Biomedical subjects

T Imamura

Publications and source records attributed to T Imamura.

At least 163 records · Page 9Linked to original sources

PubMed1999

Molecular forms of circulating adrenomedullin in patients with congestive heart failure.

In the biosynthesis of adrenomedullin (AM), an intermediate form, AM(1-52)-glycine-COOH (iAM), is cleaved from proAM and subsequently processed to a biologically active mature form, AM(1-52)-NH2 (mAM), by enzymatic amidation. We recently reported that immunoreactive AM in human plasma consists of mAM and iAM. To clarify the pathophysiological roles of mAM and iAM in heart failure, we established an assay method to specifically detect mAM, and we determined the plasma concentrations of mAM and iAM in 68 patients with congestive heart failure (CHF). The plasma mAM concentrations of the CHF patients classified as being class I or II of New York Heart Association (NYHA) functional classification were significantly greater than those of the 28 healthy controls, and a further increase was noted in the class III or IV patients. Similar increases in plasma iAM were also observed in these patients compared with controls. The increased plasma mAM and iAM in 12 patients with exacerbated CHF were significantly reduced by treatment of their CHF for 7 days. In addition, the plasma concentrations of both mAM and iAM were significantly correlated with pulmonary capillary wedge pressure, pulmonary artery pressure, right atrial pressure, cardiothoracic ratio, heart rate, and the plasma concentrations of atrial and brain natriuretic peptides in the CHF patients. Thus the plasma concentrations of both mAM and iAM were increased progressively in proportion to the severity of CHF. These results suggest that, though the role of iAM remains to be clarified, mAM acts against the further deterioration of heart failure in patients with CHF.

Adrenomedullin↗

PubMed1999

Introduction of an N-glycosylation cassette into proteins at random sites: expression of neoglycosylated FGF.

We developed a method for introducing an N-glycosylation cassette into proteins at random sites by constructing cDNAs and expressing it in mammalian cells. The protocol entails four steps: (i) generation of cDNAs that contain single, randomly-located blunt end cuts; (ii) ligation of N-glycosylation cassettes into the blunt end cuts in three-frame formats; (iii) selection of the cDNA clones encoding N-glycosylated proteins; and (iv) subcloning into an expression vector for transfection and expression in mammalian cells. This method was evaluated using secreted fibroblast growth factor (FGF) as a model protein. Several secreted FGF cDNA clones, each containing an AsnLeuSer-coding sequence at a random site, were obtained. When these clones were expressed in mammalian cells, some of the secreted FGFs were found to be N-glycosylated. The method described here should also be applicable for random introduction of functional oligopeptide/polypeptide cassettes into virtually any protein of interest.

Amino Acid Sequence↗

PubMed1999

The C-terminal region of fibroblast growth factor-1 is crucial for its biological activity and high level protein expression in mammalian cells.

We have studied the role of the carboxyl(C)-terminus of fibroblast growth factor(FGF)-1 using prokaryotic and eukaryotic expression systems. The full-length FGF-1 protein and its mutants lacking 6- and 9-amino acids at the C-terminus IFGF-1 (Cdel6) and FGF-1 (Cdel9), respectively] could be expressed in E. coli cells at the similar levels. The deletion mutants bound very weakly to FGF receptor and to heparin, and did not stimulate DNA synthesis in BALB/c3T3 cells. In contrast to E. coli cells, in NIH3T3 transfectants and L6 transfectants, the protein expression level of FGF-1 (Cdel6) was significantly lower than that of FGF-1, and longer C-terminal deletions further decreased the protein expression levels. However, the level of transcripts in the transfectants and the level of translates in in vitro system were equivalent for all the FGF-1 constructs. Treatment with proteasome inhibitors of the NIH3T3 transfectants expressing FGF-1(Cdel6) increased the protein level six-fold. The results indicate that the C-terminus of FGF-I is crucial for its biological activity and high-level expression in mammalian cells and suggest that deletion of the C-terminus of FGF-1 induces its post-translational degradation by proteasome system.

3T3 Cells↗

PubMed1999

Characterization of fibroblast growth factor-6 expressed by Chinese hamster ovary cells as a glycosylated mitogen for human vascular endothelial cells.

The gene for fibroblast growth factor (FGF)-6/hst-2 was originally identified by its close homology with the FGF-4/hst-1 gene. Aside from its ability to transform cultured fibroblasts, the characteristics of FGF-6 protein have only been studied using a simple preparation from E. coli. In the present study, we expressed FGF-6 cDNA in CHO cells and characterized the resultant protein. We found that CHO cells secreted several forms of the FGF-6 polypeptide, and that there were multiple N-terminal modifications. The longest form (18-kDa) contained the sequence, SerProAlaGlyAlaArg, as its N-terminus, which was consistent with the signal peptide cleavage site predicted from its primary structure. The core polypeptide was primarily modified by heterogeneous N-glycans that were sialylated to a small degree; among them, biantennary structures were found to predominate. Moreover, possible O-glycosylation was also detected. N-glycosylated FGF-6 potently induced DNA synthesis and proliferation of human vascular endothelial cells, whereas in the absence of N-glycosylation, FGF-6 mitogenicity was substantially diminished. The results clearly indicate that FGF-6 expressed by mammalian cells is a glycosylated mitogen for vascular endothelial cells and further suggests that N-glycosylation plays a key role in determining the mitogenicity of FGF-6.

Animals↗

PubMed1999

Distinct regulation of myoblast differentiation by intracellular and extracellular fibroblast growth factor-1.

We studied the role of fibroblast growth factor (FGF)-1 in the physiology of myoblast differentiation. We found that, while endogenous FGF-1 in L6-10 rat myoblasts did not suppress the progress of differentiation, the addition of FGF-1 to the culture medium suppressed it. Moreover, L6-10 cells stably transfected with full length FGF-1 undergo enhanced differentiation. The latter was well correlated with myogenin expression and myotube formation. Constitutive expression of a mutant FGF-1 (FGF-1U) that lacked a nuclear localization signal, promoted the differentiation of the myoblasts even more strongly. Furthermore, the expression of FGF-1U in an inducible expression system enhanced myogenin expression promptly. In L6-10 transfectants expressing a dominant-negative mutant of FGF receptor, stable transfection of FGF-1 promoted differentiation as it did in parent cells. Studies with FGF receptors and MAP kinase suggest that both are involved in the effect of FGF-1 when it is supplemented to culture medium but not during the effect of endogenous FGF-1 synthesized in cells. We conclude that intracellular (endogenous) and extracellular (exogenous) FGF-1 have differential effects on the regulation of myogenic differentiation of L6-10 cells.

Amino Acid Sequence↗

PubMed1999

[Selective peripheral denervation for spasmodic torticollis involving the levator scapulae muscle].

Patients with laterocollis or rotatory type torticollis tend to show abnormal contraction of the levator scapulae muscle and the scalene muscles. These muscles are innervated from the anterior branches of the cervical spinal nerves. Because of this, the traditional Bertrand operation dealing with posterior branches does not adequately affect the symptoms of laterocollis. The authors report selective denervation of the levator scapulae muscle in three patients and discuss its rationale. All the three patients underwent denervation of both the C1-C6 posterior spinal rami and the branches from the C3 and C4 anterior rami to the levator scapulae muscle. We added myotomy of the scalene muscle in one patient, and denervation of the omohyoid muscle which is innervated from the ansa cervicalis and the descending branch of the hypoglossal nerve. The pre/post-operative Tsui scores were 12/4, 15/1, and 14/3 respectively. There were no complications. We conclude that selective peripheral denervation of the levator scapulae muscle is safe and effective in the treatment of laterocollic type torticollis.

Adult↗

PubMed1999

[Comparison of the therapeutic effects and acid suppression of H2-receptor antagonist and proton pump inhibitor in patients with gastric body ulcer--a prospective controlled trial].

We studied, the rate and the affecting factor of ulcer healing of patients with gastric body ulcer treated by H2-receptor antagonist (H2-RA) and proton pump inhibitor (PPI). Gastric acidity was also examined using 24 hr pH monitoring. No difference was observed between the affecting factor of ulcer healing and healing rate (94.7% and 94.9%) among the patients treated by H2-RA or PPI. The average time below pH3 during treatment with H2-RA or PPI were 17.4 +/- 4.3 hr and 23.0 +/- 1.5 hr, respectively. Acid suppression was superior in PPI treated group than in H2-RA group. From these findings, we concluded that H2-RA had sufficient therapeutic efficacy in treating gastric body ulcer.

Anti-Ulcer Agents↗

PubMed1999

Structural and functional analysis of the control region of the human DNA topoisomerase II alpha gene in drug-resistant cells.

We have previously shown that the DNA topoisomerase II alpha (topo II alpha) gene is down-regulated in VP16/VM26-resistant cells at the transcriptional level. To determine the DNA elements responsible for down-regulation, the transcriptional activities of luciferase reporter constructs containing various lengths of the promoter sequences were investigated by transient transfection of two resistant cell lines, KB/VP2 and KB/VM4. The transcriptional activities of the full-length promoter (-295 to +85) and of three deletion constructs (-197, -154 and -74 to +85) were significantly down-regulated in resistant cells. In contrast, the transcriptional activity of the minimal promoter (-20 to +85) in resistant cells was similar to that in parental KB cells. Furthermore, introduction of a mutation in ICE1 abolished the down-regulation of the topo II alpha promoter activity in drug-resistant cells. In vivo footprinting analysis of topo II alpha gene promoter revealed several specific protein-binding sites, a GC box, ICE1, ICE2 and ICE3. In vivo footprinting analysis also identified a cluster of hypersensitive sites. However, there was no marked difference in protein-binding sites between parental and resistant cells. To confirm our previous results, we have established the VP16-resistant cell lines T12-VP1 and T12-VP2 from T12 cells derived from human bladder cancer T24 cells stably transfected with the chloramphenicol acetyltransferase reporter gene driven by the topo II alpha gene promoter. The expression to topo II alpha was down-regulated in both cell lines. We also found that CAT gene expression was significantly decreased to one-fifth of that in T12 parental cells. These results suggest that the expression of the topo II alpha gene requires the binding of multiple factors to the core promoter and is down-regulated at the transcriptional level, probably through binding of a negative factor to ICE1 in drug-resistant cells.

Antigens, Neoplasm↗

PubMed1999

[Benign clear cell tumor of the lung diagnosed by transbronchial biopsy].

A 26-year-old woman had an abnormal shadow on chest X-ray films during a general medical examination. A chest roentgenogram showed a nodular shadow 8 mm in diameter in the middle field of the right lung. Transbronchial biopsy specimens revealed that the coin lesion was a benign clear cell tumor. Benign clear cell tumors of the lung are rare; only 21 cases, including the present case, have been reported in Japan. Although the diagnosis in most of the cases reported required an open thoracotomy, for our patient the diagnosis was based solely on the findings of a transbronchial biopsy.

Adult↗

PubMed1999

Disappearance of complete atrioventricular block after chemotherapy for malignant lymphoma: a case report.

A 77-year-old man with malignant lymphoma presented with dizziness and exertional dyspnea. Physical examination revealed marked bradycardia (36 beats/min). Twelve-lead electrocardiography showed complete atrioventricular block with narrow QRS escape beats. Gallium scintigraphy demonstrated significant abnormal uptake in the heart. Transesophageal echocardiography showed a thick interatrial septum with increased echogenecity. He underwent chemotherapy under external temporary pacing with a suspected diagnosis of complete atrioventricular block secondary to cardiac invasion of malignant lymphoma. Atrioventricular conduction progressively improved and the complete atrioventricular block disappeared. He is currently well and has required no cardiac pacing for 6 months. We conclude that complete atrioventricular block may be reversible in some patients with malignant lymphoma, even in the elderly.

Aged↗

PubMed1998

A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens.

Coprocessing of kininogens by a mixture of human mast cell tryptase and neutrophil elastase was explored as a potential substitute for the kallikrein-dependent pathway for kinin generation during inflammation. Tryptase easily excised bradykinin from the synthetic heptadecapeptide, ISLMKRPPGFSPFRSSR, but was unable to produce significant amounts of kinin by proteolysis of kininogens. However, a mixture of tryptase and elastase released bradykinin from each protein with a yield comparable to that of human plasma kallikrein. Significantly, neither plasma nor tissue kallikrein was able to effectively process N-chlorosuccinimide-oxidized high molecular weight kininogen, an effect attributed to the oxidation of a methionine residue upstream from the N terminus of the kinin domain. In support of these results the model heptadecapetide, ISL(MO)KRPPGFSPFRSSR, was also resistant to hydrolysis by either kallikrein. In contrast, the release of bradykinin from oxidized peptide or protein substrates by the tryptase/elastase mixture was not altered. Because kininogen modification may occur at inflammatory sites, as a result of the oxidative burst of recruited neutrophils and macrophages, these results suggest an alternative pathway for kinin production and the necessity for the novel utilization of two specific proteinases known to be released from these cells during inflammatory episodes.

Amino Acid Sequence↗

PubMed1998

An alternatively spliced fibroblast growth factor (FGF)-5 mRNA is abundant in brain and translates into a partial agonist/antagonist for FGF-5 neurotrophic activity.

We detected in the brain and then cloned two novel, short forms of human and mouse fibroblast growth factor (FGF)-5 mRNA, which were designated human FGF-5S (hFGF-5S) and mouse FGF-5S (mFGF-5S), respectively. Genomic analysis indicated that mFGF-5S and authentic mFGF-5 mRNAs were transcribed from a single gene; hFGF-5S and mFGF-5S mRNAs were generated by excluding the second exon of the respective FGF-5 genes, and the alternatively spliced mRNAs encoded for 123- and 121-amino acid proteins, respectively. Indeed, a neuron-like cell line expressing mFGF-5S mRNA secreted a protein of the expected size and with FGF-5 antigenicity. In PC12 cells, expression of hFGF-5 or exposure to hFGF-5 protein induced differentiation. Neither expression of hFGF-5S, alone, nor co-expression of hFGF-5S with hFGF-5 induced significant differentiation. At high concentrations, hFGF-5S protein partially antagonized FGF-5 activity, whereas by itself, hFGF-5S exerted very weak neurotrophic activity. hFGF-5S protein binds to FGF receptor (FGFR)-1 on PC12 transfectants and partially inhibits hFGF-5-induced tyrosine phosphorylation of FGFR-1 and an FGFR substrate, but it also induces phosphorylation by itself. These results suggest that FGF-5S is a naturally expressed partial agonist/antagonist of FGF-5 neurotrophic activity in the brain and that its effects are exerted in part at the level of the receptor.

Alternative Splicing↗

PubMed1998

Strong suppression of feeding by a peptide containing both the nuclear localization sequence of fibroblast growth factor-1 and a cell membrane-permeable sequence.

Earlier studies have shown that fibroblast growth factor (FGF)-1 in the brain regulates feeding behavior. In the present study, food intake in rats was strongly suppressed by an infusion into the lateral cerebroventricle of a synthetic peptide (26 amino acids) which contains both the N-terminal nuclear localization sequence (NLS) of FGF-1 and a recently identified membrane-permeable sequence. When the NLS motif in the peptide was destroyed by mutations of two lysine residues, the mutant peptide failed to affect eating. The results suggest that the NLS of FGF-1 plays an important role in FGF-1-induced feeding suppression and they introduce a novel compound for feeding regulation.

Amino Acid Sequence↗

PubMed1998

Opposing actions of prostaglandins and oxytocin determine the onset of murine labor.

Prostaglandins (PGs) have been recently proven essential for parturition in mice. To dissect the contributions of the two cyclooxygenase (COX) isoforms to the synthesis of PGs during pregnancy, we have characterized the parturition phenotype of COX-1-deficient mice. We find that mice with targeted disruption of the COX-1 gene have delayed parturition resulting in neonatal death. Results of matings of COX-1-deficient females with COX-1 intact males, and blastocyst transfer of COX-1-deficient or -intact embryos into wild-type foster mothers, proved necessity and sufficiency of maternal COX-1 for the normal onset of labor. COX-1 expression is induced in gravid murine uterus and by in situ hybridization; this induction is localized to the decidua. Measurement of uterine PGs further confirmed that COX-1 accounted for the majority of PGF2alpha production. To evaluate the interaction of PGs with oxytocin during murine labor, we generated mice deficient in both oxytocin and COX-1. Surprisingly, the combined oxytocin and COX-1-deficient mice initiated labor at the normal time. COX-1-deficient mice demonstrated impaired luteolysis, as evidenced by elevated serum progesterone concentration and ovarian histology late in gestation, and delayed induction of uterine oxytocin receptors. In contrast, simultaneous oxytocin and COX-1 deficiency restored the normal onset of labor by allowing luteolysis in the absence of elevated PGF2alpha production. These findings demonstrate that COX-1 is essential for normal labor in the mouse, with a critical function being to overcome the luteotrophic action of oxytocin in late gestation.

Animals↗

PubMed1998

Permeable FGF-1 nuclear localization signal peptide stimulates DNA synthesis in various cell types but is cell-density sensitive and unable to support cell proliferation.

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G1 cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G1 cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.

3T3 Cells↗

PubMed1998

Sensitive determination of methomyl in blood using gas chromatography-mass spectrometry as its oxime tert.-butyldimethylsilyl derivative.

A sensitive, selective and reliable method was developed to determine methomyl ¿methyl-N-[(methylcarbamoyl)oxy]-thioacetimidate¿, a carbamate insecticide in human blood, using gas chromatography-mass spectrometry. Dimethylglyoxime served as an internal standard (I.S.). Methomyl in the blood was converted to its oxime form by sodium hydroxide. The solution made acidic with hydrochloric acid was poured into a column packed with Extrelut. Methomyloxime and I.S. were eluted from the column with a mixture of dichloromethane-ethyl acetate-chloroform (65:25:10), transformed to tert.-butyldimethylsilyl derivatives, and analyzed by gas chromatography-mass spectrometry in the electron impact mode. The calibration curves were linear in the concentration range from 1 ng/g to 100 ng/g and 100 ng/g to at least 5000 ng/g. The lower limit of detection was 0.5 ng/g. The absolute recoveries were 72-93% and within-day coefficients of variation were 3.1-5.6% at blood concentrations of 10 and 1000 ng/g. Two practical forensic applications are described.

Acetamides↗

PubMed1998

Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.

Activin Receptors, Type I↗

PubMed1998

Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome.

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.

Animals↗