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Biomedical subjects

T Fujii

Publications and source records attributed to T Fujii.

At least 145 records · Page 8Linked to original sources
PubMed2001

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase.

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.

Animals↗
PubMed2001

Cytokine and immunogenetic profiles in Japanese patients with adult Still's disease. Association with chronic articular disease.

OBJECTIVE: To determine cytokines and MHC class II alleles in Japanese patients with adult Still's disease (ASD) and clarify the association between those profiles and chronic articular disease. METHODS: Of 35 patients with ASD (13 men, 22 women, mean age at onset 34.0 yr), 17 (49%) had chronic arthritis (>6 months, chronic articular ASD) and 18 (51%) lacked chronic arthritis (systemic ASD). Cytokines and cytokine receptors in sera were measured by ELISA. Correlations of each cytokine with disease activity or C-reactive protein (CRP) were determined. MHC class II alleles were examined by polymerase chain reaction methods. RESULTS: In chronic articular ASD, female gender was more frequent and liver dysfunction and myalgia were rarer than in systemic ASD. In active disease, the white blood cell count was lower, but total IgG was greater in patients with chronic articular ASD than in those with systemic ASD. Tumour necrosis factor (TNF) alpha, soluble TNF receptor 2 and interleukin (IL)-18 were increased in both types of ASD, even in remission. Soluble IL-2 receptors, IL-4 and IL-18 levels were correlated with disease activity or CRP value only in chronic articular ASD. Interferon gamma and IL-8 remained increased only in chronic articular ASD, even when disease activity, including IL-6 and CRP, was low. DRB1*1501 (DR2) and DRB1*1201 (DR5) alleles were more frequent in chronic articular than in systemic ASD, whereas DQB1*0602 (DQ1) was frequently observed in both types of ASD. CONCLUSION: The present study suggests that ASD with chronic articular disease has distinct clinical, cytokine and immunogenetic profiles.

Adult↗
PubMed2001

Alteration of glutathione reductase expression in the female reproductive organs during the estrous cycle.

The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.

Animals↗
PubMed2001

Are DNA ploidy and epidermal growth factor receptor prognostic factors for untreated ovarian cancer? A prospective study.

To identify prognostic factors for untreated ovarian cancer, DNA ploidy, proliferative index (P.I.) and epidermal growth factor receptor (EGFR) expression were analyzed in a prospective series of 40 patients with ovarian cancer and 7 patients with borderline malignant ovarian tumor followed up for 5 years or more (median, 77 months). The frequency of aneuploid cells was 53.8% (21/39) in ovarian cancer and 14.3% (1/7) in borderline malignancy. There was no significant association between DNA ploidy and the clinicopathologic findings, in which aneuploid ovarian cancer was more common among advanced tumors. The S-phase fraction and P.I. value were higher in the patients with aneuploid tumors (p = 0.076). EGFR expression was detected in 76.9% (30/39) of ovarian cancers and 42.9% (3/7) of borderline malignant ovarian tumors, and the mean EGFR level was 5.8 +/- 12.1 (range: 0-49.5) and 28.3 +/- 71.1 (range: 0-189.4) fmol/mg protein, respectively. There was no correlation between EGFR expression and DNA ploidy, P.I., and clinicopathologic findings analyzed. The 5-year survival rate in patients with aneuploid tumors was significantly worse in patients with ovarian cancer (p = 0.0165, log-rank test). No significant relationship was shown between P.I., EGFR expression, and 5-year survival. Cox multivariate analysis showed that DNA ploidy, P.I., and EGFR expression are not associated with the risk of death (p = 0.5917, p = 0.9924, and p = 0.6840, respectively), although clinical stage shows a significant relationship (p = 0.0027). Our data showed that DNA ploidy is significantly related to the prognosis by univariate analysis, but DNA ploidy, P.I., and EGFR expression were not independent prognostic factors for the untreated ovarian cancer.

ErbB Receptors↗
PubMed2001

Successful laparoscopic treatment of ileo-cecal endometriosis producing bowel obstruction.

Bowel endometriosis manifesting with ileus is difficult to diagnose, often requiring laparotomy for diagnosis and treatment. We report here a case of ileo-cecal endometriosis causing bowel obstruction. A diagnosis of intestinal endometriosis with menstruation-associated bowel symptoms was made, and the patient was successfully treated by laparoscopic ileo-cecal resection.

Endometriosis↗
PubMed2001

Human leukocyte antigen-G-expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood.

PROBLEM: To better understand the role of human leukocyte antigen (HLA)-G in regulating the T helper (Th)1/Th2 cytokine balance, one of key conditions in determining the fate of pregnancy, we asked whether the presence of HLA-G protein altered the release of cytokines from both decidual mononuclear cells and peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: The amounts of cytokines released from decidual mononuclear cells and PBMCs were compared in the presence or absence of HLA-G-expressing cells. RESULTS: When cocultured with HLA-G-expressing cells, the amounts of tumor necrosis factor-alpha and interferon-gamma released from decidual mononuclear cells and PBMCs were decreased, while the amounts of interleukin (IL)-4 from PBMCs was increased, with IL-4 release from decidual mononuclear cells being unchanged. CONCLUSIONS: Upon contact with HLA-G, decidual mononuclear cells, and PBMCs as well, modulate their ability to release cytokines in a way that may shift the Th1/Th2 balance towards relative Th2 dominance, suggesting a role for HLA-G in maintaining pregnancy.

Adult↗
PubMed2001

Polymorphism of human leukocyte antigen-E gene in the Japanese population with or without recurrent abortion.

PROBLEM: The aim of this study was to investigate the gene frequencies and shared alleles of human leukocyte antigen (HLA)-E gene in Japanese couples with or without recurrent abortion. METHOD OF STUDY: Polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis was carried out to detect polymorphism in exon 3 of the HLA-E gene in 30 Japanese couples with recurrent abortion and 38 normal Japanese couples with proven fertility. RESULTS: No point mutation was detected in exon 3 of HLA-E in both recurrent aborters and normal controls. HLA-EG and HLA-ER alleles were detected with frequencies of 66.7% and 33.3% in couples with recurrent abortion and 69.2% and 30.8 in normal couples, respectively. The gene frequency of HLA-EG was higher than that of HLA-ER, which is contrary to that found in Caucasian, African-American and Hispanic people but similar to Chinese people. The frequency of each allele was not significantly different between recurrent aborters and normal controls. The number of shared alleles between each couple with recurrent abortion is not significantly different from that with normal controls. CONCLUSION: Allele frequencies of HLA-E were suggested to be different in Asian people from those in other ethnic people. In light of no specific distribution pattern in recurrent aborters, HLA-E polymorphism does not seem to play a role in the pathogenesis of recurrent abortion.

Abortion, Habitual↗
PubMed2001

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

Animals↗
PubMed2001

Influence of timing and magnitude of arterial wave reflection on left ventricular relaxation.

The influence of timing and magnitude of arterial wave reflection (WR) on afterload-dependent relaxation was evaluated in patients with a variety of heart diseases (group 1, age < 30 yr; group 2, age > 40 yr) and in dogs. While both femoral arteries were compressed (FC), WR returned just after the dicrotic notch (early diastole) in group 1 but before the dicrotic notch (late systole) in group 2. The time constant of the left ventricular pressure decay (tau) was shortened during FC in group 1, whereas it was prolonged in group 2. In dogs, a constriction of the thoracic aorta induced a late systolic augmentation of WR with a prolongation of tau (cf. group 2), whereas constriction of the lower abdominal aorta induced an early diastolic augmentation of WR with a shortening of tau (cf. group 1). With aortic constriction, coronary flow increased, and there was a close correlation between the peak change in backward aortic pressure and that in coronary flow regardless of the timing of WR. Thus the time at which WR returns during the cardiac cycle may have an important effect on left ventricular relaxation and coronary flow.

Adult↗
PubMed2001

Generation of periventricular leukomalacia by repeated umbilical cord occlusion in near-term fetal sheep and its possible pathogenetical mechanisms.

Periventricular leukomalacia (PVL) is a major cause of cerebral palsy. However, pathogenetic mechanisms of PVL have not been fully understood. Although it has been postulated that umbilical cord compression is related to the development of PVL, no animal experiments clearly demonstrated an association of umbilical cord occlusion with 'periventricular' white matter lesions. The purpose of this study is to determine whether umbilical cord occlusions could produce periventricular white matter lesions in fetal sheep and to examine how changes in fetal cardiovascular and metabolic variables are related to the induction of brain damage. Fourteen near-term fetal sheep underwent umbilical cord occlusion (3-min total cord occlusions 5 times at 5-min intervals). Dissections performed 24 h after cord occlusion revealed that periventricular white matter lesions were produced in 7 out of 14 sheep fetuses. According to the pattern of brain damage, we classified the fetal sheep into three groups: 5 fetuses with dominant lesions in the periventricular white matter (group I), 4 fetuses with brain lesions in the cerebral cortex and thalamus (group II) and 5 fetuses with no or minimal brain lesions (group III). Group I showed higher blood pressure and higher plasma lipid peroxide levels before cord occlusion compared to the other groups, while group II showed systemic hypotension during cord occlusion. No significant differences in changes in pH, PaCO2, PaO2 and heart rate were found between the three groups. It is speculated that PVL might be produced by an association of preexisting chronic circulatory instability with an acute episode of severe repetitive cord occlusion.

Animals↗
PubMed2001

Particulate matter induces cytokine expression in human bronchial epithelial cells.

The present study was designed to determine cytokines produced by primary human bronchial epithelial cells (HBECs) exposed to ambient air pollution particles (EHC-93). Cytokine messenger RNA (mRNA) was measured using a ribonuclease protection assay and cytokine protein production by enzyme-linked immunosorbent assay. Primary HBECs were freshly isolated from operated lung, cultured to confluence, and exposed to 10 to 500 microg/ml of a suspension of ambient particulate matter with a diameter of less than 10 microm (PM(10)) for 2, 8, and 24 h. The mRNA levels of leukemia inhibitory factor (LIF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1alpha, and IL-8 were increased after exposure to PM(10), and this increase was dose-dependent between 100 (P < 0.05) and 500 (P < 0.05) microg/ml of PM(10) exposure. The concentrations of LIF, GM-CSF, IL-1beta, and IL-8 protein measured in the supernatant collected at 24 h increased in a dose- dependent manner and were significantly higher than those in the control nonexposed cells. The soluble fraction of the PM(10) (100 microg/ml) did not increase these cytokine mRNA levels compared with control values and were significantly lower compared with HBECs exposed to 100 microg/ml of PM(10) (LIF, IL-8, and IL-1beta; P < 0.05), except for GM-CSF mRNA (P = not significant). We conclude that primary HBECs exposed to ambient PM(10) produce proinflammatory mediators that contribute to the local and systemic inflammatory response, and we speculate that these mediators may have a role in the pathogenesis of cardiopulmonary disease associated with particulate air pollution.

Aged↗
PubMed2001

Relationship between doppler-derived left ventricular diastolic function and exercise capacity in patients with myocardial infarction.

Doppler echocardiographic indices of left ventricular (LV) diastolic function are widely used to evaluate the cardiac function of patients with cardiac disease. However, there have been few reports about the relationship between Doppler indices and exercise capacity and so 44 patients with myocardial infarction were investigated by cardiopulmonary exercise testing and 2-D and Doppler echocardiography. Diastolic performance was assessed using Doppler transmitral flow velocity and pulmonary venous flow velocity. The ratio of peak E wave velocity and peak A wave velocity (E/A) correlated with peak oxygen consumption (peak Vo2) (R=0.72), and there was a negative correlation between the deceleration time of E velocity (Dct) and peak Vo2 or anaerobic threshold (AT) (R=-0.65, -0.62, respectively). The ratio of peak S wave velocity and peak D wave velocity (S/D) negatively correlated with peak Vo2 (R=-0.58). Left ventricular ejection fraction did not correlate to exercise capacity. These results suggest that the Doppler echocardiographic indices of LV diastolic function correlate with exercise capacity in patients with mild cardiac dysfunction.

Aged↗
PubMed2001

An independent non-neuronal cholinergic system in lymphocytes.

Acetylcholine (ACh) is a well characterized neurotransmitter occurring throughout the animal kingdom. In addition, both muscarinic and nicotinic ACh receptors have been identified on lymphocytes of various origin, and their stimulation by muscarinic or nicotinic agonists elicits a variety of functional and biochemical effects. It was thus initially postulated that the parasympathetic nervous system may play a role in modulating immune system function. However, ACh in the blood has now been localized to lymphocytes; indeed expression of choline acetyltransferase (ChAT), an ACh synthesizing enzyme, has been shown in human blood mononuclear leukocytes, human leukemic T-cell lines and rat lymphocytes. Stimulation of T-lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh and increased expression of mRNAs encoding ChAT and ACh receptors. The observation that M3 muscarinic receptor stimulation by ACh and other agonists increases the intracellular free Ca2+ concentration and upregulates c-fos gene expression strongly argues that ACh, synthesized and released from T-lymphocytes, acts as an autocrine and/or paracrine factor regulating immune function. These findings present a compelling picture in which immune function is, at least in part, under the control of an independent lymphoid cholinergic system.

Acetylcholine↗
PubMed2001

Non-neuronal neurotransmitters and neurotrophic factors in amniotic epithelial cells: expression and function in humans and monkey.

Human amniotic epithelial cells (HAEC) are formed from epiblasts on the 8th day after fertilization. Because they lack major histocompatibility complex (MHC) antigen, human amniotic tissue transplantation has been used for allotranplantation to treat patients with lysosomal diseases. We have provided evidence that HAEC have multiple functions such as synthesis and release of acetylcholine (ACh) and catecholamine (CA) as well as expressing mRNA coding for dopamine receptors and dopamine (DA) transporter (DAT). On the other hand, we showed that monkey amniotic epithelial cells (MAEC) synthesize and release CA and posses DA receptors and DAT. Detection of muscarinic actylcholine receptors indicates the presence of an autocrine mechanism in HAEC. Recently, we found that HAEC have neurotrophic function in conditioned medium from HAEC, indicating the presence of a novel neurotrohpic factor that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors as well as neurotransmitters to the amniotic fluid, suggesting an important function in the early stages of neural development of the embryo. This review will focus on the neuropharmacological aspects of HAEC and MAEC in relation to the physiology of amniotic membrane.

Acetylcholine↗
PubMed2001

Treatment of model soils contaminated with phenolic endocrine-disrupting chemicals with laccase from Trametes sp. in a rotating reactor.

An enzymatic treatment system for the remediation of sand contaminated with endocrine-disrupting chemicals (EDCs) was studied. Laccase from Trametes sp. (Laccase Daiwa) decreased the amounts of nonylphenol, octylphenol, bisphenol A and ethynylestradiol (synthetic estrogen) adsorbed on sea sand (2 micromol g(-1)) in a test tube with shaking. The phenolic endocrine-disrupting chemicals might have polymerized via enzymatic conversion to their phenoxy radicals. The optimum pH for the enzymatic treatment was approximately 5. A rotating reactor was used for scaling up the enzymatic treatment. The reaction rate increased by rotating the reactor. The optimum speed of revolution was 10-15 rpm for the treatment of nonylphenol. The amounts of octylphenol, bisphenol A, and ethynylestradiol also decreased enzymatically in the reactor. Our enzymatic treatment system with a rotating reactor will be useful for the treatment of soil highly polluted with phenolic EDCs.

Journal Article↗
PubMed2001

Purification and properties of two malate dehydrogenases from Candida sp. N-16 grown on methanol.

Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of Vmax(oxaloacetate)/Vmax(malate) and Kcats (555 s(-1) for oxaloacetate, 481 s(-1) for NADH) of MDH-M2 were higher than the ratio (1.37) of Vmax and Kcats (241 s(-1) for oxaloacetate, 271 s(-1) for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD+ and the activity of MDH-M1 by oxaloacetate.

Candida↗
PubMed2001

[Expression and intracytoplasmic signal transduction pathway of fibroblast growth factor (FGF)-10 in human cervical cancer cell lines].

Fibroblast growth factor (FGF) -10 is a new member of the FGF family initially reported in Japan. It is mainly synthesized by mesenchymal cells and acts on epithelial cells in a paracrine manner. FGF-10 actions are dependent on their binding to the iiib form of FGF receptor 2 (FGFR2) iiib, also known as keratinocyte growth factor receptor (KGFR). FGF-10 has high amino acid homology to keratinocyte growth factor (KGF) and plays an important role in fetal limb and lung development and skin wound healing. In the present study, the expression of FGF-10 and FGFR2 iiib messenger RNA (mRNA) in two different human uterine cervical cancer cell lines (CaSki and ME-180) were examined. Both CaSki and ME-180 cells expressed FGFR2 iiib mRNA, while only CaSki cells expressed FGF-10 mRNA and protein. Recombinant FGF-10 (1 ng/ml) increased the growth rate of ME-180 cells and also enhanced mitogen-activated protein kinase (MAPK) phosphorylation of the cells. These data indicate that FGF-10 may directly promote the growth of squamous cell cancer in the uterine cervix via the MAPK pathway.

Cell Line↗