Biomedical subjects
Publications and source records attributed to K Inoue.
Roots of Prunus armeniaca yielded a new A-type proanthocyanidin whose structure was assigned as ent-epiafzelechin-3-O-p-hydroxybenzoate-(4alpha-->8, 2alpha-->O-->7)-epiafzelechin (1). The structure of 1 was determined through extensive 1D and 2D NMR studies.
We identified a novel mutation, L322P, in a patient with X-linked adrenoleukodystrophy (ALD) who underwent bone marrow transplantation (BMT). An identification of the ALD gene mutation enabled us to employ an approach not dependent on the use of radioisotopes for detecting mixed chimerism. This assay could show more than 99.0% of the patient's peripheral white blood cells were replaced by the donor's cells.
The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40 degrees C. Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0-0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H alpha signals of the residues in the beta-sheet showed a negative correlation with the 'folding' shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the beta-sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the 'folding' shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggest that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.
This study assessed the diagnostic potential of the actigraph, the Continuous Performance Test, and the Matching Familiar Figures Test in diagnosing attention-deficit hyperactivity disorder (ADHD). Twenty boys previously diagnosed with ADHD and 52 controls were examined. By these measures the boys with ADHD were differentiated from the controls with sensitivity and specificity above 75%. We were able to classify ADHD into eight subtypes by combining the scores of the actigraph and the CPT: "hyperactive-impulsive", "hyperactive-inattentive", "impulsive-inattentive", "hyperactive", "impulsive", "inattentive", "mixed", and "unspecified" type. These classifications may be useful in diagnosing ADHD.
1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.
1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.
1. The effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods. 2. Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes. 3. In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 microM) but not alpha,beta meATP (up to 100 microM) evoked a rise in [Ca2+]i. 4. In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 microM of alpha,beta meATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype. 5. In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 microM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise. 6. These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogeneously expressed P2X2 or P2X3.
We report here that a patient with relapsed AML after allogeneic bone marrow transplantation achieved and maintained complete remission (CR) after effective donor leukocyte transfusion (DLT), without the occurrence of GVHD and marrow aplasia, for more than 21 months. This continuous CR maintenance is mainly due to the application of DLT at molecular relapse that was diagnosed by monitoring minimal residual disease (MRD) by the quantitation of WT1 (Wilms tumor gene) expression levels (WT1 assay). The present case demonstrates that early application of DLT at molecular relapse is essential for the improvement of the efficacy of DLT for relapsed AML after BMT.
BACKGROUND: Nitric oxide (NO) has diverse activities under physiological and pathophysiological conditions in many types of cells. In cultured hepatocytes, NO is produced by inducible NO synthase (iNOS) in response to interleukin (IL)-1beta. Cis-controlling elements and transcription factors which were involved in iNOS gene expression in hepatocytes have been unclear. RESULTS: We measured the transcriptional activity of the human iNOS gene promoter fused to the firefly luciferase gene in primary cultured rat hepatocytes. The luciferase assay of 5' deleted promoters revealed that the region from -365 to the transcription initiation site is required for the promoter activity of the iNOS gene. Mutations of a CCAAT/enhancer-binding protein (C/EBP)-binding site, namely the A-activator-binding site (AABS), and a nuclear factor (NF)-kappaB-binding site within this region, markedly decreased the promoter activity. Transfection of C/EBPbeta liver-enriched activator protein (LAP) or NF-kappaB (RelA + p50) activated the iNOS promoter, and transfection of LAP and NF-kappaB further activated it synergistically. In addition, either mutation of AABS and the NF-kappaB-binding site markedly reduced the basal promoter activity and the transactivation by LAP, NF-kappaB, and a combination of LAP and NF-kappaB. Electrophoretic mobility shift assays showed that C/EBPbeta was bound to AABS. CONCLUSION: These results demonstrate that C/EBPbeta may involve iNOS gene expression synergistically with NF-kappaB in primary cultured rat hepatocytes.
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The genes of the botulinum neurotoxin A (BoNT) complex are clustered in a locus consisting of two divergent polycistronic operons, one containing the non-toxic, non-haemagglutinin (NTNH) component and bontA genes, the other containing the haemagglutinin (HA) component genes. The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein. A recombinant Clostridium botulinum A strain that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing botR/A under the control of its own promoter. The transformed strain produced more BoNT/A and associated non-toxic proteins (ANTPs) and the corresponding mRNAs than the non-transformed strain. Partial inhibition of botR/A by antisense mRNA resulted in lower levels of BoNT/A, NTNH and HA70 and the levels of the corresponding mRNAs. Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons. These results show that botR/A activated transcription of the genes encoding BoNT/A and ANTPs in C. botulinum A by interacting directly with the region promoter, and that the homologous genes in C. botulinum B, C and D presumably have the same function.
Exercise induced chromosomal damage was evaluated in trained and untrained subjects, who performed treadmill running at 85% of maximal oxygen uptake for 30 min. The subjects had their peripheral blood taken before, immediately after and 30 min after the running test for the analysis of lymphocyte chromosomal damage that was evaluated by micronucleus assay. The blood samples were also subjected to X-ray irradiation in vitro to examine the modification of exercise induced chromosomal damage by a secondarily induced oxidative stress. Spontaneous chromosomal damage in lymphocytes did not significantly increase at least until 30 min after the running both in the trained and untrained subjects. However, the X-ray-induced chromosomal damage was significantly enhanced at 30 min after the running in the untrained group, but not in the trained group. The ratio of X-ray-induced/spontaneous chromosomal damage also tended to increase after the running only in the untrained group. These preliminary results suggest that intensive exercise induced very slight chromosomal damage only in the untrained group, which could be intensified by the secondarily induced oxidative stress.
We utilized helical computed tomography (CT) in three infants (aged 1 to 5 months) with tracheomalacia after repair of esophageal atresia. Helical CT was used for preoperative diagnosis and operative planning. At the time of aortosternopexy, it is often difficult to determine the direction of suture traction to yield the largest tracheal lumen. The helical CT was useful not only for diagnosis of tracheomalacia but also for operative planning of aortosternopexy.
1. Two different types of genetic polymorphisms in each of CYP2C18 and CYP2C19 genes were examined and compared with respect to their frequencies in distribution in liver DNA of 39 Japanese and 45 Caucasians. 2. Individuals who were classified into CYP2C19m1 (as detected with SmaI digestion) in exon 5 of CYP2C19 gene were found to display a CYP2C18m1 polymorphism (as detected with DdeI digestion) in the 5'-flanking region of CYP2C18 gene in Japanese and Caucasian populations. The Japanese subjects who were classified into CYP2C19m2 (as detected with BamHI digestion) in exon 4 of CYP2C19 gene were found to have a CYP2C18m2 genetic polymorphism (as detected with Tsp509I digestion) in exon 2 of CYP2C18 gene. None of the Caucasians had the CYP2C18m2 nor CYP2C19m2 alleles. 3. Frequencies in two types (C416T in exon 3, A1061C in exon 7) of CYP2C9 genetic polymorphism were found to be independent to those of CYP2C18 and CYP2C19 genetic polymorphisms in these samples. 4. Thus, the results suggest that the CYP2C18 gene is localized very closely to the CYP2C19 gene on the same human chromosome.
1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144.Ile359), and two heterozygous Cys allele (Cys144.Ile359) and Leu allele (Arg144.Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K(m)'s for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7-hydroxylation of S-warfarin, but not R-warfarin, by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower Vmax's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wild-type and Cys allele CYP2C9, although the K(m)'s are not very different in liver microsomes of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.
Seven cases with Creutzfeldt-Jakob disease (CJD) located in the basin of the Fuji river (Fuji area) in Japan were examined genetically and clinicopathologically. The onset of the disease was between 1989 and 1995. All cases were from different families, although 3 cases were family members of previously reported CJD patients. They had clinical and/or neuropathological features, corresponding to subacute spongiform encephalopathy. Five of the 7 cases, including the 3 familial cases, had the E200K mutation in the gene encoding prion protein (PRNP). It is suggested that there is a small cluster of CJD patients with a founder effect of the E200K mutation in the Fuji area, because the incidence of CJD with the E200K mutation appears to be much higher in this area than other areas in Japan. The disease penetrance of the 5 cases with the E200K mutation seems to be low, and they may have an age-related incidence in the Fuji area. These findings support the hypothesis that the phenotypes of CJD patients with the PRNP mutations are linked to the position of the mutation, but not related to ethnic or environmental factors.
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It has been shown that hypophysectomy protects aflatoxin B1 (AFB1) hepatocarcinogenesis and the prevention of apoptosis is a critical process for tumorigenesis. In this paper, we analyzed the cell death of AFB1-induced rat hepatoma Kagura-2 (K2) cells elicited by an estrogen antagonist, tamoxifen (TAM), and transforming growth factor-beta1 (TGF-beta1) to elucidate the function of endocrine factors in AFB1 hepatocarcinogenesis. TAM and TGF-beta1 induced a typical apoptosis in K2 cells. The apoptotic cell death was efficiently suppressed by glucocorticoids (GCs), but not by other steroid compounds including 17beta-estradiol (E2). Cyclo-oxygenase (COX) inhibitors such as aspirin (ASP) and indomethacin (IND) also inhibited the apoptosis, while inhibitory effects of general lipoxygenase (LOX) inhibitors such as nordihydroguaiaretic acid (NDGA) and 5,8,11-eicosatrienoic acid (ETI) were not observed. TAM and TGF-beta1 enhanced the release of [3H]arachidonic acid (AA) from pre-labeled K2 cells, which was inhibited by dexamethasone (DEX). Furthermore, cytosolic phospholipase A2 (cPLA2) activity in K2 cells treated with TAM for 2 h was higher than that in the control. Prostaglandin J2 (PGJ2) and delta12-PGJ2, AA metabolites formed in the COX pathway, induced K2 cell death. These results suggest that AA metabolites are involved in apoptotic K2 cell death elicited by TAM and TGF-beta1, and GCs could act as a tumor promoter in AFB1 hepatocarcinogenesis through the prevention of apoptosis induced by AA metabolites formed in vivo.