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Biomedical subjects

K Nagata

Publications and source records attributed to K Nagata.

At least 217 records · Page 12Linked to original sources
PubMed1999

Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats.

Amino Acid Sequence↗
PubMed1999

Purification and characterization of two amine N-sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from liver cytosols of male rabbits.

Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS-PAGE. AST-RB1 efficiently catalyzed N-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3, 6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.

Amines↗
PubMed1999

Molecular cloning, chromosome mapping and characterization of the mouse CRTH2 gene, a putative member of the leukocyte chemoattractant receptor family.

We have cloned the mouse CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) gene encoding a putative leukocyte chemoattractant receptor, of which human homologue is expressed selectively in Th2 but not in Th1 clones among T cell clones. The deduced amino-acid sequence of mouse CRTH2 bears 77% identity with its human homologue. Phylogenetic analysis suggested that both mouse and human CRTH2 are closely related to the N-formyl peptide receptor and the C5a receptor among leukocyte chemoattractant receptors. The mouse CRTH2 gene was mapped on chromosome 19c with FISH, where no other genes for leukocyte chemoattractant receptors are mapped. RT-PCR analysis revealed that mouse CRTH2 mRNA is expressed in various cell lineages, including both hematopoietic and non-hematopoietic cell lines. Expression was also observed in liver, lung, kidney, brain, heart, thymus, and spleen. These results suggest that mouse CRTH2 functions in a variety of cells, making the effects of CRTH2 pleiotropic.

Amino Acid Sequence↗
PubMed1999

Hepatocyte nuclear factor 4-mediated activation of rat CYP3A1 gene and its modes of modulation by apolipoprotein AI regulatory protein I and v-ErbA-related protein 3.

CYP3A1 gene (P450/6betaB) encodes testosterone 6beta-hydroxylase (EC 1.14.14.1) in rats. The promoter region of CYP3A1 gene contains three binding sites for nuclear factors: 6betaB-A (-105 to -86), 6betaB-B (-139 to -118), and 6betaB-C (-164 to -145). The 6betaB-A site shows a high degree of similarity to a consensus sequence of the binding site of hepatocyte nuclear factor 4 (HNF-4) and also to the 6betaA-A site on the rat CYP3A2 gene promoter region. Our previous study suggested an involvement of the 6betaA-A site in the basal transactivation of CYP3A2 gene using HepG2 cells. In the present study, transactivation through the 6betaB-A and 6betaA-A sites of CYP3A1 and CYP3A2 genes has directly been shown by coexpression of HNF-4 and CYP3A1 or CYP3A2 promoter-reporter fused genes. Similar experiments further showed that nuclear factor binding at the 6betaB-B site hampered HNF-4-mediated transactivation of CYP3A1 gene. Recombinant apolipoprotein AI regulatory protein I (ARP-1) and v-ErbA-related protein 3 (EAR-3) are shown to suppress HNF-4-mediated activation at the 6betaB-B site without competition of HNF-4.

Animals↗
PubMed1999

Selective expression of a novel surface molecule by human Th2 cells in vivo.

The search for reliable marker molecules discriminating between human Th1 and Th2 cells identified a gene encoding a novel member of the G protein-coupled leukocyte chemoattractant receptor family, which is selectively expressed in Th2 but not Th1 lineage cells, thereby named CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). Studies with anti-CRTH2 mAbs demonstrated that CRTH2 was expressed in a small population (0.4-6.5%) of CD4+ T cells in fresh PBMCs of healthy adults, but no remarkable expression was seen in B cells and NK cells. In some cases, CD8+ T cells ( approximately 3.5%) expressed CRTH2. Phenotypes of CD4+ T cells expressing CRTH2 were CD45RA-, CD45RO+, and CD25+, similar to those of Ag-activated effector/memory T cells. Freshly isolated CRTH2+ CD4+ T cells produced Th2- but little or no Th1-type cytokines upon stimulation with PMA and ionomycin. In addition, an allergen-induced proliferative response in fresh PBMCs was significantly and selectively reduced by subtracting CRTH2+ cells. Together, these results indicate that CRTH2 is selectively expressed in vivo in an activated state of Th2 cells including allergen-responsive Th2 cells, suggesting its pivotal roles in ongoing Th2-type immune reactions.

Adult↗
PubMed1999

The fate of the compressed deformed spinal cord after decompressive surgery: MR imaging and histopathological findings in experimental studies.

The authors conducted a study in which they applied the spinal cord compression-decompression model in rabbits to investigate the morphological changes and histopathological findings in the deformed spinal cord over a long-term period after performing decompressive surgery. Throughout the experimental period, mangnetic resonance (MR) images were obtained frequently; after obtaining a final MR image, the spinal cord was dissected and underwent histological examination. Immediately after decompressive surgery, axial T1-wieighted MR imaging revealed an increase in the cross-sectional area of the spinal cord during the 1st and 2nd weeks. The spinal cord area achieved a peak at an average of 5.9 weeks after decompressive surgery, when it displayed isointensity on T1- and high-intensity on T2-weighted images. The main histological findings were spongy changes in the white matter, which persisted for 4 months postsurgery. There was a significant correlation between the presurgical spinal cord area and the postsurgical decreased number of motor neuron cells. Based on the MR imaging and histopathological studies, although the deformed spinal cord that underwent compression for 3 months was immediately restored morphologically after decompressive surgery, the change in quality in the spinal cord persisted at least 4 months.

Journal Article↗
PubMed1999

Antiviral activity of Sanicula europaea L. extracts on multiplication of human parainfluenza virus type 2.

The antiviral activity of Sanicula europaea L. extracts against human parainfluenza virus type 2 (HPIV-2) was examined. The extract prepared from the leaves of the plant and a fraction separated from the crude extract with gel filtration chromatography were found to inhibit HPIV-2 replication without any toxic effect on Vero cells. The acidic fraction obtained from the crude extract of S. europaea leaves was found to be the most active fraction with plaque inhibition assay at non-cytotoxic concentrations. Unfortunately, antiviral activity was not detected in the molecules purified from the crude ethanol extract of Sanicula leaves.

Animals↗
PubMed1999

Basilar artery occlusion due to spontaneous basilar artery dissection in a child.

Basilar artery occlusion (BAO) causing brainstem infarction occurred in a 7-year-old boy without any basic disorders. A diagnosis of BAO due to basilar artery dissection (BAD) was suspected at angiography, and this was confirmed by gadolinium-enhanced magnetic resonance imaging (MRI). These investigations clearly showed all the typical diagnostic signs such as a pseudolumen, double lumen and intimal flap, and a pseudolumen in resolution. The spontaneous healing of the dissection was clearly demonstrated during 10 months of follow-up. We stress that BAD can occur in young children and that combined diagnosis with gadolinium-enhanced MRI and angiography is conclusive for diagnosis of dissecting aneurysms. Wider use of these combined diagnostic methods will allow the detection of less severe basilar artery dissection, thus extending the spectrum of presentation and prognosis.

Aortic Dissection↗
PubMed1999

Persistent patent pseudolumen of ruptured dissecting aneurysm involving the posterior inferior cerebellar artery after proximal clipping.

In the majority of cases of ruptured vertebral artery dissecting aneurysm after proximal clipping, the dissected pseudolumen persists for a very short time, probably because re-entry from the pseudolumen is minimal. Recent reports have indicated a high risk of rebleeding of dissecting aneurysms involving the posterior inferior cerebellar artery (PICA) after proximal clipping, probably due to excessive retrograde flow from the distal vertebral artery into both the PICA and the pseudolumen. We describe an extremely rare case of ruptured dissecting aneurysm involving the PICA with persistent patent pseudolumen after proximal clipping. The present case was assumed to have developed a moderate retrograde flow just sufficient to maintain the patent pseudolumen in the chronic stage. Neointimal formation is suggested to be a possible mechanism by which the pseudolumen is stabilized for a very long period.

Aortic Dissection↗
PubMed1999

Bombyxin secretion in the adult silkmoth Bombyx mori: sex-specificity and its correlation with metabolism.

Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.

Journal Article↗
PubMed1999

Terminal deletion of the long arm of chromosome 9 in acute promyelocytic leukemia with a cryptic PML/RAR alpha rearrangement.

Deletions of the long arm of chromosome 9 (9q-) are rare aberrations specifically found in acute myeloid leukemia (AML). Here we describe the first case of acute promyelocytic leukemia (APL) with a terminal 9q deletion as a sole abnormality. Chromosome analysis of the bone marrow cells showed 46,XX,del(9)(q22) in all 20 metaphases. Fluorescence in situ hybridization (FISH) analysis with painting probes of chromosomes 15, 17, and 9 revealed only two normal chromosomes 15 and 17, normal chromosome 9, and del(9)(q22). FISH with cosmid DNA probes flanking the breakpoints of t(15;17) did not show the retinoic acid receptor alpha (RAR alpha)/PML fusion signal usually generated on the der(17) t(15;17). However, rearrangement of the RAR alpha gene and expression of the PML/RAR alpha chimeric transcript were identified by Southern blot and reverse transcriptase-polymerase chain reaction analyses, respectively. These results suggested that the PML/RAR alpha fusion gene was generated by submicroscopic interstitial insertion of the RAR alpha gene into the PML gene. Therefore, 9q- was interpreted as a secondary aberration following the PML/RAR alpha rearrangement. The patient died during induction therapy because of intracerebral hemorrhage. Considering other reported cases of APL with 9q-, 9q- may be an adverse prognostic factor in APL as observed in AML with t(8;21).

Adult↗
PubMed1999

Bicuculline-resistant, Cl- dependent GABA response in the rat spinal dorsal horn.

Receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian central nervous system (CNS), have been divided into three subtypes. GABA(A) receptor is a ligand-gated chloride channel that is competitively antagonized by bicuculline, whereas GABA(B) receptor regulate Ca2+ or K+ channels through G proteins. Recently, GABA(C) receptor has been identified in mammalian and fish retina. Unlike GABA(A) receptors, the GABA(C) receptor is a bicuculline-resistant chloride channel that is selectively activated by cis-4-aminocrotonic acid (CACA), and antagonized by imidazole-4-acetic acid (I4AA) and to some extent by picrotoxin. We report here that bicuculline-resistant GABA responses mediated by chloride channels are also expressed in substantia gelatinosa (SG) neurons in the dorsal horn, which receive predominantly nociceptive inputs from periphery. The GABA responses are, however, not mimicked by CACA nor affected by I4AA, but abolished by picrotoxin. Moreover, these responses are modulated by benzodiazepines (flunitrazepam) and barbiturates (thiopental), although GABA(C) responses are not affected. Thus, the pharmacological characteristics of the GABA responses observed in SG neurons are distinct from those responses mediated by the known GABA receptors. These differences may reflect the presence of receptor subunits unique to SG neurons.

Animals↗
PubMed1999

Neuronal nicotinic acetylcholine receptors: a new target site of ethanol.

Whereas a variety of neuroreceptors and ion channels have been demonstrated to be affected by ethanol including GABAA receptors, NMDA receptors, non-NMDA glutamate receptors, 5-HT3 receptors and voltage-gated calcium channels, neuronal nicotinic acetylcholine receptors (nnAChRs) have recently emerged as a new target site of ethanol. The nnAChRs are different from the muscle type nicotinic AChRs with respect to their molecular architecture and pharmacology. This article briefly reviews the structure, distribution and function of nnAChRs for which a considerable amount of information has been rapidly accumulated during the past 5-10 years. The potent and unique action of ethanol on nnAChRs has been unveiled only during the past few years. Most recent developments along this line of ethanol action are discussed in this paper.

Animals↗
PubMed1999

Binding affinity of GM3 lactone for influenza virus.

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.

Animals↗
PubMed1999

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide.

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

Amino Acid Sequence↗
PubMed1999

Anisatin modulation of the gamma-aminobutyric acid receptor-channel in rat dorsal root ganglion neurons.

1. Anisatin, a toxic, insecticidally active component of Sikimi plant, is known to act on the GABA system. In order to elucidate the mechanism of anisatin interaction with the GABA system, whole-cell and single-channel patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. 2. Repeated co-applications of GABA and anisatin suppressed GABA-induced whole-cell currents with an EC50 of 1.10 microM. No recovery of currents was observed after washout with anisatin-free solution. 3. However, pre-application of anisatin through the bath had no effect on GABA-induced currents. The decay phase of currents was accelerated by anisatin. These results indicate that anisatin suppression of GABA-induced currents requires opening of the channels and is use-dependent. 4. Anisatin suppression of GABA-induced currents was not voltage dependent. 5. Picrotoxinin attenuated anisatin suppression of GABA-induced currents. [3H]-EBOB binding to rat brain membranes was competitively inhibited by anisatin. These data indicated that anisatin bound to the picrotoxinin site. 6. At the single-channel level, anisatin did not alter the open time but prolonged the closed time. The burst duration was reduced and channel openings per burst were decreased indicating that anisatin decreased the probability of openings.

Affinity Labels↗
PubMed1999

HSF3 is a major heat shock responsive factor duringchicken embryonic development.

The expression of heat shock genes in vertebrates is regulated mainly at the level of transcription by four heat shock transcription factors (HSFs 1-4). Avian cells express at least three HSFs (HSFs 1-3). HSF1 is rapidly activated by even mild heat shock, while HSF3 is activated only by severe heat shock. In contrast, HSF2 is not activated by heat stress and has been speculated to have developmental functions. Here, we examined the temporal and spatial profiles of changes in the levels of these three HSFs in various tissues during chicken development. We found that HSF3 was almost constantly expressed in various tissues during early to late chicken development. The expression of HSF1 was equally high in most tissues early in development and thereafter declined to different levels in a tissue-dependent manner. Thus, HSF3 became the dominant heat-responsive factor in all tissues examined. The magnitude of heat shock response determined by Northern blotting did not always correlate with the level of HSF1 expression, suggesting that not only HSF1 but also HSF3 may be a major factor mediating stress signals to heat shock gene expression in the chicken. In addition, the high-level and ubiquitous expression of HSF2 as well as HSF1 and HSF3 in early embryogenesis suggested the involvement of these factors in all developmental processes.

Animals↗