Biomedical subjects

J Fujii

Publications and source records attributed to J Fujii.

At least 19 recordsLinked to original sources

PubMed1995

Induction of nitric oxide synthase and concomitant suppression of superoxide dismutases in experimental colitis in rats.

Reactive oxygen species are thought to play an important role in some bowel diseases. In order to evaluate the participation of nitric oxide and superoxide in such diseases, we examined the expression of nitric oxide synthase (NOS) and superoxide dismutase (SOD) as well as their activities in whole excised colons of rats with colitis induced by intralumenal administration of 2,4,6-trinitrobenzenesulfonic acid. A marked increase in the inducible form of NOS mRNA was detected and NOS activity was coincidentally augmented in the group administered unbuffered TNBS (pH 1.0), in which severe inflammation was revealed by microscopic examination and myeloperoxidase activity of invading neutrophils in the tissues. The levels of the Mn- and Cu,Zn-SOD proteins as well as SOD activity were suppressed, although expression of the Mn-SOD mRNA was enhanced in colitis tissues. The elevation of NOS activity and the suppression of SOD activity occurred concomitantly at the stage of severe inflammation. This would increase peroxynitrite formation from superoxide and nitric oxide and enhance the tissue damage in experimental colitis.

Animals↗

PubMed1995

Organization of the human N-acetylglucosaminyltransferase V gene.

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc transferase V), which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-6-D-mannoside, is an important enzyme regulating the branch formation in complex-type, N-linked oligosaccharides. It has been reported that the enzymic activity of GlcNAc transferase V increases after viral transformation and the enzymic product is closely related to the metastasis of tumors. We previously reported the purification, cDNA cloning and chromosomal mapping of human GlcNAc transferase V. In this study, we describe the isolation of genomic clones encoding human GlcNAc transferase V and the structure of the gene. The human GlcNAc transferase V gene is divided into 17 exons, and the open reading frame is encoded by exons 2-17, spanning 155 kb. Analysis of the 5'-untranslated regions of mRNAs from various cells showed multiple sequences depending on the cell types. The promoter region of the GlcNAc transferase V gene was characterized by searching for any consensus sequences matching those for transcription-factor binding. The consensus sequences for a TATA box, AP-1, AP-2, and some other transcription factors were found in the 5'-upstream region of exon 1, and consensus sequences for LF-A1, HNF1-HP1, liver-restricted transcription factors and other factors were also found in intron 1. Chloramphenicol acetyltransferase fusion plasmids with either the 5'-upstream region of exon 1 or intron 1 were constructed and transfected into COS-1 cells. Promoter activities of both DNA fragments were detected, indicating that transcription starts within this region. These data suggest that the human GlcNAc transferase V gene employs a multiple promoter system for its transcription, and gene expression may therefore be regulated in tissue-specific and cell-type-specific manners.

Base Sequence↗

PubMed1995

Involvement of Ser-451 and Ser-452 in the catalysis of human gamma-glutamyl transpeptidase.

The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.

Amino Acid Sequence↗

PubMed1995

Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes.

Aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily, and participate in the reduction of a wide range of carbonyl compounds. We have purified aldose reductase from rat lens and raised antiserum against it in rabbits. Immunoblot analyses using this antibody showed that a significant amount of aldose reductase was expressed in cell lines derived from hepatomas while it was negligible in normal hepatocytes. Elevated expression of aldose reductase was also observed in cancerous lesions of 3'-methyl-4-dimethyl-aminoazobenzene (3'-Me-DAB)-induced hepatocarcinomas. Expression of aldose reductase mRNA was confirmed in these cells by Northern-blot analysis, suggesting that the induction occurred at the stage of gene transcription. The level of aldehyde reductase, however, did not change in cancerous tissue or in the cell lines. The viability of hepatoma cells in the presence of 3-deoxyglucosone and glyceraldehyde was decreased by an aldose reductase inhibitor, ONO-2235 (5-[1Z,2E)-2-methyl-3-phenylpropenylidene]-4-oxo-2-thioxo -3- thiazolidineacetic acid). Taken together, induction of aldose reductase gene expression during hepatocarcinogenesis may render cancer cells resistant to various toxic carbonyl compounds produced during metabolism or administered as anti-cancer drugs.

Aldehyde Reductase↗

PubMed1995

Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity.

S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC50 of SNAP for GPx was 2 microM at 1 h of incubation and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 microM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N omega-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.

Animals↗

PubMed1995

Human gamma-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit.

Mutant human gamma-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the alpha-amino group of the gamma-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the gamma-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates gamma-glutamyl enzyme formation.

Aspartic Acid↗

PubMed1995

Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases.

Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site.

Alanine↗

PubMed1995

Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.

We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay.

Amino Acid Oxidoreductases↗

PubMed1995

[A study on the usefulness of stress assessment based on the THP health measurement--comparison with the general health questionnaire].

Although the "Total Health Promotion Plan (THP)" drawn up by the Labor Ministry includes a self-administered stress checklist (THP-SC) as a health measurement, THP-SC has not been utilized in the way that it had been planned. Hence, we studied the usefulness of this list by comparing it with the General Health Questionnaire (GHQ). We made a questionnaire that included both THP-SC and the 60-item GHQ, and collected answers from 261 white-collar workers (male, average age 43.7). The GHQ was evaluated using the Goldberg's method. When analyzing THP-SC, the number of answers indicating stress was counted, and the respondents were divided into two groups based on the cut-off points of 75 percentile or 90 percentile. In the 60-item GHQ, 48 respondents out of 243 with valid answers had a higher score than the cut-off point, and in the 12-item GHQ, 77 out of 256 had a higher score. The average number of THP-SC was 5.67 +/- 3.19. Concerning the relationship with GHQ, many items of the 'A' section of THP-SC were significant. The comparison also suggested that four or more items checked in 'A' section or seven or more items checked in the whole list would mean that a respondent may be under stress.

Adult↗

PubMed1995

In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme: identification of glycation sites.

We have reported that the enzyme which reduces 3-deoxyglucosone (3-DG), a major intermediate and a potent cross-linker in the Maillard reaction, is identical with aldehyde reductase [Takahashi, M., Fujii, J., Teshima, T., Suzuki, K., Shiba, T., & Taniguchi, N. (1993) Gene 127, 249-253]. The enzyme purified from normal rat liver was found to be partially glycated as judged by binding to a boronate column and reactivity to anti-epsilon-hexitol lysine IgG. Sites of in vivo glycation of rat liver aldehyde reductase were identified by sequencing of digested peptides labeled with NaB[3H]4 and by mass spectrometry. The major glycated sites were lysines 67, 84, and 140. The glycated enzyme had low catalytic efficiency (kcat/Km) as compared to the nonglycated form. In streptozotocin-induced diabetic rats, the glycated form was significantly increased in kidneys. Because the enzyme plays a role in detoxifying 3-DG formed through the Maillard reaction in vivo, glycation of aldehyde reductase and reduction of its activity may result in the metabolic imbalance under diabetic conditions.

Aldehyde Reductase↗

PubMed1995

Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.

A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.

Amino Acid Sequence↗

PubMed1995

Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells.

We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗

PubMed1995

Growth retardation in human cervical dysplasia-derived cell lines by beta-carotene through down-regulation of epidermal growth factor receptor.

We used newly established cervical dysplasia-derived cell lines to elucidate a molecular mechanism of the preventive action of beta-carotene in cervical multi-step carcinogenesis. Liposomal beta-carotene was added to the culture medium for human cervical dysplasia cell lines, CICCN-2 from cervical intraepithelial neoplasia grade I (CIN I), CICCN-3 from CIN II, and CICCN-4 from CIN III, and human cervical carcinoma-derived cell lines such as CICCN-6, CICCN-18, and HeLa cells. beta-Carotene (10 mumol/L) induced significant growth retardation in three cervical dysplasia cell lines but not in three cervical carcinoma-derived cell lines. Binding activities of epidermal growth factor (EGF) and cellular amounts of either messenger RNA for EGF receptor gene or EGF receptor protein were all highest in CICCN-4 cells. Cell surface binding, as well as internalization, of 125I-labeled EGF was rapidly reduced after beta-carotene treatment in dysplasia cell lines and 170-kD protein bands of EGF receptor disappeared from protein immunoblots at day 3 of the treatment. Cellular amounts of EGF receptor messenger RNA remained constant until day 3 of the treatment and were substantially reduced after day 7. Chromatin condensations, morphologic evidence for apoptotic cell death, were observed at day 1 by staining. From these results, we contend that prevention of cervical carcinogenesis by beta-carotene is due to induction of apoptosis in cervical dysplastic cells, which are premalignant cells in cervical multi-step carcinogenesis, via down-regulation of EGF receptor protein.

Anticarcinogenic Agents↗

PubMed1995

Patient-hospital relationship and quality of life in elderly patients with hypertension.

To investigate the influence of the patient-hospital relationship on the quality of life (QOL) and on medication compliance in elderly patients with hypertension, we developed a comprehensive scale for assessing the patient-hospital relationship. In the first phase, an original questionnaire consisting of 40 items was designed under five fields for global assessment of the concept. We administered this questionnaire to 715 patients with cardiovascular diseases. Variations in the scores were evaluated by factor analysis. Three factors that comprised the majority of variance were derived by principal components analysis and varimax transformation. These orthogonal factors were considered to reflect "convenience of outpatient clinic", "satisfaction with clinical staff", and "communication with physician". We adopted three items under each of the three domains. Using this instrument, we assessed the patient-hospital relationship in 402 elderly outpatients with hypertension at 8 clinical centers. QOL and medication compliance were simultaneously assessed. A linear regression analysis revealed a significant positive correlation between patient-hospital relationship and QOL scores (r = 0.403, p < 0.0001), which means that patient-hospital relationship explained up to 16% of the total variability in scores on the Overall Quality of Life scale. The medication compliance was also better in patients with higher scores on the Patient-Hospital Relationship scale. These results indicate that the patient-hospital relationship is one of the substantial factors determining QOL and medication compliance in elderly patients with hypertension.

Aged↗

PubMed1995

Prediction of early development of chronic nonrheumatic atrial fibrillation.

The purpose of this study was to identify predictors of development of chronic nonrheumatic atrial fibrillation within one year of onset, thereby minimizing the risk of embolic complications and death. We retrospectively studied 137 patients with new-onset nonrheumatic atrial fibrillation. Chronic atrial fibrillation developed in 30 patients at the end of one year (chronic group). Atrial fibrillation remained paroxysmal in 107 patients (paroxysmal group). Clinical characteristics, electrocardiograms, and echocardiograms at the time of the onset of atrial fibrillation were compared in the two groups. Patients in the chronic group were significantly older than patients in the paroxysmal group (70.1 +/- 8.2 vs. 62.4 +/- 11.0 years, p < 0.01) and had a significantly higher incidence of congestive heart failure (13% vs. 3%, p < 0.05) and diabetes mellitus (37% vs. 19%, p < 0.05). The chronic group also exhibited higher cardiothoracic ratio (52.0 +/- 5.7% vs. 47.6 +/- 5.0%, p < 0.01), greater f-wave amplitude in lead V1 (1.48 +/- 0.91 vs. 1.06 +/- 0.45 mm, p < 0.05), larger left atrial dimension (41.0 +/- 6.4 vs. 34.2 +/- 7.6 mm, p < 0.01), and lower left ventricular ejection fraction (71.4 +/- 5.6% vs. 75.5 +/- 8.2%, p < 0.05). The presence of four or more of the following seven factors strongly predicted the development of chronic nonrheumatic atrial fibrillation within one year (88% to 100%): age > or = 65 years, congestive heart failure, diabetes mellitus, cardiothoracic ratio > or = 50%, f-wave amplitude > or = 2.0 mm, left atrial dimension > or = 38 mm, and ejection fraction < or = 76%.

Adult↗

PubMed1995

Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells.

Nitric oxide (NO) is believed to be an effector molecule that mediates interleukin (IL)-1 beta-induced destruction and dysfunction of pancreatic beta-cells. We have demonstrated that both exogenous NO and NO generated endogenously by IL-1 beta brought about apoptosis of isolated rat pancreatic islet cells as well as pancreatic beta-cell tumor-derived cell line HIT. This apoptosis was characterized by cleavage of DNA into nucleosomal fragments of 180-200 bp and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. The IL-1 beta-induced internucleosomal DNA cleavage occurred in a time- and dose-dependent manner. Actinomycin D, cycloheximide, and nitric oxide synthase inhibitors inhibited the DNA cleavage, which was correlated with the amount of NO produced, indicating that NO produced by HIT cells themselves could mediate the apoptosis. Furthermore, in the presence of tumor necrosis factor (TNF)-alpha, large amounts of NO were produced by IL-1 beta and DNA cleavage occurred more noticeably, although TNF-alpha alone did not generate NO. Streptozotocin (STZ), a diabetogenic reagent containing a nitroso moiety, also released NO and induced internucleosomal DNA cleavage in HIT cells. These results suggest that NO-induced internucleosomal DNA cleavage is an important initial step in the destruction and dysfunction of pancreatic beta-cells induced by inflammatory stimulation or treatment with STZ.

Amino Acid Oxidoreductases↗

PubMed1995

[Physiological significance of superoxide dismutase isozymes].

Superoxide dismutases (SODs) scavenge superoxide anion and participate in an essential role as a defense system against oxidative stress in body. Cu,Zn-SOD is localized at cytoplasm. A defect in the Cu,Zn-SOD gene has been demonstrated in some cases of familial amyotrophic lateral sclerosis. Trisomy of chromosome 21 in Down's syndrome increases the level of this isozyme and causes the disease. Inactivation of Cu,Zn-SOD by glycation under hyperglycemic conditions may also be a critical factor for diabetic complication. The expression of the second isozyme, Mn-SOD localized at mitochondrial matrix, is regulated in a complex manner by many stimulants such as interleukin-1, -6, tumor necrosis factor, lipopolysaccharide, and tumor promoters phorbol ester (TPA) and okadaic acid. This isozyme seems to work as a defense mechanism against damage during inflammatory responses. The third isozyme, extracellular SOD, is highly glycosylated and has affinity for heparin sulfate. This may participate in scavenging superoxide in plasma and, therefore, missense mutation in heparin binding domain increases the serum level of this isozyme, although the physiological role is not clearly understood yet.

Amyotrophic Lateral Sclerosis↗

PubMed1994

Cerebellar infarctions secondary to cranio-cervical anomalies: a case report.

We report a case of cerebellar infarctions which occurred in the territories of the bilateral posterior inferior cerebellar arteries. This case was complicated with cranio-cervical anomalies composed of assimilation of the atlas, atlanto-axial dislocation, and basilar impression. The 40-year-old male patient had no detectable risk factors predisposing to atherosclerotic arterial occlusion or cardiogenic embolism, and there were no angiographic findings of atherosclerosis. It was, therefore, postulated that the cerebellar infarctions were secondary to those cranio-cervical anomalies. The developing mechanism is discussed.

Adult↗