Hiroki Nanri

Increase of human MTH1 and decrease of 8-hydroxydeoxyguanosine in leukocyte DNA by acute and chronic exercise in healthy male subjects

To investigate the effects of exercise on oxidative DNA damage, we measured the levels of 8-hydroxydeoxyguanosine (8-OH-dG, 7,8,dihydro-8-oxo-deoxyguanosine), and mRNA of its sanitization enzyme, human MutT homologue (hMTH1), after mild acute exercise in healthy male subjects. The basal 8-OH-dG levels of physically active subjects were significantly lower than those of sedentary subjects. After mild exercise for 30min, the 8-OH-dG levels of the sedentary subjects significantly decreased. In correspondence with the change of 8-OH-dG levels, the hMTH1 mRNA levels of the physically active subjects were significantly higher than those of the sedentary subjects. The levels of hMTH1 mRNA of sedentary subjects significantly increased after mild exercise. Furthermore, there was a significant negative correlation between the levels of 8-OH-dG and hMTH1 mRNA. These results suggest that a mild exercise has a beneficial effect on the maintenance of low levels of 8-OH-dG probably by keeping the sanitization system at higher levels.

EFFECTS OF MILD AEROBIC EXERCISE AND A MILD HYPOCALORIC DIET ON PLASMA LEPTIN IN SEDENTARY WOMEN

1. The present study was conducted to investigate whether mild aerobic exercise and a mild hypocaloric diet, instead of severe restrictions on caloric intake, would affect weight reduction and plasma leptin concentrations.

Induction of apoptosis in placentas of pregnant mice exposed to lipopolysaccharides : Possible involvement of Fas/Fas ligand system

Abstract To explore the pathogenesis in placental dysfunction and abruptio placentae, we analyzed the occurrence of placental cell apoptosis and the role of Fas and Fas ligand (L) in that process in an inflammatory placental dysfunction model of pregnant mice, using lipopolysaccharides (LPS). In the present study, Day 13 pregnant mice were injected i.p. with LPS (50 μg/kg) or saline as a control, and the placentas were isolated at various time points after the injection. Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of 180–200 base pairs (bp), which is regarded as a hallmark of apoptosis. The intensity of the bands increased time-dependently, reaching a maximum level at 12 h after LPS injection. In accord with the biochemical data, histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) revealed that nuclei positive for double-stranded DNA breaks were found in decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. The number of positive nuclei was maximized at 12 h after LPS injection. As a next step, we investigated the possible involvement of Fas and Fas L in the induction of apoptosis of the placental cells after LPS injection. Western blot analysis indicated that LPS increased the expression of Fas and Fas L in the placenta by about 4-fold at 12 h and 18 h, respectively, after injection. The cells expressing Fas and Fas L were identified, using immunohistochemistry and nonradioactive in situ hybridization, as decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. Furthermore, when the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins was assessed to evaluate the relation of oxidative stress elicited by LPS to the induction of apoptosis, once again decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts were positive. Therefore, the placental dysfunction by LPS may be brought about by the Fas-mediated apoptosis of various placental cells in a paracrine/autocrine fashion, possibly under the influence of oxidative stress.

Differential induction of constitutive and inducible nitric oxide synthases by distinct inflammatory stimuli in bovine aortic endothelial cells

Exposure to various combinations of cytokines and lipopolysaccharide (LPS) has been reported to increase NO production in vascular endothelial cells. The molecular entity of the newly expressed nitric oxide synthase (NOS) in endothelial cells, however, has not yet been examined in detail. In this report, we carried out biochemical characterizations and molecular identification of NOS isoform(s) expressed in cytokine/LPS-treated bovine aortic endothelial cells (BAEC). The increased NOS activity in tumor necrosis factor-alpha (TNF-alpha)/LPS-treated BAEC was localized mainly in the cytosolic fraction and Ca2+-independent, whereas that in interferon-alpha,beta(IFN-alpha,beta)/LPS-treated BAEC was preferentially in the membrane fraction and Ca2+-dependent, suggesting that TNF-alpha/LPS increased an inducible NOS (iNOS)-like activity, and IFN-alpha,beta/LPS increased an endothelial constitutive NOS (ecNOS)-like activity. Correspondingly, the different responses to the cytokine/LPS pretreatment were demonstrated in semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for iNOS or ecNOS, that is, TNF-alpha/LPS elicited the expression of iNOS mRNA whereas IFN-alpha,beta/LPS increased that of ecNOS mRNA. A nuclear run-on transcription assay and an inhibition experiment by actinomycin D indicated that the apparent increase of ecNOS in the IFN-alpha,beta/LPS-treated BAEC was at least in part ascribed to the transcriptional activation. The nucleotide sequences of the amplified PCR products in TNF-alpha/LPS- and IFN-alpha,beta/LPS-treated BAEC were 93% and 99% identical to the corresponding regions of human hepatocyte iNOS and bovine ecNOS, respectively. These findings indicated that, in cytokine/LPS-treated BAEC, two NOS isoforms whose molecular natures were closely homologous to the conventional isoforms of iNOS and ecNOS were differently induced in response to distinct inflammatory stimuli.

Blood Pressure-Lowering Effects of Lifestyle Modification: Possible Involvement of Nitric Oxide Bioavailability

Lifestyle modification is recommended as a non-pharmacological approach to treatment of hypertension. Many investigators have reported that exercise has antihypertensive effects, and various mechanisms have been proposed to explain this phenomenon. For example, nitric oxide (NO), which may be increased by exercise, has been reported to play a crucial role in preserving vessel homeostasis both by regulating vascular tone and by exerting anti-atherosclerotic effects. NO is known to be exquisitely sensitive to inactivation by superoxide radicals. However, the relationship between the blood pressure-lowering effect of lifestyle modification and NO bioavailability remains unknown. We investigated the effects of a 12-week lifestyle modification program consisting of mild exercise and diet on changes in blood pressure, plasma nitrate/nitrite (NOx), plasma nitrotyrosine, which is the footprint of NO interaction with reactive oxygen species, and plasma extracellular-superoxide dismutase (EC-SOD). The 12-week lifestyle modification program lowered blood pressure and increased plasma NOx. When the subjects were divided into two groups according to the change of plasma nitrotyrosine as an indicator of NO bioavailability, the subjects whose plasma nitrotyrosine decreased exhibited a significant relationship between the blood pressure-lowering effect of the lifestyle modification and the increase in EC-SOD, whereas those without a decrease in plasma nitrotyrosine exhibited a significant relationship between the blood pressure-lowering effect and the increase in maximum oxygen consumption. These results indicate that the level of NO bioavailability influences the mechanism of the blood pressure-lowering effect of aerobic exercise and diet.

A weight reduction and weight maintenance program with long-lasting improvement in left ventricular mass and blood pressure*

Obesity is a major risk factor for cardiovascular disease and is associated with hypertension and increased left ventricular mass (LVM). Maintenance of reduced weight has been a matter of recent concerns in the treatment of obese subjects. This study was conducted to confirm the effect of the addition of exercise to diet on maintenance of body weight in a weight reduction program. In addition, this study was conducted to estimate whether LVM changes in parallel with a change in body weight during a long-term follow-up after a weight-reduction program. Twenty-two normotensive (NT) obese subjects and 14 mild hypertensive (HT) obese subjects ranging in age from 22 to 51 years participated in a 12-week supervised weight-reduction program involving mild exercise and a mild hypocaloric diet. After this 12-week intervention, they were advised to maintain their modified lifestyle during a 1-year follow-up period. After the 12-week intervention, the mean reductions in body weight (BW) in the NT and HT groups were 4.1 kg (P < .0001) and 5.8 kg (P < .0001), respectively. LVM in the NT and HT groups was significantly reduced from 154 g to 136 g (P < .005) and from 169 g to 152 g (P < .002), respectively. One year after intervention, the mean gains in BW in the NT and HT groups were 2.3 kg (not significant, NS) and 0.4 kg (NS), respectively. The mean gains in LVM in the NT and HT groups were 8 g (NS) and 7 g (NS), respectively. It was also shown that blood pressures in the HT group were significantly decreased after the 12-week intervention and there was no significant change in blood pressure in the HT group 1 year after intervention. In conclusion, reduced body weight was maintained for 1 year after a 12-week supervised weight-reduction program in both normotensive and mild hypertensive obese subjects. Reduced left ventricular mass was maintained for a long period in both normotensive and mild hypertensive obese subjects and lowered blood pressure was maintained in the mild hypertensive obese subjects.

Thioredoxin is associated with endotoxin tolerance in mice

Objective Oxidative stress and subsequent lipid peroxidation appear to be central to the lethal effect of lipopolysaccharide. We hypothesized that induction of an antioxidant protein, thioredoxin, would play an important role in the development of endotoxin tolerance and reduce mortality rates in lipopolysaccharide-treated mice. Design Prospective, randomized, controlled study. Setting University research laboratory. Subjects Adult, male, ddy mice. Interventions In survival-curve experiments, mice were pretreated intravenously with a low dose of Escherichia coli lipopolysaccharide (20 &mgr;g/mouse, pretreatment group) or saline (control group). A large dose of lipopolysaccharide (200 &mgr;g/mouse) subsequently was injected into the tail vein 16 hrs after pretreatment. In experiments to measure the expression of thioredoxin after lipopolysaccharide challenge, mice were injected intravenously with different concentrations of lipopolysaccharide (between 20 and 200 &mgr;g/mouse, designated the lipopolysaccharide group) or with saline (control group). Measurements and Main Results The survival rate during the 72-hr observation period was significantly higher in the pretreatment group (82%) than in the control group (30%;p = .025 by Mantel-Cox log rank analysis). After lipopolysaccharide challenge, thioredoxin concentrations in the lung, heart, and liver of mice in the pretreated group were about 1.5- to 2-fold higher than those in the control group. Enhancement in these organs became apparent about 6 hrs after lipopolysaccharide challenge and lasted until at least 24 hrs. The levels of accumulation of 4-hydroxy-2-nonenal modified proteins after a large dose of lipopolysaccharide challenge were lower in the pretreatment group than in the control group. Conclusions These results demonstrate that mice with an increased concentration of thioredoxin, induced by pretreatment with a low dose of lipopolysaccharide, had increased survival when given a subsequent high-dose challenge of lipopolysaccharide. Thus, thioredoxin is associated with the development of endotoxin tolerance in mice.

Hindlimb unloading decreases thioredoxin-related antioxidant proteins and increases thioredoxin-binding protein-2 in rat skeletal muscle

To investigate role(s) of thioredoxin-related antioxidant proteins in disuse muscle atrophy, we examined the levels of thioredoxin-1 (Trx-1), peroxiredoxin-3/SP-22 (Prx-3) and thioredoxin-binding protein-2 (TBP-2) in rat soleus muscle subjected to hindlimb unloading (HU) for 2, 4, 7 or 14 days. The muscle weight loss was initially observed on day 4. The increases in aclorein- and malondialdehyde-modified proteins, and the decreases in the levels of Trx-1, Prx-3 and Mn-SOD were observed in the late phase of muscle atrophy, whereas, the increase in mRNA expression of TBP-2, a negative regulator of thioredoxin, preceded muscle atrophy. These findings suggest that the decrease of those antioxidant proteins, particularly a marked decrease of Trx-1, may be responsible for the enhanced oxidative damage during the late phase of disuse muscle atrophy. Furthermore, the increase in TBP-2 preceding the muscle atrophy may suppress the thioredoxin-mediated redox signaling, which can be an initial trigger leading to disuse muscle atrophy.

Pertussis toxin inhibits intracellular pH changes in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Changes of intracellular pH in human neutrophils were monitored by 9-aminoacridine fluorescence. Both initial acidification and subsequent alkalinization phases induced by a chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine were dependent on the extracellular Ca2+-concentrations, and a calcium ionophore, A-23187 similarly induced the pH-changes. Pertussis toxin inhibited the pH-changes induced by the peptide while cholera toxin did not. The pH-changes induced by A-23187 were not affected by the toxins. The results suggest that the inhibitory guanine-nucleotide regulatory protein and Ca2+ are involved in the pH-changes induced by the peptide.

Cloning and sequencing of the cDNA encoding human glutaredoxin

Glutaredoxin (thioltransferase) is a small, heat-stable protein, which is involved in thiol/disulfide exchange reactions. We have isolated a cDNA that encodes glutaredoxin from a human brain cDNA library. The encoded protein contains 106 amino acids with a calculated molecular mass of 11.76 kDa and an isoelectric point of 8.09. The amino acid sequence deduced from the cDNA is more than 80% identical to those of other mammalian glutaredoxins.