive response against oxidative stress induced by moderate endurance exercise constitutes a beneficial effect of exercise.

Abstract: Protein carbonyls were studied in aging and exercise by immunoblot followed by one- or two-dimensional polyacrylamide gel electrophoresis using antibodies against 2,4-dinitrophenylhydrazones. Proteins of rat kidneys exhibited significant age-related increase in the amount of carbonyl while those of the brain and liver did not. Major carbonylated proteins in the kidney included serum albumin. In nematodes in which protein carbonyls increased with age, one of the carbonylated proteins was identified as vitellogenin, an egg-yolk protein. A possible biological significance of this protein present in abundance even after egg-laying stages is discussed in terms of protection against oxidative stress. Exhaustive exercise induced significant increase in the carbonylation of selected but unidentified proteins in the lung. This oxidative stress might be caused by xanthine oxidase in this tissue and hypoxanthine derived from ATP-depleted muscles. Exercise at high altitude caused higher carbonylation of the skeletal muscle proteins, most notably a protein likely to be actin, than that at sea level but no significant difference was observed in lipid peroxidation. These studies emphasize the value of immunoblot analysis of tissue protein carbonyls in a variety of situations where oxidative stress is likely involved.

Abstract: Recently we found that protein carbonyl content increases with age in wild-type as well as long- and short-lived Caenorhabditis elegans nematodes in inverse correlation with life span (Adachi et al., J. Gerontol. 53A, B240-B244, 1998; Yasuda et al., J. Gerontol. 54A, B47-B51, 1999). In the present study, we investigated carbonyl modification of individual proteins in young and old wild-type nematodes by two-dimensional immunoblot using antibodies against 2, 4-dinitrophenylhydrazones. A protein with apparent molecular weight of 110 kDa was found to be a major carbonylated protein in aged animals. Amino acid sequence of peptide fragments of the protein was identical to that of vitellogenin-6, a yolk protein synthesized in and secreted from the intestine during egg-laying stage. Although the function(s) of the protein in aged nematodes is unclear, we suggest that the protein may have a role to protect other cellular components from oxidation because of its metal binding capacity.

Abstract: It is known that acute physical exercise may have diverse pathophysiological consequences in various organs due to free radical formation. We have investigated whether a period of anaerobic running to exhaustion in rats results in oxidative modification of proteins in the lungs. Six rats of an exercised group (E) ran for two periods of 5 min at a speed of 30 m.min-1 followed by a recovery period of 5 min, and then by a third period of running to exhaustion. Reactive carbonyl derivatives (RCD) were measured by the Western blot technique on lungs of E and control (C) rats. In addition, the activity of glutamine synthetase (GS) was also monitored as marker of oxidative damage to proteins. This investigation revealed significant exercise-induced increases in accumulation of RCD in the lungs of the E group compared with the C group. The RCD signals were visibly stronger in proteins with molecular weight of 55 kDa and 32 kDa. The activity of GS was higher by about 30% in E rats than in C rats. The present data suggest that anaerobic exercise induces protein oxidation in the lungs.

Abstract: Both exposure to high altitude (HA) and caloric restriction (CR) may influence free radical generation. The aim of the present study was to evaluate the effects of 4 wk chronic exposure to 4,000m of HA and CR (60% of CR of control (C) rats) on protein oxidation in brain. Eighteen rats with similar body mass were assigned to CR, HA and C rats. Reactive carbonyl derivatives (RCD), marker of protein oxidation, were measured by Western blot. In addition, the activity and protein content of glutamine synthetase (GS) were determined. The body mass of C rats was significantly higher (P< 0.001) than that of HA and CR groups. The quantified signal intensity of RCD was significantly stronger in C rats than in HA and CR rats. The activity of GS was significantly increased in CR rats, while the protein content of GS was decreased in HA rats compared to C group. The data suggest that both HA and CR decreases the accumulation of RCD in the brain, however the mechanism of the decrease seems to be different during HA and CR.

Abstract: Differences in trabecular and cortical bone loss have been demonstrated clinically, but differences in bone loss at different skeletal sites remain unclear. We examined regional variations in bone loss histomorphometrically in two strains of mice in which osteopenia progresses spontaneously: tiptoe-walking Yoshimura (twy) mice (from 4 to 37 weeks of age) and senescent ICR mice (from 4 to 88 weeks of age). Morphometrical measurements were obtained to investigate the changes with age in trabecular bone area and anterior cortical bone width in the lumbar vertebral body, trabecular bone area in the tibia, bone area in the parietal bone, and the cortical index in the humerus. Results showed that, in twy mice, trabecular turnover was higher than in ICR mice, and bone loss progressed in the following order: tibial trabecular bone, lumbar trabecular bone, parietal bone, lumbar anterior cortical bone, and the humerus. In ICR mice, bone formation declined after 60 weeks. Bone loss progressed in tibial trabecular bone and the parietal bone at 60 weeks of age, followed by lumbar trabecular bone, lumbar anterior cortical bone, and the humerus at 88 weeks of age. Bone loss varied at each site and between the two mouse strains, with different bone turnover rates. The findings of the present study indicate that special attention should be paid to regional variations in the progression of bone loss associated with differences in pathologic features.

Abstract: Accumulation of altered proteins in old animals has been ascribed to slower turnover of proteins. Since proteasomes can be regarded as the major proteolytic enzymes responsible for the degradation of the majority of cellular proteins, we examined age-related changes of 20S and 26S proteasomes in the liver of young (8-10-month-old), middle-aged (15-18-month-old) and old (25-28-month-old) Fischer 344 male rats. The two forms of proteasomes were separated by glycerol gradient centrifugation. Fluorogenic peptides were used as substrates to evaluate three types of peptidase activities. The ratio of peptidase activities in the 20S proteasome vs. those in the 26S form did not appear to change with age. Unstimulated chymotrypsin-like activity found only in the 26S form decreased by 30% in the old rats as compared with that in the young ones, while no change in the activity was observed during aging when stimulated by sodium dodecyl sulfate. The trypsin-like activity declined significantly by 17% to an apparently similar extent in both 20S and 26S forms. The peptidylglutamyl peptide hydrolyzing activity exhibited gradual decrease with age, resulting in 60% lower value in the old rats as compared with the young animals. These changes are considered to account for the age-related extension of half-life of proteins. Since the amount of total proteasomes measured by immunoblot did not appear to change with age, posttranslational modifications or subunit replacement is possibly responsible for the decrease in the activities.

Abstract: We examined in vivo the effect of dietary fats and oils with different peroxidizability on protein carbonyl content, the presumed index of free radical-mediated protein oxidation. For 15.6 months, SHRSP (stroke-prone spontaneously hypertensive strain) rats were fed a diet supplemented with lard, safflower oil, perilla oil or fish oil/soybean oil, the peroxidizability of which increases in this order. The peroxidizability of tissue lipids was positively correlated with the protein carbonyl content in skeletal muscle, but not in the brain, heart or liver. The protein carbonyl content in the lard group was higher in the brain and liver compared to the other dietary groups. These results contradict the concept that long-term feeding of easily autoxidizable fatty acids allows the accumulation of lipid peroxides to accelerate the development of the free radical diseases, and suggest that tissue protein carbonyl content is not a simple reflection of autoxidizability-related lipid peroxidation but is also influenced by other biochemical processes.

Abstract: The oxidative stress related consequences of physical training at high altitude are not known. The hypothesis was tested that physical training and exposure to high altitude have adverse effects on free radical generation and activities of antioxidant enzymes. The present results showed that 4 weeks of exercise at an altitude of 4000 m increased the activity of Mn-SOD in both white and red types of skeletal muscle. The activities of Cu,Zn-SOD, catalase, and glutathione peroxidase, as well as the level of lipid peroxidation measured by TBARS and lipid hydroperoxides, did not change significantly. In contrast, the level of reactive carbonyl derivatives measured by anti-2,4-dinitrophenylhydrazone antibodies and spectrophotometry showed an increase in both types of muscle of altitude trained rats compared with sea level trained and control groups. It was suggested that the oxidative modification of certain amino acids is due to the increasing gap between activity of SOD and peroxide scavenging enzymes, which results in increases in the number of hydrogen peroxide molecules. Thus, since the mechanism of generation and/or the mode of action of radicals resulting in lipid peroxidation and protein oxidation appears to be different in vivo, both processes should be studied during oxidative stress.

Abstract: Turnover of mRNAs could be influenced not only by the synthesis of different mRNA species but also by the altered levels of mRNA-degrading enzymes such as RNases and their endogenous inhibitors. In the present work we evaluated possible age-related changes in the mRNA levels of pancreatic as well as L type RNase inhibitors in five different tissues of the BDF1, SAMR1 and SAMP8 using Northern blots. The mRNA levels varied depending on the tissues and mouse strains studied. In certain instances such as the RNase L inhibitor mRNA levels in the lung of SAMP8, there was a statistically significant (P < 0.05) increase of 40% if we compared the young (3 months old) and old (18 months old) animals. These changes could possibly contribute to a certain extent to the already lower levels of mRNAs due to decreased transcriptional activities in aged animals.

Abstract: Protein carbonyls are reported to increase in aging and in pathologies such as Alzheimer's disease and ischemic injury. Detailed study of this important issue has, however, been hampered by lack of an appropriate method to identify individual carbonylated proteins. We describe here an immunoblot method to identify individual carbonylated proteins. We describe here an immunoblot method to investigate protein carbonyls reactive to 2,4-dinitrophenyl hydrazine. Rabbit polyclonal antibodies against 2,4-dinitrophenyl hydrazine were used to study the proteins derivatized by the reagent in one- or two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting. More than 25 proteins with high carbonyl contents were clearly demonstrated in two-dimensional immunoblot of rat tissue soluble proteins. The method could detect concentrations as low as 1 pmol of carbonyls. The signals were mostly abolished by prior treatment of tissue proteins with sodium borohydride to reduce carbonyls. Fragments generated by V8 protease digestion of a single protein exhibited signal intensities of varying degrees, indicating that carbonylation is not uniform in different amino acid sequences. Proteins treated with glucose or aldehydes gave rise to positive signals, suggesting that the finding of carbonyls in tissue proteins is not necessarily an indication of direct oxidation of side chains of amino acid residues.

Abstract: It has been demonstrated that the amount of ubiquitin (Ub)-protein conjugates increases in the whole brain homogenates of old mice. To determine the specificity of the accumulation of the conjugates, we further investigated age-related changes in Ub-protein conjugates in other tissues (heart, liver, and kidney), and also in various regions (cerebrum, cerebellum, brain stem, and medulla oblongata) of the brain. The amount of high-molecular-weight Ub-protein conjugates (HMW, > 60KDa) increased remarkably in all regions of the brain examined, especially in the cerebellum, of senescent mice, but not much in other tissues. No appreciable difference in the size of free Ub pool was found between young and old mice except in the medulla oblongata. This suggests that the increase in Ub-protein conjugates is not simply due to a rise in the free Ub pool. The activity of isopeptidases in homogenates was estimated by measuring rates of deubiquitination of endogenous Ub-protein conjugates under ATP-depleted conditions. In the brain stem and medulla oblongata, they decreased significantly with age. In the cerebrum and cerebellum, however, no significant difference was observed between young and old animals. These results partially explain the increases observed in medulla and brain stem, but cannot explain the increases in cerebrum and cerebellum.

Abstract: The effects of peroxisome proliferators on the levels of rat serum proteins were examined. The proliferators reduced the levels of several proteins in various ways with respect to proliferator-specificity and the time courses of the changes. The identification of three proteins by amino acid sequencing showed that they were functionally unrelated. This diversity suggests that several primary and secondary effects of the peroxisome proliferators caused the various changes in the levels of several serum proteins. Among these, apolipoprotein E was the most but transiently down-regulated by the two proliferators tested. Northern blots of the rat liver mRNA suggested that the primary step of the down-regulation was at pre-translation.

Abstract: Cerebral deposition of beta-amyloid peptide (beta A) is a hallmark of Alzheimer's disease. Concentration of beta A could play a critical role in the rate of amyloid deposition. It is therefore of considerable importance to identify proteases involved in processing of beta A. 125I-labeled synthetic beta A specifically cross-linked to a single protein with M(r) = 110,000 in cytosol fractions from rat brain and liver. This protein was identified as insulin degrading enzyme (IDE) since the labeling of the 110 kDa protein was completely blocked by an excess of insulin, and anti-IDE monoclonal antibodies precipitated the labeled protein. Purified rat IDE effectively degraded beta A.

Abstract: Progress in molecular biological investigations of aging in Japan is overviewed. Emphasis is put on studies which, in the author's opinion, appear to have considerable relevance to a definition of aging, i.e. functional decline of cells and tissues with advancing age. (1) Changes in the nuclear DNA, most significantly in methylation, and deletions of mitochondrial DNA have been shown to occur with age. (2) Various aspects of protein metabolism have been investigated, i.e. the fidelity of translation, accumulation of altered enzymes, oxidative damage, and half-lives and degradation of proteins. (3) Japanese researchers have made significant contributions to the understanding of the chemical structure and the mechanisms of generation of paired helical filaments in Alzheimer's disease. Also, studies on beta-amyloid peptide are noteworthy. (4) Murine models of aging were developed and molecular biological investigations on them are progressing. Nematodes and fruit flies are also used as models.(5) Changes in gene expression with age have attracted considerable interest.

Abstract: Protein histidine kinase (Motojima, K., and Goto, S. (1993) FEBS Lett. 319, 75-79) and phosphatase in rat liver extract were characterized. The histidine kinase was recovered mostly in the membrane and the phosphatase in the soluble fraction. The kinase and its substrate 36-kDa protein (P36) were co-solubilized from the membrane under conditions in which most of the other kinases, and their substrate proteins were not solubilized. The solubilized kinase and P36 were co-eluted after high pressure liquid chromatography gel filtration, showing an apparent molecular mass of 70-75 kDa. They were also co-eluted after ion exchange chromatography. These characteristics, together with its complete resistance to genistein, indicate that the rat liver histidine kinase is not cognate to the yeast enzyme (Huang, J., Nasr, M., Kim, Y., and Matthews, H.R. (1992) J. Biol. Chem. 267, 15511-15515). The phosphatase that dephosphorylates histidyl-phosphorylated P36 was also studied using rat liver subcellular fractions and in vitro phosphorylated P36 as the substrate. The characteristics of the phosphatase, that is, 1) Mg2+ requirement for activity, 2) apparent molecular mass of 45 kDa by high performance liquid chromatography gel filtration, and 3) resistance to 100 microM okadaic acid, suggest that the primary phosphatase active in vitro is protein phosphatase 2C.

Abstract: Removal of a peroxisome proliferator from the diet triggered the degradation of peroxisomes and induced the transient expression of a 220 kDa soluble protein in rat liver. The 220 kDa protein was purified by conventional methods and analyzed by amino acid sequencing. A total of 99 amino acid residues in 4 lysylendopeptidase-digested peptides completely matched those in rat fatty acid synthase. The transient induction of fatty acid synthase mRNA during peroxisome degradation was confirmed by Northern blotting.

Abstract: A novel protein kinase is induced in rat liver plasma membrane by the administration of peroxisome proliferators. A 36 kDa protein (P36) on the membrane was rapidly phosphorylated in vitro by the kinase and the phosphorylated amino acid was identified as phosphohistidine. Histidine phosphorylation of P36 was activated in vitro by recombinant Ras protein and GTP; both decreased Michaelis constant (Km) for ATP from 1.25 to 0.25 microM. The novel histidine kinase, products of which have been overlooked due to their acid lability, may participate in cellular signaling and peroxisome proliferators may perturb the pathway.

Abstract: We investigated age-related changes in the ubiquitination of histones in mouse brains. By Western blot analysis using rabbit anti-ubiquitin antibodies, histone H2A and H2B were found to be the only proteins that are ubiquitinated in the acid-extractable nuclear proteins of both young (4-month-old) and senescent (29-month-old) mouse brains. The proportion of ubiquitinated histones was about 30% higher in senescent mice than in young ones. When total brain homogenates were analyzed similarly using somewhat different age groups of the animals, the proportion in the old animals was about 2-fold higher than that in the young.

Abstract: The changes in protein phosphorylation during induction and deinduction of peroxisome proliferation in rat liver by three types of proliferators were studied by in vitro phosphorylation assay. Among the variously phosphorylated proteins, an increase during induction and a decrease during deinduction in phosphorylation of P100, a cytosolic protein having a molecular weight of 100 kDa, was most remarkable. The time course of enhancement of phosphorylation by the administration of the proliferators, however, was not parallel with proliferation of peroxisome but with increase in the liver DNA content. Amino acid sequencing of the protein indicated the identity of its N-terminal 17 amino acid residues with those of elongation factor 2 (EF2). Increase in the amount of EF2 by peroxisome proliferators was confirmed by immunoblotting and this was almost parallel with peroxisome proliferation, suggesting that both increase in the amount of EF2 and some changes in phosphorylation activities account for a large increase in in vitro phosphorylation of EF2 by the administration of peroxisome proliferators.

Abstract: Administration of clofibrate in rat results in down-regulation of several liver proteins and a vast induction of peroxisomal proteins. One protein was identified as BiP/GRP78 using antibodies and cDNA cloning. The level of mRNA was reduced by the drug.

Abstract: The effect of clofibrate, a hypolipidemic drug and a potent peroxisome proliferator, on expression of a nonperoxisomal transthyretin (prealbumin) gene was studied using rats fed clofibrate for various periods. Upon feeding a diet containing clofibrate, the level of transthyretin mRNA was down-regulated, reaching 20% of the control level, in an almost reciprocal time course to that of increases in the levels of peroxisomal mRNAs. Studies on expression of other steroid hormone regulated genes suggest that clofibrate may down-regulate several but not all types of steroid hormone regulated mRNA expression.

Abstract: A cDNA clone encoding ribonuclease inhibitor was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a high degree of conservation of a repeated structure. The mRNA was detected in all seven tissues of rat examined, the amount being highest in the lung and lowest in the heart.

Abstract: BC-1 RNA is a brain-specific small RNA transcript of identifier sequences present in the somas and dendrites of neurons. We recently reported that the RNA is complexed with a protein(s) to form a 10 S ribonucleoprotein particle (Kobayashi, S., Goto, S., and Anzai, K. (1991) J. Biol. Chem. 266, 4726-4730). We demonstrate here that this 10 S BC-1 ribonucleoprotein particle contains a DNA-binding protein(s) (Bp-1 protein) capable of interacting with a region between split promoter sequences for RNA polymerase III within the identifier sequences. The region has short inverted repeats: a perfect octanucleotide repeat (GCGCTTGCCTAGCAAGCGC) and an imperfect heptanucleotide repeat (GCCTAGCAAGCGCAAGGC), each of which contains a GCAAG/CTTGC motif. We also demonstrate that the binding of this protein either to the array of pentamer motifs or to BC-1 RNA is mutually exclusive. The molecular masses of photo-cross-linking adducts of Bp-1 protein to a 32P-labeled GCAAG/CTTGC motif-specific probe were estimated to be about 31 and 36 kDa, indicating that two species of Bp-1 proteins may be present in the brain.

Abstract: The degradation rate of [(3)H]leucine labeled intracellular proteins was compared in hepatocytes derived from young (4-7-month-old) and aged (23-month-old) mice. Average half-lives of long-lived proteins were 40 h and 65 h in the cells from young and old animals, respectively. This observation confirms our earlier results that half-lives of foreign proteins introduced into the cells from old mice are 50-60% longer than those of younger counterparts (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133; Ishigami and Goto, 1990, Arch. Biochem. Biophys., 277, 189-195).

Abstract: Identifier sequences are transcribed to generate a brain-specific BC-1 RNA present as a ribonucleoprotein particle in the dendrites and somata of neurons. This ribonucleoprotein particle contains an identifier sequence-binding protein (Bp-1 protein). We report here the purification of BC-1 RNA and demonstrate that Bp-1 protein interacts directly with the RNA. We also demonstrate an accumulation of Bp-1 protein in the nucleus of brain cells from mouse fetus and newborns that precedes the postnatal increase in BC-1 RNA. Cytoplasmic Bp-1 protein present in a complex with BC-1 RNA increases postnatally with a concomitant decrease in nuclear Bp-1 protein. These observations suggest that Bp-1 protein may play a role(s) in the synthesis and nuclear export of BC-1 RNA.

Abstract: BC-1 RNA is a small RNA transcript of the identifier repetitive sequences present in rodent genomes. The RNA has been reported to be specific to the brain and confined to the cytoplasm. The RNA level increases during the 1st month after birth. To understand its cytoplasmic function, it seems important to examine whether BC-1 RNA is present as an RNP. It is believed that the protein component may govern the functions of BC-1 RNA in the brain cells. In the present report, we have demonstrated that BC-1 RNA is not free but complexed with proteins to form a 10 S RNP in the cytoplasm. We have also shown that the 10 S RNP is not associated with cytoplasmic structures such as polysomes/ribosomes or microsomes. The buoyant density of the RNP was 1.26 g/cm3 in metrizamide. Furthermore, some of the protein components were shown to be in direct contact with RNA, since photo-cross-linking adducts of protein to BC-1 RNA were identified upon UV irradiation of the 10 S BC-1 RNP.

Abstract: The rat chromosomal gene for uricase was cloned. The gene spans more than 20 kilobases and the coding region is divided into 8 exons by 7 introns. The organization of the rat uricase gene is so greatly different from that of the soybean gene that the difference may not have been caused only by the removal of some ancestral introns during the period of widely separated evolution.

Abstract: The rat chromosomal gene for transthyretin was cloned and its dual promoter region was analyzed by primer extension. Semi-quantitative studies by primer extension analysis to determine the transcriptional start sites and their usage in the three tissues have suggested that the rat transthyetin gene has dual promoters: a major proximal promoter which is used in the liver and in the brain but not at all in the kidney, and a minor distal promoter which is totally used in the kidney and significantly in the brain.

Abstract: Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.

Abstract: Presence of two uricase mRNA species in rabbit was revealed by cDNA cloning. They were different only in the 5'-non-coding region. Their hepatic and extra-hepatic expression in a tissue-specific manner was demonstrated by Northern blot and primer extension analyses.

Abstract: Active oxygens have been suggested to be involved in age-related alterations of organelles and molecules. In this study we investigated the influence of active oxygen on aminoacyl-tRNA synthetases partially purified from rat liver. Treatment of leucyl-tRNA synthetase with Fe3(+)-ascorbate resulted in the increased heat-lability of the enzyme. The inactivation was inhibited by radical scavengers such as mannitol and benzoate, suggesting that hydroxyl radicals are responsible for heat-labilization of the enzyme. On the other hand, a considerable part of tyrosyl-tRNA synthetase was converted to heat-labile forms without added iron and ascorbate under aerobic conditions but not under anaerobic conditions. These and other findings suggested that the heat-labilization of this enzyme is caused by active oxygens probably generated by the reaction of dioxygen and transition metal ions present in the enzyme preparations. Heat-inactivation curves of the enzymes modified as described above were similar to those observed for the enzymes from aged animals in that these enzymes exhibited higher percentages of heat-labile forms than the unmodified enzymes from young animals [Takahashi and Goto, 1987, Arch. Gerontol. Geriatr. 6, 73-82; Takahashi and Goto, 1987, Arch. Biochem. Biophys. 257, 200-206]. The present findings are consistent with the theory that active oxygens are involved in the age-related alterations of enzymes.

Abstract: We previously reported that the half-life of protein degradation in cells from old mice is about 50% longer than that in cells from young or middle-aged ones. In the present study we investigated the degradation rate of microinjected proteins (horseradish peroxidase (HRP) and ovalbumin (OVA] and pulse-labeled proteins in hepatocytes from dietary-restricted old mice. Dietary restriction was initiated when mice were 23 months of age and performed in two steps (first 80% and then 60% of the ad libitum intake), the total period being 70 days. Hepatocytes were isolated from mice fed a restricted diet and fed ad libitum. The half-lives of HRP, OVA, and pulse-labelled proteins in the hepatocytes from mice fed a restricted diet were about 40% shorter than those in the cells from mice fed ad libitum. These values were close to those in the cells of young animals. These results are discussed in relation to our previous findings that a similar regimen reduces the percentage of heat-labile enzymes accumulated in tissues of aged animals.

Abstract: Change in the degradation rate of ovalbumin (OVA) microinjected into liver parenchymal cells isolated from mice of various ages was studied. OVA was injected by osmotic lysis of pinosomes and the amount of OVA was determined by immunoblotting using purified antibody to OVA. Cellular activity as judged by rates of protein synthesis and degradation of pulse-labeled proteins was not affected by the injection. To localize injected OVA in the cells, cell extracts were fractionated by differential centrifugation and the amount of OVA and the activity of beta-D-galactosidase as a lysosomal marker enzyme of each fraction were determined. The ovalbumin was mostly found in the soluble fraction, while the beta-D-galactosidase activity was found in the particulate fraction, indicating that the ovalbumin was spread in the cytosol but was not present in the pinosomes or lysosomes. The average half-lives of OVA were 106, 113, and 164 h in the cells from young (3.5-6.5 months), middle-aged (13.5-20.0 months), and old (24.5-30.5 months) mice, respectively. Thus, the half-life of ovalbumin in the cells of senescent mice was about 50% longer than that in the cells of young or middle-aged mice. These results are in good agreement with those of our previous investigation, which showed that the half-life of inactivation of horseradish peroxidase was extended by about 50% in the hepatocytes from old mice (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133).

Abstract: cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.

Abstract: The regional distribution of metabotropic L-glutamate responses was investigated in Xenopus oocytes injected with poly(A)+-RNA from a rat brain which was separated into 3 parts: cerebrum, cerebellum and brainstem. Under voltage-clamp, oscillatory current responses were induced more in cerebellum or brainstem poly (A)+-RNA-injected oocytes, and less in cerebrum poly(A)+-RNA-injected oocytes. These results suggest that the metabotropic glutamate receptor is distributed mostly in cerebellum and brainstem.

Abstract: cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.

Abstract: We have studied degradation of proteins in liver parenchymal cells in culture obtained from mice of various ages. Horseradish peroxidase (HRP) was microinjected into the cells by the hypertonic-hypotonic method of Okada and Rechsteiner (Cell, 29 (1982) 33-41). Rate of inactivation of HRP was examined biochemically. Average functional half-lives of the enzyme were 50 h, 53 h and 75 h in the cells of young (3-8 months old), middle-aged (18-20 months old) and old (28-30 months old) mice, respectively. Thus, half-life of the inactivation of the enzyme in "old" cells was about 50% longer than that in "young" or "middle-aged" cells, suggesting that rate of protein degradation is lowered in senescent animals.

Abstract: We analyzed brain extracts from fetal or adult mice with a 32P-labeled 5'-half fragment of identifier (ID) sequence in a gel mobility shift assay. Upon digestion of extracts with RNase A prior to the binding reactions, a protein factor(s) that specifically binds to the fragment was shown to appreciably increase in amount with either of the extracts. Furthermore, the binding was competed with single-stranded ID sequences. These observations suggest that the protein factor is capable of interacting with either the genes or their small RNA transcripts.

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Abstract: The modulatory action of taurine on the glycine response was investigated in Xenopus oocytes injected with messenger RNA from mouse brainstem under voltage-clamped condition. Taurine competitively inhibited the glycine response in a dose-dependent manner. This inhibition was insensitive to bicuculline and intracellularly injected EGTA. The results suggest that taurine could directly modulate glycinergic neurotransmission in the central nervous system.

Abstract: A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.

Abstract: We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.

Abstract: The ID repetitive sequence has been reported to be transcribed as small RNA in both a brain-specific and a developmental stage-specific manner. Several brain-specific proteins required for transcription, along with RNA polymerase III, may be involved in controlling the gene activity throughout development. We analyzed extracts from the brains and livers of mice in an electrophoretic mobility shift assay. Of ID sequence-binding proteins, we detected a protein factor(s) that interacts specifically with the region between two promoter sequences for RNA polymerase III. This protein factor seems to be relevant to postnatal accumulation of the small RNA transcripts of ID sequences, since its time course of expression is consistent with that of the synthesis of the small RNA during development. A penta-nucleotide direct repeat (GCAAG) and its inverted complement (CTTGC) are both present in that region and may be involved in the binding site for the protein factor. The biological significance of the binding site and interacting protein factor(s) is discussed.

Abstract: The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

Abstract: The possibility of change in the rate of misrecognition of amino acids by rat liver tyrosyl-tRNA synthetase during aging was investigated. Frequency of misrecognition of phenylalanine vs tyrosine was determined at two temperatures by competitive assay using partially purified enzymes. At 25 degrees C, the error frequencies were 5.17 x 10(-8) and 8.24 x 10(-8) in young and old animals, respectively. These values are much below the reported error frequencies for the prokaryotic enzymes: i.e. approximately 5 x 10(-6). Although the fidelity of tyrosyl-tRNA synthetase from old animals appeared to be slightly lower, the difference was not statistically significant. At 37 degrees C, the error frequencies were increased 1.3-1.5-fold, but again the difference between young and old animals was not significant. To our knowledge, this is the first report in which fidelity of aminoacyl-tRNA synthetase of animals of various ages has been compared using natural amino acids.

Abstract: Injection of mRNA from different regions of the central nervous system (CNS) of the mouse into the Xenopus oocyte caused a difference in the relative glycine to gamma-aminobutyric acid (GABA) response of the oocyte. Glycine caused the response in oocytes injected with brainstem mRNA but hardly in oocytes injected with cerebrum mRNA and in oocytes injected with cerebellum mRNA. In contrast, GABA caused the response in oocytes injected with mRNA from each of the 3 parts of the CNS. These obvious regional differences may reflect the distribution of the receptors to each plausible neurotransmitter in the CNS.

Abstract: The influence of dietary restriction on alterations of two aminoacyl-tRNA synthetases was investigated in mouse tissues. Mice were fed a restricted diet (first 80% and then 60% of the ad libitum intake) from 23.5 months of age for 70 days. Before dietary restriction, about 35 and 25% of these enzymes in the brain and liver, respectively, were heat-labile. Dietary restriction resulted in a decrease in the percentage of heat-labile enzyme in both tissues: After 40 and 70 days of dietary restriction the percentage of heat-labile enzyme decreased to about 20 and 10%, respectively, in the brain and to undetectable or very low levels in the liver. These results suggest an interesting possibility that prolongation of the life span by dietary restriction is due to reduction in the level of altered enzyme molecules whose accumulation may be detrimental to cellular functions.

Abstract: The ID sequence is a repetitive sequence family of rodent species that is transcribed specifically as small RNA in the brain. The amount of brain-specific small RNA in mice was shown to increase greatly in the first month after birth and then to remain unchanged until senescence. This RNA was suggested to be involved in regulation of brain-specific gene expression during postnatal development.

Abstract: Age-related changes of two aminoacyl-tRNA synthetases in BDF1 mouse and Fischer 344 rat tissues were investigated. The proportions of heat-labile enzymes in the brain and liver were 5 to 10% in young animals, but 25 to 40% in old animals. The proportions began to increase markedly with increase in the mortality rate of the animals. Significant correlations were found between the proportions of heat-labile tyrosyl- and leucyl-tRNA synthetases in the brain and liver of individual animals. Moreover, the proportions of the heat-labile enzymes in the brain were also significantly correlated with those in the liver. Thus, it appears that when one enzyme is heat-labile in one tissue other enzymes are also heat-labile in the same tissue and the same enzyme is also heat-labile in other tissues. Analysis of the size-distribution of partially purified leucyl-tRNA synthetase complexes indicated that, while enzyme preparations from young animals tended to consist of complexes of larger sizes, those from older animals contained smaller complexes, a 10S complex being a major component. On heating preparations from old animals, the activity in the 10S peak disappeared most rapidly. This finding suggests that the heat-stability of the enzyme depended on its molecular form.

Abstract: A taurine response was induced in the surface membrane of the oocytes of Xenopus laevis by injection of the mRNA from the neonatal mouse brain, and the response was studied electrophysiologically. The permeability of mRNA-injected oocytes to chloride ions was increased by the application of taurine in a dose-dependent manner. The same oocyte also responded to GABA, but bicuculline suppressed only the GABA response. These results suggest a possibility that taurine could be a neurotransmitter of certain neurons as yet unknown in the central nervous system.

Abstract: RNAase inhibitor was purified to homogeneity from rat liver. The purified inhibitor was identified by measuring the activity in gel slices after polyacrylamide gel electrophoresis under non-denaturing conditions. Antibodies were prepared by immunization of guinea pigs with the inhibitor. The antibodies formed single precipitin lines with free RNAase inhibitor and RNAase A-inhibitor complex that fused in a micro-Ouchterlony test. Partially purified IgG from immunized animals, but not control animals, inhibited the activities of the free inhibitor and the inhibitor in the complex. In the latter case, the RNAase-inhibitor complex was dissociated and active RNAase was liberated.

Abstract: Age-related changes of aminoacyl tRNA synthetases were investigated in tissues from 3- to 24-month-old rats. The proportions of heat-labile enzymes in the liver, kidney and brain were 0-20% in young animals, but 15-40% in old animals. The proportions of heat-labile enzymes increase from about 15 months of age when the probability of death also increases greatly. These findings suggest that decrease in functional activities of various tissues in old animals is in some way related to the accumulation of these altered proteins.

Abstract: Age-related changes of polypeptide chain elongation factor 2 (EF-2) in mouse and rat livers were investigated. The percentage of heat-labile components in EF-2 was 5 to 10% in young animals and 15 to 45% in old ones. These results and other findings (Takahashi et al., in press) support the idea that decrease in translational activity in old animals is due, in part, to alterations of protein components of the translational system.

Abstract: A novel method was developed to estimate the translational fidelity of mammalian ribosomes in vitro with protamine mRNA of rainbow trout as template. Protamines are mixtures of basic proteins consisting of only seven types of amino acids (Arg, Ile, Val, Ser, Pro, Ala, and Gly), arginine (codon, AGR and CGN) being abundant. Taking advantage of the absence of lysine (codon, AAG) in the proteins, we determined the misincorporation of this amino acid into protamines in a cell-free translation system consisting of mouse liver ribosomes, protamine mRNA, [3H]lysine, [14C]arginine, and seven unlabeled amino acids: Ile, Val, Ser, Pro, Ala, Gly, and Met. After the reaction, translation products were analyzed by either sucrose gradient centrifugation or polyacrylamide gel electrophoresis. In the former method, radioactive protamines are mostly found on monosomes, but not on polysomes, probably because of the basic nature of the proteins. The error frequency was calculated from the molar ratio of [3H]lysine to [14C]arginine incorporated into protamines with an appropriate correction. The frequency was found to be 0.0006-0.002. This method enabled us to determine the frequency of misrecognition of purine bases at the second position of arginine codons in mRNA.

Abstract: The translational activities of ribosomes from young and old mouse livers were examined in an assay system dependent on rabbit globin mRNA. Old ribosomes showed 30-40% lower activity than young ribosomes. This observation, together with our previous findings (N. Mori, D. Mizuno and S. Goto, Mech. Ageing Dev., 10 (1979) 379-398), suggests functional deterioration of ribosomes of old animals. To examine the mechanism of the deterioration, the activities for formation of the initiation complex of 40 S ribosomal subunits in the livers of young and old mice were examined in vitro. The activity was found to be 15-20% lower in old mice (21 months old or more) than in young ones. This fact partly explains the decreased activity for ribosomal protein synthesis in old mice.

Abstract: Possible alterations in the population of poly(A)(+)mRNA during ageing were investigated by translation in vitro of poly(A)(+)mRNA from the liver, kidney and brain of male ddY mice of different ages. [35S]Methionine-labeled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography. A protein product with a molecular weight of 30 000 and isoelectric point of 6.5 was reproducibly observed only in the fluorograms of translation products of poly(A)(+)mRNA derived from the livers of senescent mice (24.5 months old). However, no age-related change was detected in the translation products of the kidney and brain. These results suggest that gene expression in liver cells changes at the level of the population of cytoplasmic poly(A)(+)mRNA during ageing.

Abstract: We have developed a novel method to examine the translational fidelity of mammalian ribosomes in vitro, where protamine mRNA was used as a template. This method enabled us to determine frequency of misrecognition of purine bases at the second position of arginine codons (AGR/AAR) in the mRNA. Using this method the fidelity of translation of ribosomes derived from mouse livers was found to remain unchanged from 2 to 29 months, the maximum life span of the animal. This conclusion is not consistent with the "error catastrophe" theory of aging. This is the first report in which translational fidelity of ribosomes of animals of various ages has been compared by an in vitro translation of a natural mRNA.

Abstract: The proportion of single stranded DNA in various tissues of mouse during aging was examined by measuring sensitivity to a single-strand specific nuclease. Brain DNA from mice of more than 15 mth old contained 3% of the single stranded regions while that from younger animals contained 2%. The DNAs from liver, kidney, heart and spleen did not show significant age-associated changes.

Abstract: Poly(adenylic acid) [poly(A)] containing cytoplasmic ribonucleic acid (RNA) in differentiating fetal calf myoblasts cultivated in vitro was examined by hybridization with radioactive poly(uridylic acid). The size distribution of the poly(A)-containing RNA after sucrose-gradient centrifugation was similar in cells before and after differentiation. There was no apparent correlation between the length of the poly(A) segment and the change in stability of messenger RNA which occurs on differentiation, nor with the polysomal or nonpolysomal localization of the RNA in the cytoplasm. The average length of the poly(A) segments in cytoplasmic RNA in the steady state was found to be dependent on the size of the RNA: the longer the RNA, the longer the average length of the poly(A) sequence. In contrast, in pulse-labeled RNa, the length of poly(A) is similar in all size classes of RNa.

Abstract: During purification of the RNase-inhibitor complex from rat reticulocytes, two forms of the complex were separated by DEAE-Sephadex chromatography. One complex was purified 10,000-fold from the cell lysate, and the other 1,000-fold. Both complexes were shown to be composed of 1 mol of RNase and 1 mol of inhibitor. Reconstitution experiments revealed that the multiplicity of the complex was primarily due to differences in the RNase. The RNases derived from the two complexes differed in their sensitivities to metal ions and hemin.

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Abstract: The function of ribosomes prepared from the liver of young (2--5 months old) and senescent (15--26 months old) mice were compared in vitro. The conclusions that can be drawn from the liver of senescent mice is 10 to 40% lower than that of young mice; (2) the fidelity of translation does not change grossly with age; (3) the thermosensitivity of translational activity of ribosomes and the activity of ribosome-associated ribonuclease do not change with age; (4) there is an age-dependent accumulation of free 40S ribosomal subunits in the cytoplasm of mouse liver. The relation of age-dependent changes in the structure and function of ribosomes is discussed.

Abstract: The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.

Abstract: The possibility of alterations in cytoplasmic RNA in mouse liver, kidney and brain during aging was investigated. The cytoplasmic RNAs in these organs gave similar profiles of optical density at 260 nm with three major peaks at 28S, 18S and 4S on sucrose gradient centrifugation. However, the ratio of the amounts of 18S and 28S RNA increased significantly with age in the brain and liver. The polyacrylamide gel electrophoretic patterns of extracts of the three tissues under both native and denaturing conditions were nearly identical regardless of the age of the animals. Since most of the minor components separated on gels were probably in vivo degradation products of ribosomal RNA, these results suggest that the extent of apparent and hidden breaks in ribosomal RNA does not change during aging.

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