Biomedical subjects

H Kato

Publications and source records attributed to H Kato.

At least 451 records · Page 25Linked to original sources

PubMed1999

Extracellular adenosine 5'-triphosphate plus activation of glutamatergic receptors induces long-term potentiation in CA1 neurons of guinea pig hippocampal slices.

The mechanism of adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) was studied pharmacologically using guinea pig hippocampal slices. Application of 10 microM ATP for 10 min transiently depressed, then slowly augmented, synaptic transmission in CA1 neurons, leading to LTP. This ATP-induced LTP was blocked by addition of an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist. Furthermore, co-application of 10 microM ATP and 100 microM L-glutamate for 10 min also induced LTP, and this effect was blocked by the use of Ca2+-free solution during drug application. These results suggest that, in CA1 neurons, a co-operative effect involving extracellular ATP and the activation of NMDA receptors, which increases intracellular Ca2+ levels, is required to trigger the intracellular biological process involved in ATP-induced LTP.

2-Amino-5-phosphonovalerate↗

PubMed1999

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

Animals↗

PubMed1999

Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Two-dimensional photon counting imaging and spatiotemporal characterization of ultraweak photon emission from a rat's brain in vivo.

The process of metabolic reactions within living cells leads to spontaneous ultraweak light emission. The development of a system for highly sensitive imaging and spatiotemporal analysis of ultraweak photon emission from a rat's brain is reported in this paper. The equipment used in this experiment consists of a two-dimensional photon-counting tube with a photocathode measuring 40 mm in diameter, a highly efficient lens system, and an electronic device to record time series of a photoelectron train with spatial information. The sensitivity and ability to extract spatiotemporal information from sequential data of a single photoelectron train were examined. The minimum detectable radiant flux density of the system was experimentally estimated to be 9.9 x 10(-17) W/cm2 with a 1-s observation time. Spontaneous photon emission was demonstrated from an exposed rat's cortex in vivo without adding any chemical agent or employing external excitation. An image of ultraweak photon emission was compared with one obtained after cardiac arrest. The intensity after cardiac arrest was depressed to approximately 60% of before that. The regional properties of time courses of emission intensity were also demonstrated, indicating the potential usefulness for spatiotemporal characterization of photon emission with mapping of physiological information such as oxidative stress. This technology constitutes a novel method, with the potential to extract pathophysiological information from the central nervous system.

Animals↗

PubMed1999

Long-term efficacy of immunization against hepatitis B virus in infants at high-risk analyzed by polymerase chain reaction.

Perinatal transmission of hepatitis B virus (HBV) is a common cause of chronic infection. In the present study, we evaluated the long-term efficacy of immunization against HBV in infants at high-risk, by using polymerase chain reaction (PCR). Two hundred and fifty-one infants received hepatitis B immunoglobulin at birth and a course of hepatitis B vaccine within 6 months of age between 1981 and 1993. Of 251 infants, 203 (81%) and 97 (39%) were followed until 1 and 4-6 years of age, respectively. HBV-DNA was detected by PCR in 74 (36%) of 203 children at 1 year of age, while the prevalence rate of children positive for HBV-DNA decreased to 14 (14%) of 97 children at 4-6 years of age, including 2 children who had the breakthrough variants of HBV. Our results indicate that most of HBV infections occur early, during the first year, and that immunization against HBV effectively protects infants at high-risk against viral transmission, at least up to 4-6 years of age.

Adult↗

PubMed1999

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection.

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 microM and the calibration curve was linear at least up to 5 microM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 microM and from 3.5 to 6.7 at 5 microM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 microM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.

Animals↗

PubMed1999

Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase.

Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH) isoform I is a member of a family of complex enzymes composed of mutually homologous alpha(1) and alpha(2) subunits, both of which account for catalytic activity, and the beta subunit. We previously demonstrated that the expression of one catalytic subunit, alpha(1), is developmentally regulated, resulting in a switching of the catalytic complex from alpha(1)/alpha(2) to alpha(2)/alpha(2) during brain development (Manya, H., Aoki, J., Watanabe, M., Adachi, T., Asou, H., Inoue, Y., Arai, H., and Inoue, K. (1998) J. Biol. Chem. 273, 18567-18572). In this study, we explored the biochemical differences in three possible catalytic dimers, alpha(1)/alpha(1), alpha(1)/alpha(2), and alpha(2)/alpha(2). The alpha(2)/alpha(2) homodimer exhibited different substrate specificity from the alpha(1)/alpha(1) homodimer and the alpha(1)/alpha(2) heterodimer, both of which showed similar substrate specificity. The alpha(2)/alpha(2) homodimer hydrolyzed PAF and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylethanolamine (AAGPE) most efficiently among 1-O-alkyl-2-acetyl-phospholipids. In contrast, both alpha(1)/alpha(1) and alpha(1)/alpha(2) hydrolyzed 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid more efficiently than PAF. AAGPE was the poorest substrate for these enzymes. The beta subunit bound to all three catalytic dimers but modulated the enzyme activity in a catalytic dimer composition-dependent manner. The beta subunit strongly accelerated the enzyme activity of the alpha(2)/alpha(2) homodimer but rather suppressed the activity of the alpha(1)/alpha(1) homodimer and had little effect on that of the alpha(1)/alpha(2) heterodimer. The (His(149) to Arg) mutant beta, which has been recently identified in isolated lissencephaly sequence patients, lost the ability to either associate with the catalytic complexes or modulate their enzyme activity. The enzyme activity of PAF-AH isoform I may be regulated in multiple ways by switching the composition of the catalytic subunit and by manipulating the beta subunit.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1999

The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association.

Mutant-type p53 (mt p53) is largely accumulated in cancer cells due to its increased stability. To elucidate the mechanism of mt p53 stabilization, we analysed the turnover of p53 mutated at codon 248 whose alteration is most frequently found in human cancers. Proteasome inhibition induced the accumulation of ubiquitinated mt p53, indicating that the ubiquitinated forms were essentially unstable and degraded by the proteasome. The presence of a small amount of the ubiquitinated mt p53 relative to the abundant non-ubiquitinated form suggested that the mt p53 ubiquitination was a rate-limiting process in the slow turnover. Two phenomena destabilizing mt p53 via the ubiquitin-proteasome degradation were proved to be independent. First, the coexpression of wild-type p53 (wt p53) promoted mt p53 destabilization as feedback regulation. Second, geldanamycin also induced mt p53 destabilization through the dissociation of the protein from hsp90 but not through the restoration of wt p53 function. Neither the mutant-specific conformation nor the N-terminal phosphorylation seemed to contribute directly to the mt p53 stabilization. Further, a two-dimensional gel electrophoresis revealed that most of the post-translationally modified mt p53 was equally subjected to ubiquitination and subsequent proteasomal degradation. These findings are evidence that mt p53 stabilization depends on the impaired ubiquitination due to both the loss of wt p53 function and the hsp90 association.

Amino Acid Sequence↗

PubMed1999

BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain.

Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library. One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2. PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody. PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c. Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha. In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.

Animals↗

PubMed1999

NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2.

Nuclear factor kappaB (NF-kappaB) is a transcription factor that controls the expression of many cellular and viral genes. The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis. Here we report that 53BP2, a protein previously identified by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system. This specific interaction was confirmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo. We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus. Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells. Neither the N-terminal nor the C-terminal of 53BP2 fused to GFP induced cell death. Interestingly, co-transfection with a p65 expression plasmid significantly inhibited 53BP2-induced cell death. The previous findings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth.

Animals↗

PubMed1999

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.

Amino Acid Sequence↗

PubMed1999

Imaging of thermal activation of actomyosin motors.

We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with rise and fall times of several ms, and locally in a region of approximately 10 micrometers in diameter with a temperature gradient up to 2 degrees C/micrometers. Temperature distribution was imaged pixel by pixel by image processing of the fluorescence intensity of rhodamine phalloidin attached to (single) actin filaments. With short pulses, actomyosin motors could be activated above physiological temperatures (higher than 60 degrees C at the peak) before thermally induced protein damage began to occur. When a sliding actin filament was heated to 40-45 degrees C, the sliding velocity reached 30 micrometers/s at 25 mM KCl and 50 micrometers/s at 50 mM KCl, the highest velocities reported for skeletal myosin in usual in vitro assay systems. Both the sliding velocity and force increased by an order of magnitude when heated from 18 degrees C to 40-45 degrees C. Temperature-pulse microscopy is expected to be useful for studies of biomolecules and cells requiring temporal and/or spatial thermal modulation.

Actomyosin↗

PubMed1999

Distinct tolerance pathways in sensitized allograft recipients after selective blockade of activation signal 1 or signal 2.

BACKGROUND: CD4-targeted therapy or blocking of CD28-B7 T-cell costimulation may produce indefinite cardiac allograft survival in presensitized rats. This study analyzes the immune events associated with tolerance pathways after the blockade of activation signal 1 (CD4 monoclonal antibody [mAb]) or signal 2 (CTLA4Ig). METHODS AND RESULTS: Lewis rats sensitized with Brown Norway skin grafts reject LBNF1 cardiac allografts in <36 hr. Animals were treated with RIB-5/2, a nondepleting CD4 mAb, or with CTLA4Ig + LBNF1 spleen cells. RIB-5/2 monotherapy uniformly produced permanent cardiac graft acceptance, whereas CTLA4Ig produced indefinite graft survival in about 50% of sensitized rats. Spleen cells (100 x 10(6)) from CD4 mAb-treated rats conferred donor-specific tolerance after transfer into new sets of recipients. This tolerant state could be then transferred with regulatory cells in an infectious manner into new cohorts of engrafted rats. In contrast, features of infectious tolerance could be detected in CTLA4Ig-treated hosts after infusion of >300 x 10(6) of splenocytes. CD4 mAb therapy abolished the transcription of both T helper (Th)1 and Th2 cytokines compared with rejecting controls. In contrast, CTLA4Ig treatment resulted in a selective sparing of Th2-type cytokines. Surviving grafts in both groups were largely protected from signs of chronic rejection. CONCLUSIONS: CD4 mAb-induced blockage of activation signal 1 or CTLA4Ig-mediated blockage of costimulatory signal 2 may induce a true transplantation tolerance in sensitized rats, as documented by permanent graft acceptance and attenuation of chronic injury. The infectious pathway operates in a cell dose-dependent manner. Th2-type deviation in the graft itself is not required for tolerance maintenance, and it does not necessarily lead to chronic injury.

Abatacept↗

PubMed1999

Arcadlin is a neural activity-regulated cadherin involved in long term potentiation.

Neural activity results in long term changes that underlie synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity. Here, we identify a novel cadherin molecule Arcadlin (activity-regulated cadherin-like protein). arcadlin mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by N-methyl-D-aspartate-dependent synaptic activity in long term potentiation. The extracellular domain of Arcadlin is most homologous to protocadherin-8; however, the cytoplasmic region is distinct from that of any cadherin family member. Arcadlin protein is expressed at the synapses and shows a homophilic binding activity in a Ca2+-dependent manner. Furthermore, application of Arcadlin antibody reduces excitatory postsynaptic potential amplitude and blocks long term potentiation in hippocampal slices. Its close homology with cadherins, its rapid inducibility by neural activity, and its involvement in synaptic transmission suggest that Arcadlin may play an important role in activity-induced synaptic reorganization underlying long term memory.

Amino Acid Sequence↗

PubMed1999

Progression-linked overexpression of c-Met in prostatic intraepithelial neoplasia and latent as well as clinical prostate cancers.

The c-met proto-oncogene encoding the receptor for the hepatocyte growth factor is expressed in several cancers. In the present study, c-met protein (c-Met) was detected in eight of 22 (36%) cases of prostatic intraepithelial neoplasia (PIN), five of 15 (33%) latent and 17 of 21 (81%) clinical prostate cancers, including seven metastatic lesions, using an immunohistochemical method. All seven (100%) metastatic lesions investigated demonstrated strong staining, and a correlation between c-Met expression and histology was observed. These results suggest a significant relationship between c-Met expression and progression of prostate neoplasms, including latent cancers.

Adenocarcinoma↗

PubMed1999

Structure of tropinone reductase-II complexed with NADP+ and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis.

Tropinone reductase-II (TR-II) catalyzes the NADPH-dependent reduction of the carbonyl group of tropinone to a beta-hydroxyl group. The crystal structure of TR-II complexed with NADP+ and pseudotropine (psi-tropine) has been determined at 1.9 A resolution. A seven-residue peptide near the active site, disordered in the unliganded structure, is fixed in the ternary complex by participation of the cofactor and substrate binding. The psi-tropine molecule is bound in an orientation which satisfies the product configuration and the stereochemical arrangement toward the cofactor. The substrate binding site displays a complementarity to the bound substrate (psi-tropine) in its correct orientation. In addition, electrostatic interactions between the substrate and Glu156 seem to specify the binding position and orientation of the substrate. A comparison between the active sites in TR-II and TR-I shows that they provide different van der Waals surfaces and electrostatic features. These differences likely contribute to the correct binding mode of the substrates, which are in opposite orientations in TR-II and TR-I, and to different reaction stereospecificities. The active site structure in the TR-II ternary complex also suggests that the arrangement of the substrate, cofactor, and catalytic residues is stereoelectronically favorable for the reaction.

Alcohol Oxidoreductases↗

PubMed1999

Elimination of Kupffer cells and nafamostat mesilate rinse prevent reperfusion injury in liver grafts from agonal non-heart-beating donors.

BACKGROUND: We hypothesized that microcirculatory disturbance was an obstacle to liver transplantation (LTx) from non-heart-beating donors (NHBDs) and that it was attributed mainly to a deterioration of sinusoidal endothelial cells (SECs) and sinusoidal narrowing. This study was designed to examine porcine orthotopic LTx using livers obtained from pretreated agonal NHBDs, and to determine whether the maintenance of the liver microcirculation would result in successful LTx from agonal NHBDs. METHODS: Pigs were allocated to five groups: (i) control group; (ii) NM group, in which grafts were rinsed with nafamostat mesilate (NM) rinse; (iii) LD group, in which Kupffer cells in grafts were eliminated by liposome-encapsulated dichloromethylene diphosphonate (L-DMDP); (iv) LDNM group, in which grafts pretreated with L-DMDP were rinsed with NM rinse; (v) heart-beating donor (HBD) group. In all groups, but the HBD group, the livers were pretreated with FK506 and prostaglandin I2 analogue, and were preserved in University of Wisconsin solution after cardiac arrest. Thereafter orthotopic LTx was performed. RESULTS: After reperfusion, it was histologically demonstrated that elimination of Kupffer cells prevented SECs deterioration and NM rinse prevented sinusoidal narrowing. The hepatic energy charge recovered in all groups except the control group. In the LDNM group, three of four recipients survived more than 7 days. CONCLUSIONS: For a successful LTx from agonal NHBDs, it is important to prevent microcirculatory disturbance caused by SEC deterioration and sinusoidal narrowing after reperfusion. Combination therapy consisting in the elimination of Kupffer cells and NM rinse prevented primary graft non-function in liver grafts from agonal NHBDs.

Adenosine Triphosphate↗

PubMed1999

Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains.

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains. These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.

Adult↗