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T Kumagai

Publications and source records attributed to T Kumagai.

At least 73 records · Page 4Linked to original sources

Crystal structures of the transposon Tn5-carried bleomycin resistance determinant uncomplexed and complexed with bleomycin.

The transposon Tn5 carries a gene designated ble that confers resistance to bleomycin (Bm). In this study, we determined the x-ray crystal structures of the ble gene product, designated BLMT, uncomplexed and complexed with Bm at 1.7 and 2.5 A resolution, respectively. The structure of BLMT is a dimer with two Bm-binding pockets composed of two large concavities and two long grooves. This crystal structure of BLMT complexed with Bm gives a precise mode for binding of the antibiotic to BLMT. The conformational change of BLMT generated by binding to Bm occurs at a beta-turn composed of the residues from Gln(97) to Thr(102). Crystallographic analysis of Bm bound to BLMT shows that two thiazolium rings of the bithiazole moiety are in the trans conformation. The axial ligand, which binds a metal ion, seems to be the primary amine in the beta-aminoalanine moiety. This report, which is the first with regard to the x-ray crystal structure of Bm, shows that the bithiazole moiety of Bm is far from the metal-binding domain. That is, Bm complexed with BLMT takes a more extended form than the drug complexed with DNA.

Acetyltransferases↗

Synthesis, SARs, and pharmacological characterization of 2-amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists.

(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest in the study of group II mGluRs. Our interest focused on a conformationally constrained form of compound 4, because it appeared that the rigid form resulted in not only selectivity for group II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on the electron distribution in the molecule) of this atom and carbon-fluorine bond energy. Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC(50) = 29.4 +/- 3.3 nM and 45.4 +/- 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC(50) = 18.3 +/- 1.6 nM and 62.8 +/- 12 nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-7: ED(50) = 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivity and PCP-induced head-weaving behavior, respectively; 4: ED(50) = >100 mg/kg and 3.0 mg/kg for PCP-induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a compound [(3)H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful ((+)-7: K(i) = 47.7 +/- 17 nM and 65.9 +/- 7.1 nM for mGluR2 and mGluR3, respectively; 4: K(i) = 23.4 +/- 7.1 nM and 53.5 +/- 13 nM for mGluR2 and mGluR3, respectively). On the basis of a successful result of compound 7, we focused on the introduction of a fluorine atom on the C6 position of compound 4. (1R,2S,5R, 6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of compound 4 or 7 ((-)-11: K(i) = 16.6 +/- 5.6 and 80.9 +/- 31 nM for mGluR2 and mGluR3, respectively). Our interest shifted to modification on CH(2) at C4 position of compound 11, since replacement of the CH(2) group with either an oxygen atom or sulfur atom yielded compound 5 or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH(2) at the C4 position of compound 11. The carbonyl group might slightly change the relative conformation of three functional groups, the amino group and two carboxylic acids, which have important roles in mediating the interaction between group II mGluRs and their ligand, compared with the CH(2) group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-Amino-6-fluoro-4-oxobicyclo[3.1. 0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028) exhibited a remarkably high degree of agonist activity for mGluR2 (K(i) = 0.570 +/- 0.10 nM) and mGluR3 (K(i) = 2.07 +/- 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed in CHO cells (K(i) = >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyclidine (PCP)-induced head-weaving behavior (ED(50) = 0.090 microg/kg) and hyperactivity (ED(50) = 0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the treatment of schizophrenia.

Administration, Oral↗

Identification of negative regulatory regions within the first exon and intron of the BCL6 gene.

Chromosomal translocations involving BCL6 gene are frequent in human B-cell lymphomas. Chromosomal breaks preferentially occur within a 3-kb region containing the first exon and intron. Recent reports have revealed that internal deletions or point mutations also are common in this region, suggesting that structural alteration of this region may be a crucial event in the development of lymphomas. In this study, we identified two regions in the BCL6 gene that negatively regulate BCL6 expression. One region, ES, is located within the first exon between nucleotides +472 and +543, and a second region, IS, is located between +783 and + 918 of the first intron. A consensus nucleotide sequence for the binding of the BCL6 protein itself was found within the ES region. An electrophoretic mobility shift assay and a co-transfection experiment using a BCL6 expression vector showed that transcription of the BCL6 gene was negatively regulated by the BCL6 gene product. The IS region which is included in the regions commonly deleted in B-cell lymphomas had a silencer activity. Structural alterations of these two regions may play roles in the deregulated expression of the BCL6 gene in B-cell lymphomas.

Cell Transformation, Neoplastic↗

Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells.

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), caspase-3-like protease activation (2-4 h), chromatin condensation and DNA ladder formation (3-4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1-3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.

Apoptosis↗

4-hydroxy-2-nonenal, the end product of lipid peroxidation, is a specific inducer of cyclooxygenase-2 gene expression.

Cyclooxygenase-2 (COX-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being upregulated by diverse stimuli. Based on the experimental evidence that oxidative stress is associated with the upregulation of COX-2, we evaluated the effect of the oxidized fatty acid metabolites on COX-2 induction in rat liver epithelial RL34 cells. Among the compounds tested, only 4-hydroxy-2-nonenal (HNE), a highly mutagenic and genotoxic aldehyde generated during oxidative stress, dramatically induced COX-2. Enhanced gene expression of COX-2 by treatment with HNE was evident as a drastic elevation of the mRNA level. We also found that intracellular glutathione status was strictly related to HNE-induced COX-2 expression. These findings suggest the presence of a signaling pathway in the cellular response mediated by locally produced lipid peroxidation products under oxidative stress.

Aldehydes↗

Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results suggest that 15-deoxy-Delta(12,14)-PGJ2 induces the expression of the rat GSTP1 gene through binding of proteins, including c-Jun, to a specific GPEI.

Animals↗

Gelatin-containing diphtheria-tetanus-pertussis (DTP) vaccine causes sensitization to gelatin in the recipients.

Gelatin-specific T cell response was performed to determine whether a series of vaccinations with gelatin-containing DTP is a primary sensitization process in gelatin allergy. Thirty-seven recipients with gelatin-containing DTP who developed adverse reactions after vaccination and eight recipients of DTP without gelatin who also developed adverse reactions were studied. In addition, 10 subjects receiving gelatin-containing vaccine and 10 subjects inoculated with non-gelatin vaccine who did not show any adverse reactions were also investigated. All subjects inoculated with gelatin-containing DTP vaccine showed positive T cell responses against gelatin, however, occurrence of adverse reactions did not correlate with T cell responses. We conclude that DTP vaccine containing gelatin induces sensitization to gelatin in the recipients, but the mechanism of local reactions remains unknown.

Child↗

The 1.5 A crystal structure of a bleomycin resistance determinant from bleomycin-producing Streptomyces verticillus.

Bleomycin (Bm)-binding protein, designated BLMA, which is a Bm resistance determinant from Bm-producing Streptomyces verticillus, was crystallized in a form suitable for X-ray diffraction analysis. The diffraction intensity data were collected up to a resolution of 1.5 A with a merging R-value of 0.054 at a completeness of 94 %. The BLMA structure, determined by the single isomorphous replacement method including the anomalous scattering effect (SIR-AS) at a resolution of 2.0 A, was refined at 1.5 A resolution. The final R-factor was 19.0 % and R(free) was 22.1 % including 91 water molecules. The crystal packing showed a dimer form, which was generated by arm exchange. The 1.5 A high-resolution experiment allowed an analysis of the side-chain disorder of BLMA. The structural comparison of BLMA with a homologous protein from Streptoalloteichus hindustanus, designated Shble protein, showed that a Ser100-Gly103 loop was farther from the groove, which is a Bm-binding site, in BLMA than in the Shble protein. Furthermore the hydrophobicity of the groove in BLMA is much lower than that in the Shble protein. The structural differences between these proteins may be responsible for the observation that a half-saturating concentration (K(1/2)) of Bm is higher for BLMA than for the Shble protein.

Acetyltransferases↗

Age-related changes in myelopoietic response to lipopolysaccharide in senescence-accelerated (SAM) mice.

The effects of in vivo lipopolysaccharide (LPS) administration on myelopoiesis were examined in senescence-accelerated (SAM) mice. Young mice injected with LPS exhibited: (a) increased femoral proliferative pool size; (b) transient reduction in femoral non-proliferative pool size and number of femoral colony forming unit-granulocyte macrophages (CFU-GMs); (c) marked increase in splenic CFU-GMs; and (d) transient increase in S-phase of femoral CFU-GMs. The responses of old mice after LPS administration differed from those of young mice in the following points: (a) no recovery of the femoral non-proliferative pool or femoral CFU-GMs, (b) less significant augmentation of the femoral proliferative pool and splenic CFU-GMs, and (c) prolonged reduction in S-phase of femoral CFU-GM. Injection of LPS into mice resulted in a hyperproduction of colony-stimulating activity (CSA) in bone followed by production of colony-inhibitory activity (CIA) in young mice and in contrast, an excessive CIA secretion from bone without an increase in CSA levels in old mice. These imbalances in the regulatory factors derived from non-hemopoietic cells in the bones may lead to an inappropriate response of myelopoiesis in aged SAM mice after LPS administration, which may play a key role in infections.

Aging↗

Strongyloides venezuelensis: heparin-binding adhesion substances in immunologically damaged adult worms.

Immunologically damaged Strongyloides venezuelensis adult worms were examined for their mucosal invasion ability and secretion of heparin-binding adhesion substances. S. venezuelensis was expelled from male Wistar rats 4 to 5 weeks after infection. Four-week-old adult worms were smaller and had fewer eggs than 1-week-old adult worms. One-week-old, 4-week-old, and 5-week-old adult worms equally established in the recipient mouse intestine when surgically implanted. Adult worms of 4 and 5 weeks of age secreted adhesion substances as much as 1-week-old adult worms. There was no difference in the heparin-binding activities and the lectin-binding profile of adhesion substances among adult worms of different ages. The rate of secretion of adhesion substances from the mouth was also identical. Heparin-binding activities were detected in crude adult worm proteins; however, proteins of 5-week-old adult worms had weaker heparin-binding activities than those of 1-week-old adult worms. Western blotting revealed that a number of heparin-binding proteins were lost in 5-week-old adult worms. A heparin-binding protein of 42. 0 kDa, which was consistently expressed in adult worms, was a possible component of heparin-binding adhesion substances which are secreted from the mouth.

Animals↗

Protective effect of riluzole on MPTP-induced depletion of dopamine and its metabolite content in mice.

The neuroprotective effects of riluzole (2-amino-6-trifluoromethoxy benzothiazole), a Na+ channel blocker with antiglutamatergic activity, MK-801, a blocker of N-methyl-D-aspartate (NMDA) receptors and monoamine oxidase (MAO) inhibitor pargyline were compared in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) levels in mice. The mice received four intraperitoneal injections of MPTP (10 mg/kg) at 1-hr intervals and then the brains were analyzed at 1, 3 and 7 days after the treatments. Dopamine and DOPAC levels were significantly decreased in the striatum from 1 day after MPTP treatments. A severe depletion in dopamine and DOPAC levels was found in the striatum 3 and 7 days after MPTP treatments. Riluzole dose-dependently antagonized the MPTP-induced decrease in dopamine and DOPAC levels in the striatum. Pargyline also protected against MPTP-induced decrease in dopamine levels in the striatum. However, this drug showed no significant change in the striatal DOPAC levels. On the other hand, MK-801 failed to protect against MPTP-induced decrease in dopamine levels in the striatum. However, MK-801 reversed the MPTP-induced decrease in DOPAC levels. These results suggest that riluzole can protect against MPTP-induced striatal dopamine and DOPAC depletion in mice. This protective effect may be caused by inactivation of voltage-dependent Na+ channels by riluzole. Furthermore, the present study suggests that the activation of NMDA receptors does not mainly contribute to MPTP-induced neurodegeneration, whereas MAO, especially MAO type B(MAO-B) plays a crucial role in MPTP-induced degeneration of the nigrostriatal dopaminergic neuronal pathway.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

Crystallization and preliminary X-ray diffraction studies of Streptomyces phospholipase A2 in a calcium-binding form.

Phospholipase A2 (PLA2) as a calcium-binding form, produced by Streptomyces violaceoruber, was crystallized in a form suitable for the diffraction analysis using the vapor diffusion method. Crystals were grown in 0.1 M Tris-HCl buffer (pH 8.5), 20 mM Ca2+ containing 50-60% (v/v) 2-methyl-2,4-pentanediol as a precipitant. They belong to the monoclinic space group P2(1), with the cell dimensions a=38.3 A, b=54.3 A, c=30.6 A, and beta=90.2 degrees. The crystals diffract the X-ray well and the diffraction intensity data were collected up to 1.6 A resolution. The crystal volume per unit mass, V(M) is 2.35 A3 Da(-1) with one molecule in the asymmetric unit, which corresponds to a solvent content of 47.7%.

Calcium↗

On chronological changes in the basic EEG rhythm in persons with Down syndrome - with special reference to slowing of alpha waves.

The authors tried to know specificity of aging in persons with Down syndrome (DS) from the aspect of electroencephalograph (EEG) frequency changes through the cross-sectional and longitudinal studies, in comparison with normal persons as well as those with mentally retardation except the Down syndrome (non-DS MR). Subjects for a cross-sectional study were 265 persons with DS, 242 with non-DS MR and 239 healthy persons, and subjects for a follow-up study were 28 persons with DS and 14 with non-DS MR, whose EEGs were recorded repeatedly once a year during 8 or 9 years. Resting EEGs from the frontal, central and occipital regions were examined through power spectrum. In the cross-sectional study, the number of subjects with DS who showed dominant component within 8 Hz band of the basic rhythm reached maximum in its appearance rate at 40-44 years of age in the occipital area, but this slowing progressed already at 30-34 years of age. While in non-DS MR, the number of subjects who showed dominant component at 8 Hz reached maximum at 45-49 years of age, and this slowing of the basic rhythm was not so clear as in DS. In the follow-up study for subjects with DS, although the lowering in EEG frequency to 8 Hz took place in various years of age individually, earlier distinct decrease of the frequency was commonly noticed. These earlier steep lowering of EEG frequency was discussed in relation to the senile signs and to the decline of brain function referring to Alzheimer disease.

Adolescent↗

Design, synthesis and structure-affinity relationships of 4-methylidenepiperidine and 4-aryl-1,2,3,6-tetrahydropyridine derivatives as corticotropin-releasing factor1 receptor antagonists.

Recently, various non-peptide corticotropin-releasing factor1 (CRF1) receptor antagonists have been reported. Structure-affinity relationships (SARs) of non-peptide CRF antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Our interest focused on the Up-Area in deriving the novel methylidenepiperidine derivatives 8-10 and 4-aryl-1,2,3,6-tetrahydropyridine derivatives 11-13 as non-peptide CRF1 receptor antagonists. Compounds 8a and 11a had moderate affinity for CRF1 receptor, but compounds 9, 10, 12 and 13 did not exhibit CRF1 receptor affinity. Modification of derivatives 11 afforded compounds 11i (CRA1001) and 11x (CRA1000), which had high affinity and selectivity for CRF1 receptors with potent anxiolytic-like and antidepressant-like properties in some experimental animal models. These findings suggest that the hydrophonic unit (Up-Area) may be useful for design of CRF1 antagonists. We report here the design, synthesis and SARs of the derivatives 8 and 11 and isosteres 9, 10, 12 and 13.

Animals↗

Liver weight of adult Japanese, especially recent weight values.

Cases of 445 adult Japanese autopsies of the Tokyo Metropolitan Medical Examiner's Office were used in this study. They were either negative for all hepatitis virus-related markers examined or had little or no histopathological hepatic changes. The maximum liver weight was observed in the fifth decade in both sexes, and after the fifth decade the liver weight decreased markedly with increasing age. The sexual difference in the liver weight was most predominant in the third to fifth decades, but the sexual difference was not marked in the older age groups. The highest liver weight to body weight ratio was observed in the fifth decade of both sexes, and a total decadal pattern of the ratio was similar to a parabola. An interesting finding was that the male liver weights in the third to fifth decades considerably increased in recent years, but the female liver weights in the third decade were almost the same despite the difference in investigation period. We suggest the data of this study may be a standard for Japanese people.

Journal Article↗

The lymphoproliferative response to enzymatically digested gelatin in subjects with gelatin hypersensitivity.

BACKGROUND: This study was designed to evaluate the immunogenic characteristics of enzymatically digested gelatin, 'FreAlagin', employing the lymphoproliferative response in subjects with gelatin hypersensitivity. Our purpose was to assess the response of primed lymphocytes to the newly developed FreAlagin and compare it to the response to conventional gelatin. METHODS: A gelatin-specific lymphocyte proliferation test (LPT) was performed in 110 children with adverse reactions to gelatin-containing vaccines, who showed positive gelatin-specific cell-mediated immunity and were thus diagnosed as having gelatin hypersensitivity. Gelatin-specific IgE was measured in all subjects. The antigenic activity of FreAlagin to lymphocytes was compared with that of conventional bovine gelatin. Positive and negative control specimens were obtained from the patients with anaphylaxis and from subjects inoculated with gelatin-free vaccine who showed no adverse reactions in order to establish the fluorometric ELISA system to determine IgE antibody to gelatin and LPT. RESULTS: The lymphocyte activity against FreAlagin was much less than that to Wako gelatin and more than half of the subjects who reacted positively to Wako gelatin had a negative LPT to FreAlagin. Although 47% of the subjects had positive LPTs to FreAlagin, all but two still had lower SIs to FreAlagin compared with Wako gelatin. CONCLUSION: We conclude that the antigenic activity of FreAlagin as measured by the cell-mediated immune response is significantly less than that of conventional bovine gelatin. However, it is still necessary to perform clinical trials to show a reduced or absent clinical reactivity to FreAlagin in sensitized patients to conventional gelatin.

Animals↗

Clinical usefulness of urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori: a collaborative study in nine medical institutions in Japan.

BACKGROUND: A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit. MATERIALS AND METHODS: A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests. RESULTS: Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA. CONCLUSIONS: The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.

Antibodies, Bacterial↗