Biomedical subjects

K Nagata

Publications and source records attributed to K Nagata.

At least 199 records · Page 11Linked to original sources

PubMed1999

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation.

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.

Amino Acid Sequence↗

PubMed1999

Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments.

To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.

3T3 Cells↗

PubMed1999

A new translocation, t(2;4;12)(p21;q12;p13), in CD7-positive acute myeloid leukemia: a variant form of t(4;12).

We describe a 41-year-old man with CD7-positive acute myeloid leukemia (AML-M0) with trilineage-myelodysplasia. Chromosome analysis of the bone marrow cells showed 46.XY.t(2;4;12) (p21;q12;p13). Cytological and clinical features of our case were quite similar to those of AML with t(4;12)(q11-12;p13). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) by using the whole-chromosome painting probes specific for chromosomes 2, 4, and 12. FISH analysis with the use of the YAC 936e2 probe, which covers the TEL gene, did not show the split signal, suggesting that a gene other than TEL was involved in the leukemogenesis of the present case. Our case with AML with t(2;4;12)(p21;q12;p13) appears to be the first case of a variant type of AML with t(4;12) (q11-12;p13).

Acute Disease↗

PubMed1999

CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s).

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T-helper- (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti-FcepsilonR1 antibody, cord blood-derived mast cells secreted factor(s) that induced Ca(2+) mobilization in CRTH2-expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell-mediated immune responses such as allergic reactions.

Basophils↗

PubMed1999

The N-terminal LIM domain negatively regulates the kinase activity of LIM-kinase 1.

LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5. 5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (DeltaLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activities in vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the DeltaLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1 in vivo.

Actins↗

PubMed1999

The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing.

The expression of heat shock genes is controlled at the level of transcription by members of the heat shock transcription factor family in vertebrates. HSF4 is a mammalian factor characterized by its lack of a suppression domain that modulates formation of DNA-binding homotrimer. Here, we have determined the exon structure of the human HSF4 gene and identified a major new isoform, HSF4b, derived by alternative RNA splicing events, in addition to a previously reported HSF4a isoform. In mouse tissues HSF4b mRNA was more abundant than HSF4a as examined by reverse transcription-polymerase chain reaction, and its protein was detected in the brain and lung. Although both mouse HSF4a and HSF4b form trimers in the absence of stress, these two isoforms exhibit different transcriptional activity; HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and hHSF4b acts as a transcriptional activator. Furthermore HSF4b but not HSF4a complements the viability defect of yeast cells lacking HSF. Moreover, heat shock and other stresses stimulate transcription of target genes by HSF4b in both yeast and mammalian cells. These results suggest that differential splicing of HSF4 mRNA gives rise to both an inhibitor and activator of tissue-specific heat shock gene expression.

Alternative Splicing↗

PubMed1999

Sulfotransferase catalyzing sulfation of heterocyclic amines.

Cytosolic sulfation of arylamines to form sulfamates is found to be mediated by sulfotransferases of three gene families (SULT1 to 3). Among them, a SULT3 form (ST3A1) showed a high selectivity for N-sulfation of N-substituted aryl and alicyclic compounds. SULT1 (phenol) and SULT2 (hydroxysteroid) sulfotransferases showed N-sulfating activities of carcinogenic heterocyclic amines. For N-hydroxyarylamine O-sulfation, SULT1 forms showed high activity. In rats, ST1C1 mediated the metabolic activation of N-hydroxyarylamines. However, the related form (ST1C2) in humans showed the negligible activity. Instead, ST1A3 showed high metabolic activating abilities among human sulfotransferases.

Amines↗

PubMed1999

Cloning and characterization of psu1(+), a new essential fission yeast gene involved in cell wall synthesis.

We have isolated a new gene, psu1(+), from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequences shows that this protein has striking homology to the SUN family of the budding yeast, hence designated Psu1 (S. pombe homologue of the SUN family). Disruption of the psu1(+) gene revealed that it is essential for growth, and the null phenotype showed the swelling of cells followed by eventual lysis. We introduced psu1(+) gene in the disruptant strain and repressed it giving resistance to 1, 3-beta-glucanase digestion. Our results suggest that Psu1 plays an essential role in cell wall synthesis in S. pombe.

Amino Acid Sequence↗

PubMed1999

Interaction of anisatin with rat brain gamma-aminobutyric acidA receptors: allosteric modulation by competitive antagonists.

Anisatin, a toxic sesquiterpene isolated from the Japanese star anise (Illicium anisatum L.), competitively inhibited the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB), a non-competitive antagonist of gamma-aminobutyric acid (GABA)A receptors, to rat brain membranes with an IC50 value of 0.43 microM. R 5135, a competitive GABA antagonist, decreased the potency of anisatin in inhibiting [3H]EBOB binding in a negatively cooperative manner. Two other competitive antagonists, SR 95531 (gabazine) and (-)-bicuculline methiodide, had similar effects. On the other hand, R 5135 exerted little influence on the potencies of the other non-competitive antagonists tested: EBOB, picrotoxinin, isopropylbicyclophosphate, and dieldrin. Thus, anisatin was clearly different from the other non-competitive antagonists in responding to the action of competitive antagonists on (GABA)A receptors. These findings suggest that the binding region of anisatin might overlap with that of the other non-competitive antagonists, but that anisatin must interact with other specific region(s).

Affinity Labels↗

PubMed1999

Coiled-coil structure-mediated dimerization of template activating factor-I is critical for its chromatin remodeling activity.

Template activating factor-I (TAF-I)alpha and TAF-Ibeta have been identified as the host factors that activate DNA replication of the adenovirus genome complexed with viral basic core proteins (Ad core). TAF-I causes a structural change of the Ad core, thereby stimulating not only replication but also transcription from the Ad core DNA in vitro. TAF-I also activates transcription from the reconstituted chromatin consisting of DNA fragments and purified histones through chromatin remodeling. Although the carboxyl-terminal region, which is highly rich in acidic amino acids, is essential for the TAF-I activity, it remains unclear how other parts are involved in its activity. The native TAF-I isolated from HeLa cells exists as either hetero- or homo-oligomer. Here, we have demonstrated by cross-linking assays that most of TAF-I exists as a dimer. Analyses using deletion mutant TAF-I proteins revealed that the amino-terminal region of TAF-I common to both alpha and beta is essential for dimerization. This region is predicted to form a coiled-coil structure. Indeed, mutations disrupting this putative structure abolished the dimerization capability and reduced the TAF-I activity in the Ad core DNA replication assay. Furthermore, we found that TAF-I mutants lacking the acidic tail act in a dominant-negative manner in this assay. These observations strongly suggest that the dimerization of TAF-I is important for its activity.

Adenoviridae↗

PubMed1999

Dynamic computed tomography predicts tumor temperature and response to thermoradiotherapy in superficial and subsurface tumors.

BACKGROUND: Dynamic computed tomography (CT) was performed on patients undergoing thermoradiotherapy for superficial or subsurface tumors, and the correlation between tumor enhancement and tumor temperature during hyperthermia was evaluated. The authors further investigated whether tumor enhancement by dynamic CT is predictive of tumor response to thermoradiotherapy. METHODS: Thermoradiotherapy was given to 26 patients. Radiotherapy consisted of 40-70 gray. Hyperthermia was conducted over 3-5 sessions, and tumor temperature was measured at each session. Dynamic CT was performed prehyperthermia (within 1 week before the initiation of hyperthermia) and midtherapy (within 1 week after 2 sessions of hyperthermia). RESULTS: A complete response (CR) was obtained in 11 patients (42%) and either a partial response or no response (non-CR) in 15 (58%). There was no correlation between tumor enhancement obtained by prehyperthermia CT and tumor temperature parameters or response. However, the deltaCTmax (maximum increased enhancement) by prehyperthermia and midtherapy CT was 39.0 +/- 18.9 HU and 26.1 +/- 14.2 HU, respectively, in CR patients, and 46.4 +/- 21.1 HU and 49.6 +/- 19.1 HU, respectively, in non-CR patients. This change in deltaCTmax at midtherapy was significantly different between groups (P < 0.01). The deltaCTmax ratio for prehyperthermia and midtherapy CT studies correlated with the average tumor temperature (P < 0.05). CONCLUSIONS: Tumor enhancement by prehyperthermia and midtherapy dynamic CT predicted tumor temperature during hyperthermia and response to thermoradiotherapy for superficial or subsurface malignancies.

Adult↗

PubMed1999

Expression of mucin 1 (MUC1) in esophageal squamous-cell carcinoma: its relationship with prognosis.

Using 2 anti-mucin 1 (MUC1) monoclonal antibodies (MAbs), DF3 and BCP8, we examined MUC1 expression immunohistochemically in 192 esophageal squamous-cell carcinomas (SCCs). In normal squamous epithelium of the esophagus, DF3 was not expressed, but BCP8 was expressed on the cell membrane, mainly in the surface layer. In esophageal SCCs, DF3 and BCP8 were expressed mainly on the cell membrane of SCC cells, but also in the cytoplasm in several cases. To analyze the correlation of MUC1 expression and the prognosis of the patients, the 192 cases were divided into 2 groups: high-expression group (HEG, > 50% of the neoplastic cells stained) and low-expression group (LEG, < 50% of neoplastic cells stained). DF3-HEG (24 patients) showed a significantly poorer survival rate than DF3-LEG (168 patients), whereas there was no significant difference in survival between BCP8-HEG (43 patients) and BCP8-LEG (149 patients). Also, in the analysis of 162 patients with advanced stage (submucosal or deeper invasion) to exclude the influence of low expression of DF3 and BCP8 in 30 patients with early stage (up to the level of muscularis mucosae), DF3-HEG (24 patients) showed significantly poorer survival than DF3-LEG (138 patients), whereas there was no significant difference in survival between BCP8-HEG (42 patients) and BCP8-LEG (120 patients). The results of our study on esophageal SCC suggest that the expression of sialyl oligosaccharides detected by DF3 is related to poor prognosis.

Carcinoma, Squamous Cell↗

PubMed1999

Functional domains of template-activating factor-I as a protein phosphatase 2A inhibitor.

Template-Activating Factor-I (TAF-I) alpha and beta, chromatin remodeling factors, were identified as the stimulatory factor for replication of the adenovirus DNA complexed with viral basic core proteins. Recently, two cellular inhibitors for protein phosphatase 2A (PP2A) have been isolated. One of these inhibitors, designated IPP2A2, is a truncated version of TAF-Ibeta. Here, it is shown using recombinant TAF-I proteins that both TAF-Ialpha and beta have the PP2A inhibitor activity. The N-terminal region but not the C-terminal acidic region, the latter of which is essential for the chromatin remodeling activity, is shown to be required for the PP2A inhibitor activity. Roles of TAF-Ialpha- and beta-specific regions, the C-terminal acidic region, and other regions of TAF-I for the PP2A inhibitor activity are also discussed.

Animals↗

PubMed1999

Impaired force-frequency relations in patients with hypertensive left ventricular hypertrophy. A possible physiological marker of the transition from physiological to pathological hypertrophy.

BACKGROUND: The extent to which force-frequency and relaxation-frequency relations (FFR and RFR, respectively) and exercise-induced adrenergic stimulation affect myocardial inotropic and lusitropic reserves has not been established in patients with left ventricular (LV) hypertrophy (LVH). METHODS AND RESULTS: We calculated the maximum first derivative of LV pressure (LV dP/dtmax) and the LV pressure half-time (T1/2) during pacing, exercise, and isoproterenol infusion in 17 patients with hypertensive LVH and 9 control subjects to investigate the influence of increases in heart rate (HR) and adrenergic stimulation on inotropic and lusitropic reserves. Group A consisted of 10 LVH patients who showed a progressive increase in the HR-LV dP/dtmax relation. Group B consisted of 7 LVH patients in whom the HR-dP/dtmax relation at physiological pacing rates was biphasic. The LV mass index was larger and the LV ejection fraction was smaller in group B than in group A (244+/-72 g/m2 versus 172+/-22 g/m2 and 55+/-18% versus 72+/-6%, respectively; both P<0.05). The increase in LV dP/dtmax was greater during exercise than pacing alone for similar increases in HR in all groups (P<0.05) (group A, 111+/-22% versus 25+/-14%; group B, 105+/-35% versus 14+/-10%; control, 111+/-24% versus 25+/-12%). T1/2 was shorter (P<0.05) during exercise than with pacing alone in all groups (group A, 41+/-6% versus 11+/-3%; group B, 38+/-9% versus 14+/-4%; control, 44+/-6% versus 12+/-5%). Isoproterenol infusion caused similar increases in LV dP/dtmax and similar decreases in T1/2 in all groups. CONCLUSIONS: The FFR was biphasic in patients with severe LVH irrespective of LV function but was preserved in patients with less severe LVH and control subjects. Importantly, the RFR and adrenergic control of both inotropic and lusitropic reserves were well preserved in all LVH patients. A biphasic FFR at physiological pacing rates may be one of the earliest markers of the transition from physiological adaptation to the pathological process in LVH patients.

Adrenergic beta-Agonists↗

PubMed1999

Experimental external fixation combined with percutaneous discectomy in the management of scoliosis.

STUDY DESIGN: An assessment of the value of external fixation with or without percutaneous discectomy for the management of scoliosis in young rabbits with induced progressive thoracic scoliosis. OBJECTIVES: To investigate in an experimental setting the effect of external fixation with or without percutaneous discectomy for the management of scoliosis, as a preliminary study to precede clinical consideration. SUMMARY OF BACKGROUND DATA: External fixation of the spine using percutaneous transpedicular screws has been used clinically for cases of traumatic spinal injury, infectious spine, or chronic low back pain caused by a disc lesion. Percutaneous discectomy for the management of scoliosis has been reported. METHODS: Thirty-two young rabbits underwent partial resection of the right lower ribs. Nine rabbits were not treated after production of scoliosis and were followed as controls. At 4-6 weeks after production of scoliosis, in 23 animals, Kirschner wires were inserted percutaneously into the T9-T10 and L1-L2 disc space, and both ends were attached to an external fixator after correction of the scoliosis. In 8 of those 23 animals, percutaneous discectomy was also performed at the apex of the caudal compensatory curvature. RESULTS: In these 23 animals, the initial correction by fixation on of the caudal vertebrae was accompanied by a derotation in the apex. Five animals treated with external fixation only and four treated with combined percutaneous discectomy survived with external fixation until the age of 17 weeks and were followed to the natural cessation of the curve progression, at which the fixation was removed and a final assessment was made. The mean progression of curvature was 15.8 degrees in the group of five animals with external fixation only, and 33.8 degrees in the controls. In the group of four animals treated with supplementary percutaneous discectomy, however, the treated disc space became rigid, and the mean progression of curvature after removal of the fixation was only 5.3 degrees. CONCLUSIONS: The results of the current study suggest the potential for external fixation to allow for derotation and, when combined with percutaneous discectomy, to offer a feasible method of managing scoliosis in the human adolescent. This study was a preliminary experimental study; further experimental studies are planned to develop this novel technique.

Animals↗

PubMed1999

Reorganization of the primary afferent termination in the rat spinal dorsal horn during post-natal development.

To study the reorganization of the primary afferent input in the spinal dorsal horn during post-natal development, synaptic responses evoked by large Abeta and fine Adelta afferents were recorded from substantia gelatinosa (SG) neurons in slices obtained from immature (post-natal days 21-23) and mature rats (post-natal days 56-60). Threshold stimulus intensities and conduction velocities (CVs) of Abeta and Adelta afferents were determined by intracellular recordings of the antidromic action potentials from dorsal root ganglion (DRG) neurons isolated from immature and mature rats. In immature rats, excitatory postsynaptic currents (EPSCs) were elicited by stimulation sufficient to activate Abeta afferents in the majority of SG neurons (64.9%, 24 of 37 neurons), while most EPSCs observed in mature rats were elicited by stimulation of Adelta afferents (62.5%, 25 of 40 neurons). These observations suggest that the primary afferents innervating SG neurons were reorganized following maturation; Abeta afferents were the predominant inputs to the SG neurons in the immature state, thereafter Adelta afferents were substituted for the Abeta afferents to convey sensory information to the SG neurons. This relatively slow reorganization of the sensory circuitry may correlate with slow maturation of the SG neurons and with a delay in the functional connections of C afferents to the SG neurons.

Animals↗

PubMed1999

Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart.

BACKGROUND: The myocardial protective role of heat shock protein (HSP) has been demonstrated. Recently, we reported that ischemia/reperfusion induced a significant activation of heat shock factor (HSF) 1 and an accumulation of mRNA for HSP70 and HSP90. We examined the role of reactive oxygen species (ROSs) in the induction of stress response in the ischemic-reperfused heart. METHODS AND RESULTS: Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element. Induction of mRNA for HSP70 and HSP90 was examined by Northern blot analysis. Repetitive ischemia/reperfusion, which causes recurrent bursts of free radical generation, resulted in burst activation of HSF1, and this burst activation was significantly reduced with either allopurinol 1 mmol/L (an inhibitor of xanthine oxidase) or catalase 2x10(5) U/L (a scavenger of H2O2). Significant activation of HSF1 was observed on perfusion with buffer containing H2O2 150 micromol/L or xanthine 1 mmol/L plus xanthine oxidase 5 U/L. The accumulation of mRNA for HSP70 or HSP90 after repetitive ischemia/reperfusion was reduced with either allopurinol or catalase. CONCLUSIONS: Our findings demonstrate that ROSs play an important role in the activation of HSF1 and the accumulation of mRNA for HSP70 and HSP90 in the ischemic-reperfused heart.

Allopurinol↗

PubMed1999

The human genome has only one functional hsp47 gene (CBP2) and a pseudogene (pshsp47).

Among all the species investigated to date, only in humans is hsp47 reported to exist as two separate genes. Here we examined whether hsp47 forms a gene family, and if so, how many genes constitute the family. Cloning and sequencing of human hsp47 cDNA revealed that only one gene, identical to CBP2, was transcribed. No transcript corresponding to colligin, which was reported to be a human homologue of hsp47, was found. Genomic southern hybridization using the exon III fragment of mouse hsp47 as a probe, however, showed two bands for several restriction enzyme digests. We cloned and sequenced the gene corresponding to the extra band and found that a pseudogene (pshsp47) existed in the human genome. We have mapped this pseudogene to chromosome 9p12-p13 by fluorescent in situ hybridization (FISH) using a 3.5kb genomic fragment containing the entire pshsp47 sequence as a probe. These results suggested that functional hsp47 exists as CBP2, not as colligin, and a highly conserved pseudogene is present in the human genome.

Base Sequence↗