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Biomedical subjects

J Fujii

Publications and source records attributed to J Fujii.

At least 91 records · Page 5Linked to original sources
PubMed1997

The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor.

Glutathione peroxidase (GPx) was inactivated by S-nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide donor (Asahi, M., Fujii, J., Suzuki, K., Seo, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Taniguchi, N. (1995) J. Biol. Chem. 270, 21035-21039). The structural basis of the inactivation was studied. We also show that 3-morpholinosydnonimine N-ethylcarbamide, a peroxynitrite precursor, as well as synthetic peroxynitrite also inactivated bovine GPx. The degree of incorporation of a sulfhydryl reagent, n-octyldithionitrobenzoic acid, into GPx decreased after pretreatment with SNAP as evidenced by mass spectrometry. To identify the modification site of this enzyme by SNAP, both SNAP-pretreated and untreated GPxs were reacted with n-octyldithionitrobenzoic acid and digested with lysylendopeptidase, and the resulting peptides were subjected to mass spectrometry. This technique identified a bridge between two peptides, one of which contains Sec45 at the catalytic center and Cys74, and the other contains Cys91. Although there are two possible combinations, selenocysteine 45 (Sec45) and Cys91 or Cys74 and Cys91, the tertiary structure of GPx indicates that a cross-link between Sec45 and Cys91 is more feasible. This is consistent with the experimental evidence that SNAP specifically inactivates GPx, in which Sec45 forms the catalytic center. Thus, we conclude that SNAP mainly oxidized Sec45 to form a selenenyl sulfide (Se-S) with a free thiol, leading to the inactivation of the enzyme. These data suggest that nitric oxide and its derivatives directly inactivate GPx in a specific manner via the production of a selenenyl sulfide, resulting in an increase in intracellular peroxides that are responsible for cellular damage.

Amino Acid Sequence↗
PubMed1997

Growth suppression and induction of heat-shock protein-70 by 9-cis beta-carotene in cervical dysplasia-derived cells.

The aim of the present study was to determine to what extent 9-cis beta-carotene, one of the most abundant naturally-occurring cis-isomers of beta-carotene, can inhibit the growth of cervical dysplasia-derived cells in comparison with all-trans beta-carotene. We found that 9-cis beta-carotene was dose-dependently more effective than all-trans beta-carotene. Both carotenes induced the intracellular accumulation of heat-shock protein-70 (HSP70), and the treated cells showed morphological changes indicative of apoptosis. The results of the present study strongly suggest that the induction of HSP70 by beta-carotene might be involved in beta-carotene-mediated suppression of the cell growth through apoptosis.

Cell Division↗
PubMed1997

TGF-beta1 triggers oxidative modifications and enhances apoptosis in HIT cells through accumulation of reactive oxygen species by suppression of catalase and glutathione peroxidase.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional polypeptide that is related to the progression of chronic pancreatitis. However, the mechanism of beta-cell damage by TGF-beta1 is unknown. Treatment with TGF-beta1 enhanced internucleosomal DNA cleavage caused by exogenous hydrogen peroxide in a hamster pancreatic beta-cell line (HIT). TGF-beta1 also induced protein oxidation, assessed by measuring carbonyl groups in proteins, and was involved in reactions that lead to lipid peroxidation. This eventually destructs membrane lipids and forms malondialdehyde. We have investigated its effects on two major antioxidative enzymes, catalase and glutathione peroxidase (GPx). TGF-beta1 suppressed mRNA expression as well as reduced the activities of catalase and GPx. The decrease in the catalase and GPx activities in TGF-beta1-treated cells resulted in an increase in intracellular peroxides as judged by flow cytometric analysis using a peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate. These data suggest that the augmented production of reactive oxygen species by TGF-beta1 through suppression of antioxidative enzymes may cause cellular damage and consequent apoptosis and induce pancreatitis or diabetes.

Adenoma, Islet Cell↗
PubMed1997

The effect of nitric oxide synthase inhibitor on form-deprivation myopia.

PURPOSE: Form-deprivation myopia (FDM) is believed to result principally from actions of substances that modulate information processing in the retina. We used a chick model to investigate what role nitric oxide (NO), a gaseous neuromodulator, might play in the development of FDM. METHODS: We injected different concentrations of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) (30 ml) into the left eyes and the same volume of saline into the right eyes of 6-day-old chicks. Both eyes of most chicks were occluded for 6 days with translucent goggles. After removal of the goggles, the refraction was measured by retinoscopy and the axial lengths with an A-mode ultrasound. In some chicks we measured the concentration of NOx (nitrite and nitrate) in the retina. A few chicks, not wearing occluders after injection of L-NAME and saline, ERG and refraction, were examined 6 days after the treatment. RESULTS: In chicks that wore occluders, refractive error and axial length were significantly less affected in eyes injected with L-NAME (180, 360, or 540 mM) compared to control (right) eyes. ERG changes were reversible, except in eyes injected with the highest concentration (540 mM) of L-NAME. The eyes of chicks, injected with L-NAME and reared without occlusion, had normal refractive values. After 6 days of form deprivation, the concentration of NOx in the retina of eyes injected with L-NAME (180 mM) was significantly less than the concentration in eyes injected with saline. CONCLUSIONS: The injection of L-NAME before occlusion of developing chick eyes leads to reversible modifications in retinal function and inhibits the development of form-deprivation myopia.

Animals↗
PubMed1997

Genetic defect of the sodium pump-defective mutant Nap-1 from the marine Vibrio alginolyticus.

The marine bacterium, Vibrio alginolyticus, has a respiratory chain-linked Na(+)-translocating NADH-quinone reductase (NQR). Among several mutant cells defective in Na+ pump activity, Nap1 was a very stable mutant and a spontaneous revertant could not be isolated from Nap1. Using genetic information from the recently sequenced nqr operon, the genetic defects in Nap1 were examined, and the sodium pump-defective mutant Nap1 was found to be caused by the insertion of a 1.2 kbp DNA fragment into the C-terminal region of nqr6 gene.

Amino Acid Sequence↗
PubMed1997

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.

The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.

Air Conditioning↗
PubMed1997

Increase in erythrocyte volume in patients with chronic atrial fibrillation.

Non-anemic macrocytosis is occasionally observed in patients with a history of alcoholism or chronic liver disease. Recently we reported that patients with atrial fibrillation (AF) had larger erythrocytes than normal subjects. In the present study, we examined the temporal changes in hematologic measurements during the development of AF in 88 male patients (chronic AF: 49, paroxysmal AF: 39), and compared alcohol intake among the groups. The mean corpuscular volume of erythrocytes was significantly greater in chronic AF patients, both before (98.1 +/- 6.1 (SD) fl p < 0.05) and after onset (within 6 months: 98.4 +/- 7.1 fl p < 0.05; 1 year: 98.6 +/- 6.2 fl p < 0.01), compared with 300 age-matched male control subjects with sinus rhythm (95.9 +/- 3.6 fl). Hematocrit increased significantly with development of AF in both chronic and paroxysmal AF patients (p < 0.005). Mean daily alcohol consumption did not differ significantly among the control, chronic AF and paroxysmal AF groups. Our findings indicate that erythrocyte size is already larger in patients with chronic AF before the condition develops.

Adult↗
PubMed1997

Roles of purine nucleotides and adenosine in enhancing NOS II gene expression in interleukin-1 beta-stimulated rat vascular smooth muscle cells.

The production of nitric oxide (NO) by vascular smooth muscle cells (VSMC) is stimulated by interleukin-1 beta (IL-1 beta). This is enhanced in a dose-dependent manner by ADP, although it alone failed to induce nitrite accumulation. Purine nucleotides and their nonhydrolizable analogues as well as adenosine also exhibit variable enhancing effects. This enhanced nitrite formation was due to induction of the NO synthase (NOS II) gene as judged by Northern hybridization using an NOS II specific probe and by Ca2+ independency of the NOS II activity. 8-(p-Sulfophenyl)-theophylline, a blocker of adenosine receptors, suppressed the enhanced NO production by adenosine and ADP to the level of that with IL-1 beta alone. These data indicate that activation of the adenosine receptor on VSMC may enhance production of NOS II by modulating a signal transducing pathway of IL-1 beta. Although cAMP is a candidate as the second messenger, it was not significantly elevated by either ADP or adenosine treatment in IL-1 beta-stimulated cells. This mechanism might be stimulated under conditions with release of various purine and their derivatives.

Adenosine↗
PubMed1997

[Trends in the prescriptions for antihypertensive drugs in elderly patients].

We compared trends in the prescription of antihypertensive drugs in elderly patients with those in middle-aged patients. We analyzed prescriptions given to 141 patients with hypertension who were 65 years old or older (mean age, 73 years) and to 511 patients with hypertension who were 64 years old or younger (mean age, 59 years). The numbers of drugs prescribed did not differ between the elderly and the non-elderly patients. In both groups, about 50% of patients were given prescriptions for one antihypertensive drug, about 40% were given prescriptions for two drugs, and about 10% were given prescriptions for three drugs. Calcium antagonists were the agents most commonly prescribed as monotherapy (51.4% and 56.4% in the non-elderly and elderly respectively; n.s.); followed by beta-blockers (32.2% and 25.6%; n.s.). Diuretics were prescribed less often to non-elderly patients than to elderly patients (5.1% and 10.0%; p < 0.01). When prescriptions for more than one antihypertensive agent were included in the analysis, calcium antagonists were still the most commonly prescribed agents (63.8% and 66.4%; n.s.), followed by the beta-blockers (53.3% and 66.4%; p < 0.05). Diuretics were less commonly prescribed to non-elderly patients (13.5% and 21.3%; p < 0.005), while ACE inhibitors (19.0% and 11.6%; p < 0.005) and beta-blockers (52.3% and 44.0; p < 0.05) were more commonly prescribed to non-elderly patients. When we classified the data according to the time of the first visit, we found that patients whose first visit was earlier were more likely to have been given a prescription for a diuretics, whether they were elderly or non-elderly. Calcium antagonists were the most commonly prescribed agents in all time periods studied, followed by beta-blockers. Comparison of our results with those of a similar study done at our clinic in 1990 showed that the use of calcium antagonist monotherapy doubled in both groups (non-elderly: 26.4% in 1990 to 51.4% in 1995, p < 0.001; elderly: 29.5% in 1990 to 56.4% in 1995, p < 0.001) and that the use of diuretics declined markedly (non-elderly; 20.3% to 5.1%, p < 0.001; elderly: 30.2% to 10.0%, p < 0.001). A similar trend was seen when prescriptions for more than one antihypertensive drug were included in the analysis.

Aged↗
PubMed1997

[Brain lesions in rabbits given an intravenous injection of verotoxin 2 and protection by anti-VT2 antibody].

Magnetic resonance imaging was obtained to determine the effects of VT2 toxemia on the rabbit's CNS. The first lesion was noted at 24 h in the hypothalamic area of all experimental animals. The rabbits accompanied with the brain stem lesion were dead within 6 days. We examined the integrity of cerebrospinal fluid-brain barrier (CBB) using a tracer. The tracer was detected throughout the cytoplasm of the ependymal cell layer covering the third ventricle after intrathecal injection of the tracer, which means a deterioration of CBB. Furthermore, we examined whether anti-VT2 antibody injected intrathecally protects rabbits from brain damage. All the rabbits survived when they were given an intrathecal injection of rabbit anti-VT2 antibody 2h before the intravenous injection of VT2.

Animals↗
PubMed1997

[Experimental study on the pathogenesis of mitral annular calcification: calcium deposits in mitral complex lesions induced by vagal stimulation in rabbits].

Cervical vagal stimulation in rabbits frequently causes peculiar mitral complex lesions which are detected by deposition of colloidal carbon. The present study examined the effects of vagal stimulation on the calcium (Ca) contents in the mitral complex lesions. Anesthetized rabbits were forced into the supine position with electrocardiographic monitoring. The animals were divided into those with vagal stimulation (n = 37), and those without manipulation as controls (n = 25). Colloidal carbon (1 ml) was intravenously injected into the animals on the next day. All animals were sacrificed after 1 week. After perfusion of the heart by heparinized saline, Ca content per g myocardium in the mitral annulus, papillary muscle, free wall or apex in the left ventricle was measured by the atomic absorption method. Immediately after vagal stimulation, bigeminy due to premature ventricular contractions was observed in 76%, and systolic murmur was heard in 30%. Mitral complex lesions detected by carbon deposits were found in 73% of the rabbits with vagal stimulation. Ca content in the mitral annulus or papillary muscle was significantly greater than that in the free wall or apex (p < 0.001). Ca content in the mitral annulus was significantly greater in the rabbits with vagal stimulation than in the control group (p < 0.001). In the former group, Ca content in the mitral annulus in animals with mitral complex lesions was significantly greater than that in those without mitral complex lesions (p < 0.01). These results suggest that these mitral complex lesions might provide an experimental model of mitral annular calcification, and that the autonomic nervous system and arrhythmia might be involved in the mechanism of this calcification.

Animals↗
PubMed1996

Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction.

Several reducing sugars brought about apoptosis in isolated rat pancreatic islet cells and in the pancreatic beta-cell-derived cell line HIT. This apoptosis was characterized biochemically by inter-nucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatin condensation and apoptotic body formation. N-Acetyl-L-cysteine, an antioxidant, and aminoguanidine, an inhibitor of the glycation reaction, inhibited this apoptosis. We also showed directly that proteins in beta-cells were actually glycated by using an antibody which can specifically recognize proteins glycated by fructose, but not by glucose. Furthermore, fluorescence-activated cell sorting analysis using dichlorofluorescein diacetate showed that reducing sugars increased intracellular peroxide levels prior to the induction of apoptosis. Levels of carbonyl, an index of oxidative modification, and of malondialdehyde, a lipid peroxidation product, were also increased. Taken together, these results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress mainly through the glycation reaction, which may explain the deterioration of beta-cells under conditions of diabetes.

Acetylcysteine↗
PubMed1996

Quinazoline derivatives suppress nitric oxide production by macrophages through inhibition of NOS II gene expression.

We have found three novel quinazolidine derivatives which inhibit the formation of nitrite dose-dependently in a murine macrophage cell line, RAW264.7. The decreased nitrite formation was due not to the inhibition of nitric oxide synthase activity but to suppression of NOS II mRNA and protein expression. In rat vascular smooth muscle cells (VSMC), however, these compounds rather enhanced NOS II mRNA. These compounds also prevented LPS-stimulated heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) gene expression in RAW264.7 cells, but again not in VSMC. The three quinazolidine derivatives specifically inhibit gene expression of NOS II, HO-1 and COX-2 only in macrophage cells, indicating that they are selective inhibitors of inducible gene expression in macrophages.

Animals↗
PubMed1996

Glycation and inactivation of sorbitol dehydrogenase in normal and diabetic rats.

Sorbitol dehydrogenase (SDH) is involved in the polyol pathway, which plays an important role in the pathogenesis of diabetic complications. We have measured the tissue distributions of SDH mRNA, both the immunoreactive enzyme levels and the enzyme activity. SDH mRNA was especially abundant in liver, kidney and testis. Both the activity and enzyme content are high in liver and kidney but not in testis. The discrepancy between mRNA and immunoreactive enzyme levels and the activity of SDH observed in testis was also seen in livers of streptozotocin-induced diabetic rats. SDH was found to exist in both glycated and non-glycated forms, with larger amounts of the glycated protein in the diabetic liver. Moreover, after incubation of purified enzyme with glucose or fructose, its activity was markedly decreased. These results indicate that glycation causes a decrease in SDH activity in liver under diabetic conditions. The same post-transcriptional event might occur to decrease the activity of SDH in testis in normal animals.

Animals↗
PubMed1996

Induction of apoptotic cell death by methylglyoxal and 3-deoxyglucosone in macrophage-derived cell lines.

Production of 2-oxoaldehyde compounds increase during hyperglycemic conditions and is cytotoxic to susceptible cells. We have investigated the effects of methylglyoxal and 3-deoxyglucosone at physiological concentrations on monocytic leukemia U937 cells and other cell lines. Both ladder formation of DNA and nuclear fragmentation were observed in the cells treated with these agents, indicating that apoptotic cell death was induced. The fluorescent intensity of an oxidation sensitive dye (2',7'-dichlorofluorescin) was increased in U937 cells but not in other cells in which apoptosis was not induced. The levels of intracellular glutathione, however, were only slightly changed. Apoptosis and intracellular oxidant levels were enhanced by buthionine sulfoximine, an inhibitor of glutathione biosynthesis, and partially blocked by N-acetylcysteine, an antioxidant. Thus, it is conceivable that elevation of intracellular oxidant stress is a cause of the apoptosis induced by cytotoxic 2-oxoaldehyde compounds.

Apoptosis↗
PubMed1996

Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis.

In about 20-25% of cases of familial amyotrophic lateral sclerosis (FALS) patients have mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The mechanism through which the mutations in the SOD1 gene cause ALS still remain unknown. We performed pulse-chase experiments using a system for the transient expression of human SOD1 in COS7 cells to examine whether the Ala4Thr mutation, which we previously reported, decreases the stability of SOD1. The expression vector (pEF-BOS) carrying the wild-type or mutant (Ala4Thr) human SOD1 cDNA was transfected into COS7 cells, and transiently expressed human SOD1 was then metabolically radiolabeled. Half-lives of the wild-type and the Ala4Thr mutant SOD1 were determined to be 78 h and 18 h, respectively. These results suggest that the Ala4Thr mutation in SOD1 decreases the stability of SOD1 and that this instability may play an important role in the pathogenesis of the degeneration of motor neurons in FALS.

Amyotrophic Lateral Sclerosis↗
PubMed1996

The structural organization of the human skeletal muscle ryanodine receptor (RYR1) gene.

The RYR1 gene encoding the Ca2+ release channel of human skeletal muscle sarcoplasmic reticulum has been cloned and exon/intron boundaries have been determined, together with a minimum of 30 bp of intron sequence flanking each splice junction. The gene contains 106 exons, of which two are alternatively spliced. The length of the gene, determined by the alignment of 16 genomic phage clones, a cosmid clone, and several long polymerase chain reaction products, is approximately 160 kb. Exons range from 15 to 813 bp, while introns range from 85 to about 16,000 bp. Analysis of the gene has confirmed published errors in the human RYR1 cDNA and confirmed the structure of two alternatively spliced exons. The numbering of the nucleotides comprising the RYR1 cDNA and the numbering of amino acids encoded by them were corrected to account for these earlier errors and omissions. Analysis of 2.4 kb of the 5' upstream sequence indicated the presence of a CCAAT box and several Sp1 binding sites between nucleotides -200 and -60 bp, flanking the proposed transcription start site at -130 bp. Several other potential transcription factor binding sites were identified throughout the 5' sequence. Knowledge of the structure of the RYR1 gene will provide an invaluable resource for the discovery of mutations in the gene that are causal of human malignant hyperthermia and central core disease.

Amino Acid Sequence↗