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T Nishino

Publications and source records attributed to T Nishino.

At least 37 records · Page 2Linked to original sources
PubMed2001

Dissection of the regional roles of the archaeal Holliday junction resolvase Hjc by structural and mutational analyses.

Hjc is an archaeal DNA endonuclease, which resolves the Holliday junction in the presence of divalent metals. Combined with mutational analyses, the x-ray structure of the Pyrococcus furiosus Hjc crystal grown in the presence of ammonium sulfate revealed a positively charged interface, rich in conserved basic residues, and the catalytic center (Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K. (2001) Structure 9, 197-T204). This structural study also suggested that the N-terminal segment and some loops of Hjc play crucial roles in the cleavage of DNA. However, a structural view of the interaction between these regions and DNA remains elusive. To clarify the regional roles of Hjc in the recognition of the Holliday junction, further structural and biochemical analyses were carried out. A new crystal form of Hjc was obtained from a polyethylene glycol solution in the absence of ammonium sulfate, and its structure has been determined at 2.16-A resolution. A comparison of the two crystal structures has revealed that the N-terminal segment undergoes a serious conformational change. The site-directed mutagenesis of the sulfate-binding site within the segment caused a dramatic decrease in the junction binding, but the mutant was still capable of cleaving DNA with a 20-fold lower efficiency. The kinetic analysis of Hjc-Holliday junction interaction indicated that mutations in the N-terminal segment greatly increased the dissociation rate constants of the Hjc-Holliday junction complex, explaining the decreased stability of the complex. This segment is also responsible for the disruption of base pairs near the junction center, through specific interactions with them. Taken together, these results imply that, in addition to the secondary effects of two basic loops, the flexible N-terminal segment plays predominant roles in the recognition of DNA conformation near the crossover and in correct positioning of the cleavage site to the catalytic center of the Hjc resolvase.

Amino Acid Sequence↗
PubMed2001

Vertical density matrix algorithm: a higher-dimensional numerical renormalization scheme based on the tensor product state ansatz.

We present a new algorithm to calculate the thermodynamic quantities of three-dimensional (3D) classical statistical systems, based on the ideas of the tensor product state and the density matrix renormalization group. We represent the maximum-eigenvalue eigenstate of the transfer matrix as the product of local tensors that are iteratively optimized by the use of the "vertical density matrix" formed by cutting the system along the transfer direction. This algorithm, which we call vertical density matrix algorithm (VDMA), is successfully applied to the 3D Ising model. Using the Suzuki-Trotter transformation, we can also apply the VDMA to 2D quantum systems, which we demonstrate for the 2D transverse field Ising model.

Journal Article↗
PubMed2001

Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration.

Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4-monohydroxy or 3,4-dihydroxy B-ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3-oxygenation. However, it exhibited virtually no PPO activity toward non-chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO-catalyzed reactions and conclude that the enzyme is a chalcone-specific PPO specialized for aurone biosynthesis.

Benzofurans↗
PubMed2001

Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II.

Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO.

Aged↗
PubMed2001

Crystal structure of the archaeal holliday junction resolvase Hjc and implications for DNA recognition.

BACKGROUND: Homologous recombination is a crucial mechanism in determining genetic diversity and repairing damaged chromosomes. Holliday junction is the universal DNA intermediate whose interaction with proteins is one of the major events in the recombinational process. Hjc is an archaeal endonuclease, which specifically resolves the junction DNA to produce two separate recombinant DNA duplexes. The atomic structure of Hjc should clarify the mechanisms of the specific recognition with Holliday junction and the catalytic reaction. RESULTS: The crystal structure of Hjc from the hyperthermophilic archaeon Pyrococcus furiosus has been determined at 2.0 A resolution. The active Hjc molecule forms a homodimer, where an extensive hydrophobic interface tightly assembles two subunits of a single compact domain. The folding of the Hjc subunit is clearly different from any other Holliday junction resolvases thus far known. Instead, it resembles those of type II restriction endonucleases, including the configurations of the active site residues, which constitute the canonical catalytic motifs. The dimeric Hjc molecule displays an extensive basic surface on one side, which contains many conserved amino acids, including those in the active site. CONCLUSIONS: The architectural similarity of Hjc to restriction endonucleases allowed us to construct a putative model of the complex with Holliday junction. This model accounts for how Hjc recognizes and resolves the junction DNA in a specific manner. Mutational and biochemical analyses highlight the importance of some loops and the amino terminal region in interaction with DNA.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8.

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.

Adenosine Triphosphatases↗
PubMed2001

Enzymatic formation of aurones in the extracts of yellow snapdragon flowers.

The yellow coloration of snapdragon (Antirrhinum majus) flowers is mainly provided by the 6-glucosides of aureusidin and bracteatin. However, the biochemical mechanism of aurone biosynthesis is not well understood. In this study, we have identified aurone-biosynthesizing activity in the extracts of yellow snapdragon flowers. Incubation of 2',4',6',4-tetrahydroxychalcone (THC) with an enzyme preparation in the presence of H(2)O(2) caused the enzymatic formation of a single product, aureusidin, without the formation of a previously proposed 2-(alpha-hydroxybenzyl)coumaranone intermediate. The formation of aureusidin from THC was specifically observed with yellow flowers as well as aurone-accumulating flowers of other colors. The pH optimum for the enzymatic formation of aureusidin was around 5.4. Stoichiometric studies showed that one mole of aureusidin formation was accompanied by the consumption of one mole of oxygen with no detectable consumption of H(2)O(2), which may work as an enzyme activator. The oxidative formation of aureusidin from THC could be explained in terms of the action of a single enzyme, an internal monooxygenase catalyzing the 3-hydroxylation and oxidative cyclization of THC. Incubation of 2',4',6',3,4-pentahydroxychalcone (PHC) with an enzyme yielded both aureusidin and bracteatin at an approximate molar ratio of 6:1. In this case, H(2)O(2) was not required for enzyme activity but rather inhibited the reaction. The 4'-glucosides of THC and PHC could also act as substrates for the formation of the 6-glucosides of aurones. These results suggest that aureusidin can be produced from either THC or PHC, whereas bracteatin is not produced through the 5'-hydroxylation of aureusidin but arise solely from PHC.

Journal Article↗
PubMed2001

Validating cortical surface analysis of medial prefrontal cortex.

This paper describes cortical analysis of 19 high resolution MRI subvolumes of medial prefrontal cortex (MPFC), a region that has been implicated in major depressive disorder. An automated Bayesian segmentation is used to delineate the MRI subvolumes into cerebrospinal fluid (CSF), gray matter (GM), white matter (WM), and partial volumes of either CSF/GM or GM/WM. The intensity value at which there is equal probability of GM and GM/WM partial volume is used to reconstruct MPFC cortical surfaces based on a 3-D isocontouring algorithm. The segmented data and the generated surfaces are validated by comparison with hand segmented data and semiautomated contours, respectively. The L(1) distances between Bayesian and hand segmented data are 0.05-0.10 (n = 5). Fifty percent of the voxels of the reconstructed surface lie within 0.12-0.28 mm (n = 14) from the semiautomated contours. Cortical thickness metrics are generated in the form of frequency of occurrence histograms for GM and WM labelled voxels as a function of their position from the cortical surface. An algorithm to compute the surface area of the GM/WM interface of the MPFC subvolume is described. These methods represent a novel approach to morphometric chacterization of regional cortex features which may be important in the study of psychiatric disorders such as major depression.

Adult↗
PubMed2001

Quantitative determination of gelatinase activity among enterococci.

Gelatinase, hemolysin and aggregation substance have all been reported to be virulence factors of enterococci. In this study, gelatinase production was investigated in isolates of Enterococcus faecalis (n=93), E. faecium (n=49) and E. avium (n=36) recovered from hospitalized patients. Gelatinase was detected in 45% of E. faecalis isolates, but could not be detected in E. faecium and E. avium. Gelatinase activity was then measured by radial diffusion for the 42 gelatinase-positive E. faecalis isolates. To convert gelatinase activity into proteinase K activity, a standard curve was produced by placing different concentrations of proteinase K into wells in the gelatine plate. Gelatinase activity per E. faecalis colony ranged from 2.6 x 10(-7) to 2.2 x 10(-5) microg/ml, proportionate to the activity of proteinase K. An approximately 84-fold difference in gelatinase concentration was observed between the colony producing the highest amount and that producing the lowest amount. This method may be useful for determining the virulence of given isolates in relation to gelatinase production as it is quick, easy and inexpensive to perform.

Adult↗
PubMed2001

Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides.

The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.

Citrulline↗
PubMed2001

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis.

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR METHOD: Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.

Adrenomedullin↗
PubMed2001

Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.

BACKGROUND: Anaphylatoxin C5a mediates inflammatory responses through interaction with a specific C5a receptor (C5aR), the expression of which is thought to be restricted to peripheral blood leukocytes. Although the presence of C5aR on cultured mesangial cells and tubular epithelial cells has recently been documented, the tissue distribution of C5aR in diseased kidney has not yet been determined. METHODS: Immunohistochemistry and nonradioactive in situ hybridization for C5aR were performed in 34 tissue samples of kidneys from patients with various renal diseases, including 4 with minimal change nephrotic syndrome (MCNS), 5 with membranous nephropathy (MN), and 25 with mesangial proliferative glomerulonephritis (mesGN; 15 patients with IgA nephropathy, 5 with non-IgA mesGN, and 5 with lupus nephritis). Normal portions of surgically resected kidney served as the control. RESULTS: In normal kidneys, C5aR protein was detected in tubular epithelial cells, while C5aR mRNA was detected in a few glomerular cells, tubular epithelial cells, and vascular endothelial and smooth muscle cells. In MCNS, the distribution of C5aR protein and mRNA was similar to that in normal kidneys. In MN and mesGN, C5aR protein and mRNA were detected in mesangial cells, glomerular epithelial and endothelial cells, Bowman's capsule cells, tubular cells, infiltrating cells, and vascular endothelial and smooth muscle cells. The glomerular expression of C5aR mRNA and protein correlated positively with the degree of mesangial hypercellularity and mesangial matrix expansion in mesGN. In the tubulointerstitium, interstitial expression of C5aR mRNA correlated positively with the degree of tubular atrophy and interstitial broadening in mesGN. Furthermore, the interstitial expression of C5aR mRNA correlated positively with the level of serum creatinine. CONCLUSIONS: Our results indicate that renal cells produce C5aR and that activation of C5a/C5aR pathway on renal cells may be involved in tissue injury in mesGN.

Adolescent↗
PubMed2001

Effects of minor surgery and endotracheal intubation on postoperative breathing patterns in patients anaesthetized with isoflurane or sevoflurane.

We studied the effects of minor surgery and endotracheal intubation on postoperative breathing patterns. We measured breathing patterns and laryngeal resistance during the periods immediately before intubation (preoperative) and immediately after extubation following minor surgery (postoperative) in eight patients anaesthetized with sevoflurane and eight patients anaesthetized with isoflurane, breathing spontaneously through a laryngeal mask airway at a constant end-tidal anaesthetic concentration (1.0 MAC). In both sevoflurane-anaesthetized and isoflurane-anaesthetized patients, expiratory time was reduced and inspiratory and expiratory laryngeal resistance increased after surgery. In sevoflurane-anaesthetized patients, occlusion pressure (P0.1) increased without changes in inspiratory time (T(I)). Occlusion pressure did not change and T(I) was greater in isoflurane-anaesthetized patients after surgery. Minor surgery may have a small but significant influence on breathing and increased laryngeal resistance following endotracheal intubation may modulate these changes. The difference in breathing pattern between sevoflurane and isoflurane may be a result of different responses of the central nervous system to different anaesthetics in the presence of increased laryngeal resistance.

Adult↗
PubMed2001

Immunocytochemistry of glutathione S-transferase in taste bud cells of rat circumvallate and foliate papillae.

Immunocytochemistry was used to investigate the distribution of cells reacting with specific antibodies against glutathione S-transferase (GST) mu and pi in rat circumvallate and foliate taste buds; the findings were confirmed by Western blotting. Double immunofluorescence staining for protein gene product (PGP) 9.5 and GST subunits allowed the classification of taste bud cells of both papillae into: (i) cells immunoreactive to either PGP 9.5 or GST subunit antibody; (ii) cells immunoreactive to both antibodies; and (iii) cells that did not react with either of these antibodies. Immunoelectron microscopy revealed that most GST subunit-immunoreactive cells seemed to be either type II or type III cells based on their ultrastructure. Since PGP 9.5 is now widely used as a marker for type III cells in mammalian taste buds, it seems reasonable to believe that most GST subunit-immunoreactive cells are type II cells. Whether cells immunoreactive for both PGP 9.5 and GST subunits constitute a small subpopulation of type III cells or whether they are intermediate forms between type II and III cells is under investigation. No type I cells reacted with antibodies against GST subunits in the present study. GST subunits in taste bud cells may participate in xenobiotic metabolism of certain substances exposed to taste pits, as already shown for olfactory epithelium.

Animals↗
PubMed2001

X-ray absorption spectroscopic analysis of the high-spin ferriheme site in substrate-bound neuronal nitric-oxide synthase.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving N(omega)-hydroxy-L-arginine, an enzyme-bound intermediate. The N(omega)-hydroxy-L-arginine- and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance signals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the N(omega)-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the N(omega)-hydroxy-L-arginine- and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 A. Cu(2+) inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the N(omega)-hydroxy-L-arginine-bound form and only slightly affected by the divalent cation inhibitor of constitutive NOS.

Animals↗
PubMed2001

The effect of epidural anesthesia on respiratory distress induced by airway occlusion in isoflurane-anesthetized cats.

UNLABELLED: The role of afferent information from the chest wall in the genesis of dyspnea is not fully elucidated. We have developed an animal model for the study of airway occlusion (AO) and proposed new concepts of minimum alveolar anesthetic concentration for AO (MACAOR) and the duration from the start of AO to the onset of the positive motor response (DOCCL) to evaluate respiratory distress quantitatively. We examined the effects of thoracic epidural anesthesia on respiratory distress by using our animal model. Adult cats (n = 24) were anesthetized with isoflurane, and an epidural catheter was placed after T9 laminectomy. After determination of MACAOR, DOCCL was measured. Animals were then randomly assigned into three groups: the EPD Group (n = 12) received epidural 1% lidocaine (0.4 mL/kg), IM saline (0.4 mL/kg), and saline infusion. The IM Group (n = 6) received epidural saline (0.4 mL/kg), IM 1% lidocaine (1 mL/kg), and saline infusion. The PHE Group (n = 6) received epidural 1% lidocaine (0.4 mL/kg) and IV phenylephrine (0.5-1 microg. kg(-1). min(-1)) to maintain a stable arterial blood pressure. DOCCL and MACAOR were measured in each animal at 15 min after the administration of drugs. Plasma lidocaine concentrations were measured before and after epidural or IM injection. DOCCL was significantly longer after epidural injection in all groups than before the injection. Although there was no significant difference in the values of MACAOR between before and after the epidural injection in the EPD Group, the IM administration of lidocaine in the IM Group significantly reduced MACAOR. Plasma concentrations of lidocaine were similar in all groups at all measurement points. Our data indicate that thoracic epidural anesthesia using 1% lidocaine significantly reduced respiratory distress induced by AO. This effect is most likely caused by a systemic effect of lidocaine rather than by reduced afferent information from the chest wall. IMPLICATIONS: Thoracic epidural anesthesia reduced respiratory distress induced by airway occlusion. This effect is most likely caused by the systemic effect of lidocaine, rather than by the reduced afferent information from the chest wall.

Anesthesia, Epidural↗