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Biomedical subjects

R Inoue

Publications and source records attributed to R Inoue.

At least 55 records · Page 3Linked to original sources
PubMed2000

Reduced IFNgamma production in response to IL-12 stimulation and/or reduced IL-12 production in atopic patients.

BACKGROUND: Several studies have shown that interleukin (IL)-4 and interferon-gamma (IFNgamma) are important for the regulation of IgE production and that IL-12 induces IFNgamma. OBJECTIVE: IFNgamma production in response to IL-12 stimulation and IL-12 production were investigated in peripheral blood mononuclear cells (PBMCs) of atopic patients with various levels of serum IgE. METHODS: Cytokine production from PBMCs was measured following stimulation with a nonspecific stimulator (phytohemagglutinin: PHA), a specific stimulator (Der f 1) and IL-12 in 17 healthy controls and 23 atopic patients with various serum IgE levels. RESULTS: The IFNgamma production by IL-12-stimulated PBMCs in the atopic group was lower than that in the control group. Furthermore, the serum IgE level was negatively correlated with IFNgamma production by PBMCs stimulated with IL-12 (P < 0.001), and with IL-12 production by PBMCs stimulated with Der f 1 (P < 0.001). Although the IFNgamma concentrations by PHA-stimulated PBMCs were correlated with those by IL-12-stimulated PBMCs, there were differences in several patients. CONCLUSIONS: Our results indicated that atopic patients may have some abnormality in the IL-12-IFNgamma loop. It was shown that the elevation in IgE levels in atopic patients may be due to reduced IFNgamma production in response to IL-12 stimulation and/or due to reduced IL-12 production.

Adolescent↗
PubMed2000

Characterization of human polymorphic DNA repair methyltransferase.

The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare.

Blotting, Northern↗
PubMed2000

Temperature-sensitive gating of cation current in guinea pig ileal muscle activated by hyperpolarization.

The temperature dependence of hyperpolarization-activated current (I(h)) was investigated in freshly isolated guinea pig ileal smooth muscle cells, using the nystatin-perforated whole cell recording technique. Hyperpolarizing pulses (-50 to -120 mV) from -40 mV evoked time-dependent inward rectifying currents with a reversal potential of -33 mV and a slow activation time course well approximated by a single exponential. The properties of these currents, such as steady-state variables, dependence on external K, modification by norepinephrine, and blockade by Cs or ZD-7288, coincide well with those of the "classical" I(h) discovered in the sinoatrial node. Raising the temperature (range: 22-33 degrees C) accelerated the activation time course of this I(h) and shifted its 50% activation potential positively (12 mV/10 degree) with much less change in the maximum conductance. Based on a simple closed-open model, this can be explained by a high temperature dependence of the opening rate constant (temperature coefficient: 3.4). The activation profile of reconstructed I(h) at 36 degrees C suggests that a considerable overlap could occur between the ranges of I(h) activation and physiological membrane potential.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Intracellular ATP slows time-dependent decline of muscarinic cation current in guinea pig ileal smooth muscle.

The effects of intracellular nucleotide triphosphates on time-dependent changes in muscarinic receptor cation currents (I(cat)) were investigated using the whole cell patch-clamp technique in guinea pig ileal muscle. In the absence of nucleotide phosphates in the patch pipette, I(cat) evoked every 10 min decayed progressively. This decay was slowed dose dependently by inclusion of millimolar concentrations of ATP in the pipette. This required a comparable concentration of Mg(2+), was mimicked by UTP and CTP, and was attenuated by simultaneous application of alkaline phosphatase or inhibitors of tyrosine kinase. In contrast, a sudden photolytic release of millimolar ATP (probably in the free form) caused a marked suppression of I(cat). Submillimolar concentrations of GTP dose dependently increased the amplitude of I(cat) as long as ATP and Mg(2+) were in the pipette, but, in their absence, GTP was ineffective at preventing I(cat) decay. The decay of I(cat) was paralleled by altered voltage-dependent gating, i.e., a positive shift in the activation curve and reduction in the maximal conductance. It is thus likely that ATP exerts two reciprocal actions on I(cat), through Mg(2+)-dependent and -independent mechanisms, and that the enhancing effect of GTP on I(cat) is essentially different from that of ATP.

Adenosine Triphosphate↗
PubMed2000

Long-term study of a female hyper-IgM immunodeficiency.

Hyper-IgM immunodeficiency (HIM) is an immunological disorder characterized by normal or elevated serum IgM levels, and reduced serum IgG and IgA levels, due to the disruption of immunoglobulin class switching in B cells. X-linked hyper-IgM is caused by the defective expression of the CD40 ligand on activated T cells, which induces immunoglobulin class switching along with some cytokines, such as interleukin 4, by the signal transduction of CD40 in B cells. We report on a Japanese girl who initially showed low serum IgM, IgG and IgA levels like patients with common variable immunodeficiency; however, in the course of time, serum IgG levels became reduced and serum IgM levels increased, resulting in the typical immunoglobulin profile of HIM. Neutropenia, one of the features of X-linked HIM, was not observed. In spite of extremely low serum IgG levels, she did not show any predisposition to severe infection, even without gammaglobulin replacement therapy. No mutation of the CD40 ligand or CD40 was detected. Sequencing of the complementarity-determining region of immunoglobulin heavy-chain genes in peripheral B lymphocytes revealed that they were all in frame, and insertion of the N region was detected. These results indicate that the heavy-chain gene rearrangement in the patient's B cells is intact. Non-X-linked HIM has heterogeneous pathogenetic mechanisms, and some groups may show the resistance to infection at the healthy donor level. The underlying defects in non-X-linked HIM might be specifically involved in class switching.

Adolescent↗
PubMed2000

T-cell-specific expression of kinase-defective Eph-family receptor protein, EphB6 in normal as well as transformed hematopoietic cells.

Although most kinase-defective growth factor receptor proteins are associated with pathogenic conditions, a kinase-defective Eph-family receptor protein, EphB6, is expressed in normal human tissues. We generated monoclonal antibodies specific for human EphB6 to characterize its expression on human hematopoietic cells. A very small population of normal human peripheral white blood cells (0.57 +/- 0.07%, n = 12) expressed EphB6. The EphB6-positive cells were CD2+, CD7+, CD3+ and CD4+ or CD8+ lymphocytes, but they did not express CD19 or CD11b. In human bone marrow, only 1.5 +/- 0.19% of lymphocytes expressed EphB6. Compared with the expression in peripheral lymphocytes, prominent expression of EphB6 protein was demonstrated in CD4+CD8+ double-positive mouse thymocytes. The T-cell lineage-specific expression was strictly conserved in human leukemia/lymphoma cells. Among T-cell-derived leukemia cells, the expression level of EphB6 seemed to decrease with maturation of the cells. These results suggest that EphB6 expression is regulated in T-cell development.

Animals↗
PubMed2000

Feasibility of high-dose chemotherapy without stem cell support as a first-line treatment for non-Hodgkin's aggressive lymphoma: a pilot study.

A regimen which incorporates cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) is the standard treatment for patients with non-Hodgkin's lymphoma (NHL), but it has not been effective in patients with aggressive NHL who are at high risk. The aim of the present trial was to investigate the feasibility of high-dose chemotherapy (HDC) without stem cell support as a first-line treatment. The primary endpoint was a complete remission rate. The second endpoint was survival. Fourteen patients with aggressive NHL entered the study and were treated according to the K93 protocol (3 cycles of CHOP, high-dose etoposide and ifosfamide, and high-dose methotrexate) Eleven patients (79%) achieved complete remission (CR) and two (14%) achieved partial remission (PR). Overall survival (OS) after five years was 79%. The actuarial five year disease free survival (DFS) for the eleven cases of CR was 75%. During chemotherapy, grade IV hematologic toxicity was observed in all patients and grade IV non-hematologic toxicity in only one patient, who experienced oral ulcers. Peripheral blood stem cell (PBSC) apheresis was performed in eight cases. One harvesting was enough to provide an adequate number of CD34+ cells for the subsequent PBSC transplantation (PBSCT). In conclusion our study confirmed the efficacy of the K93 protocol in obtaining a good response (CR + PR) rate and a very good DFS rate in most cases of aggressive NHL, with acceptable toxicity. This regimen may improve the outcome in cases of aggressive NHL without stem cell support. It seems worthwhile to conduct a randomized controlled study comparing the K93 protocol with the standard CHOP regimen.

Adult↗
PubMed2000

[First documentation of transfusion-associated babesiosis in Japan].

A 40-year-old man received blood transfusion in December 1998 because of gastric bleeding from a peptic ulcer. One month later, he developed febrile hemolytic anemia. Administration of high doses of glucocorticoid significantly reduced the hemolysis, but did not cure the disease. To investigate the cause of the hemolysis, the patient was transferred to our hospital in May 1999. Giemsa-stained peripheral blood smears showed Babesia parasites in the red blood cells (RBC), and PCR analysis confirmed the presence of Babesia microti DNA. The parasitemia disappeared hematologically after 2 weeks of quinine and clindamycin therapy. However, parasite DNA was still detectable in the RBC. Although treatment with oral atovaquone was given for 2 weeks, parasitemia and febrile hemolysis recurred within a month after the last treatment. Fortunately, complete remission was obtained after a second 12-week course of therapy with quinine and clindamycin. PCR analysis revealed asymptomatic Babesia infection in one of eight samples from the original blood donor. The initial steroid therapy given to the patient without an accurate diagnosis seemed to have delayed augmentation of the specific antibodies (IgG) against Babesia microti, thus prolonging the parasitemia after the initial acute stage of babesiosis.

Adult↗
PubMed1999

Mutations of the IL-12 receptor beta2 chain gene in atopic subjects.

Interleukin-12(IL-12) promotes cell-mediated Th1 responses and production of IFN-gamma that downregulates IgE production. The signal of IL-12 is transduced through the IL-12 receptor (IL-12R) and Stat4. Twenty-four of 75 atopic individuals with high levels of IgE showed insufficient IFN-gamma production by peripheral blood mononuclear cells (PBMCs) following stimulation with IL-12 but not that with phytohemagglutinin (PHA). Interestingly, 10 of the above 24 subjects were found to be heterozygous for truncated (2496 del 91) or missense (1577 A to G and 2799 A to G) mutations of IL-12R beta2 chain gene (IL-12R beta2). Insufficient phosphorylation of Stat4 was also demonstrated in these 10 individuals. This is the first report showing that reduced IFN-gamma production following IL-12 stimulation is associated with the heterozygous IL-12R beta2 mutations in atopic subjects.

Adolescent↗
PubMed1999

Electrophysiological actions of a novel K(+) channel opener MCC-134 on rabbit portal vein smooth muscle.

The effects of a newly synthesized K(+) channel opener, 1-[4-(1H-imidazol-1-yl)benzoyl]-N-methylcyclobutane-carbothioamide (MCC-134) on membrane currents and intracellular Ca(2+) mobilization were investigated in rabbit portal vein smooth muscle cells. Under voltage-clamped conditions, MCC-134 dose-dependently induced K(+)-selective currents (I(MCC); EC(50) 5.3 microM) showing little desensitization but fast deactivating properties on washout of drugs. I(MCC) was completely blocked by 10 microM glibenclamide, not affected by iberiotoxin (500 nM), charybdotoxin (200 nM) or apamin (500 nM), and inhibited by nonspecific K(+) channel blockers, tetraethylammonium (1-10 mM), 4-aminopyridine (0.1-1 mM) and Ba(2+) (0.01-0.1 mM). Intracellularly applied nucleotide diphosphates (1 mM) were effective at maintaining I(MCC) (apparent potency; ADP<==GDP falling dotsIDP<UDP), whereas intracellular ATP exhibited a biphasic, i.e., augmentative and inhibitory effect(s) on I(MCC). Single channel activities of about 15 pS (40/140 mM extra-/intracellular K(+)) fully accountable for macroscopic I(MCC) were activated by MCC-134. MCC-134, at a concentration as high as 100 microM, suppressed voltage-dependent Ca(2+) and noradrenaline-induced cation currents, and concomitant elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)).

Adenosine Diphosphate↗
PubMed1999

Predominant distribution of nifedipine-insensitive, high voltage-activated Ca2+ channels in the terminal mesenteric artery of guinea pig.

We have found nifedipine-insensitive (NI), rapidly inactivating, voltage-dependent Ca2+ channels (current, NI-I(Ca)) with unique biophysical and pharmacological properties in the terminal branches of guinea pig mesenteric artery, by using a whole-cell mode of the patch-clamp technique. The fraction of NI-I(Ca) appeared to increase dramatically along the lower branches of mesenteric artery, amounting to almost 100% of global I(Ca) in its periphery. With 5 mmol/L Ba2+ as the charge carrier, NI-I(Ca) was activated with a threshold of -50 mV, peaked at -10 mV, and was half-activated and inactivated at -11 and -52 mV, respectively, generating a potential range of constant activation near the resting membrane potential. The NI-I(Ca) was rundown resistant, was not subject to Ca(2+)-dependent inactivation, and exhibited the pore properties typical for high voltage-activated Ca2+ channels; Ba2+ is approximately 2-fold more permeable than Ca2+, and Cd2+ is a better blocker than Ni2+ (IC(50), 6 and 68 micromol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca2+ channels such as omega-conotoxins GVIA and MVIIC (each 1 micromol/L) and omega-agatoxin IVA (1 micromol/L) were ineffective at inhibiting NI-I(Ca), whereas nimodipine partially (10 micromol/L; approximately 40%) and amiloride potently ( approximately 75% with 1 mmol/L; IC(50); 107 micromol/L) blocked the current. Although these properties are reminiscent of R-type Ca2+ channels, expression of the alpha(1E) mRNA was not detected using reverse transcriptase-polymerase chain reaction. These results strongly suggest the predominant presence of NI, high voltage-activated Ca2+ channels with novel properties, which may be abundantly expressed in peripheral small arterioles and contribute to their tone regulation.

Animals↗
PubMed1999

Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca(2+) influx activated in response to stimulation of G(q) protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features, compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca(2+) release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca(2+) influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and for Ca(2+) release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca(2+) release. In fact, TRP7 expression did not affect capacitative Ca(2+) entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn(2+) influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca(2+) at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid revealed an essential role of intracellular Ca(2+) in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced cation currents in various native systems.

Amino Acid Sequence↗
PubMed1999

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.

Apoenzymes↗
PubMed1999

Tissue factor pathway inhibitor-2 is a novel mitogen for vascular smooth muscle cells.

A mitogen for growth-arrested cultured bovine aortic smooth muscle cells was purified to homogeneity from the supernatant of cultured human umbilical vein endothelial cells by heparin affinity chromatography and reverse-phase high performance liquid chromatography. This mitogen was revealed to be tissue factor pathway inhibitor-2 (TFPI-2), which is a Kunitz-type serine protease inhibitor. TFPI-2 was expressed in baby hamster kidney cells using a mammalian expression vector. Recombinant TFPI-2 (rTFPI-2) stimulated DNA synthesis and cell proliferation in a dose-dependent manner (1-500 nM). rTFPI-2 activated mitogen-activated protein kinase (MAPK) activity and stimulated early proto-oncogene c-fos mRNA expression in smooth muscle cells. MAPK, c-fos expression and the mitogenic activity were inhibited by a specific inhibitor of MAPK kinase, PD098059. Thus, the mitogenic function of rTFPI-2 is considered to be mediated through MAPK pathway. TFPI has been reported to exhibit antiproliferative action after vascular smooth muscle injury in addition to the ability to inhibit activation of the extrinsic coagulation cascade. However, structurally similar TFPI-2 was found to have a mitogenic activity for the smooth muscle cell.

Animals↗
PubMed1999

Clinical evaluation of SITA: a new family of perimetric testing strategies.

PURPOSE: To perform a clinical comparison of the Humphrey Full Threshold strategy with its intended replacement, SITA Standard, regarding testing time, reproducibility, and measured threshold sensitivity in normal subjects and glaucoma patients. METHODS: Thirty-eight healthy volunteers and 80 patients with primary open-angel glaucoma underwent central 30-2 testing with both Full Threshold and SITA strategies using a Humphrey HFA 2 Model 740 perimeter. The testing time, reproducibility, and measured threshold sensitivity of the two strategies were compared. RESULTS: The testing time for measurements with SITA was 56% lower in normal subjects and 45% lower in glaucoma patients than with Full Threshold. Cases having higher mean sensitivity or cases requiring longer testing time for Full Threshold had a greater percentage reduction in testing time for SITA. The test-retest variability studied in normal subjects was lower, but not significantly so, with SITA (2.9%) than with Full Threshold (3.4%). The mean sensitivities of SITA were higher (1 dB) than those of Full Threshold not only in normal subjects but also in glaucoma patients. This higher sensitivity in SITA was found irrespective of the order of the two tests both in normal subjects and glaucoma patients. Analysis by each testing point revealed that the difference was not dependent on the test point position or the sensitivity at the point. CONCLUSION: SITA has greater patient acceptability than the Full Threshold strategy. However, the difference in sensitivity can be considerable in a serial comparison of one patient's fields tested by Full Threshold and SITA.

Adolescent↗
PubMed1999

Psychological impact of informed consent in hospitalized cancer patients. A sequential study of anxiety and depression using the hospital anxiety and depression scale.

Patients with cancer receive an explanation of their disease and the recommended treatment when they are asked to give informed consent (IC). In the course of this process patients suffer severe distress, including anxiety and depression, but physicians tend to underestimate it. The goal of this study was to reveal the magnitude of such stress and any changes to this during the IC process by means of the Hospital Anxiety and Depression (HAD) scale, a self-assessment scale. Of 171 in-patients newly diagnosed with lung cancer, 119 were assessable for serial HAD scale scores on admission, immediately after the IC process, and at 1 and again at 2 weeks after the IC. Both anxiety and depression scores increased significantly immediately after IC. Female patients had significantly higher anxiety and depression scores than males at 1 week after the IC. The patients with poor performance status demonstrated high anxiety scores on admission and immediately after the IC, and substantial depression persisted longer in these patients. The prevalence of high scores of more than 11 (judged as adjustment disorder or more severe state) immediately after the IC was 50% for anxiety and 31% for depression. The prevalence decreased significantly within 1 or 2 weeks, but 41% and 14% of the patients still showed high anxiety and depression scores, respectively. Physicians should be aware of these facts and pay special attention to their patients' psychological distress in routine clinical practice.

Adjustment Disorders↗