Biomedical subjects

M Ikeda

Publications and source records attributed to M Ikeda.

At least 667 records · Page 37Linked to original sources

PubMed1997

Tranilast inhibits the growth of rat mesangial cells.

We investigated the effects of tranilast on the growth of cultured rat mesangial cells. The number of mesangial cells increased fivefold during a 5-day incubation in RPMI 1640 with 20% fetal bovine serum. The number of cells was significantly lower in the presence of tranilast than in its abscence. Tranilast (0 approximately 500 microM) inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis of rat mesangial cells cultured in RPMI 1640 medium containing 0.5% fetal bovine serum in a dose-dependent manner. The inhibition of DNA synthesis by tranilast was not affected by the presence of indomethacin (1 microg/ml) or N(G)-monomethyl-L-arginine (0.5 mM). Tranilast did not stimulate nitrite oxide synthesis in PDGF-stimulated cells. Mitogen-activated protein kinase activity in mesangial cells was significantly increased by exposure to PDGF, while the effect was significantly suppressed in the presence of tranilast. The present study revealed that tranilast inhibits the growth of rat mesangial cells, independently of nitric oxide or prostacycline synthesis.

Animals↗

PubMed1997

Characterization of Ral GDP dissociation stimulator-like (RGL) activities to regulate c-fos promoter and the GDP/GTP exchange of Ral.

Ral GDP dissociation stimulator-like (RGL) has been identified to be a possible effector protein of Ras. RGL shares 50% amino acid identity with Ral GDP dissociation stimulator and contains the CDC25-like domain in the central region and the Ras-interacting domain in the C-terminal region. Since the modes of activation and action of RGL have not yet been clarified, in this paper we have analyzed the functions of RGL. In COS cells, RGL interacted with RasG12V/E37G (a Ras mutant in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf, but not with RasG12V/T35S which bound to Raf. Raf did not inhibit the binding of RGL to RasG12V/E37G under the condition that Raf inhibited that of RGL to RasG12V. Expression of either RGL or Raf into NIH3T3 cells slightly activated c-fos promoter, while coexpression of both proteins greatly stimulated the c-fos promoter activity. RGL stimulated the GDP/GTP exchange of Ral and this action was enhanced by the post-translational modification of Ral. However, RGL was not active on Ras, Rac, CDC42, Rap, or Rho. Furthermore, this action of RGL to stimulate the GDP/GTP exchange of Ral was dependent on Ras in COS cells. These results suggest that RGL constitutes another Ras-signaling pathway which is distinct from the Raf pathway and indicate that the RGL pathway regulates the c-fos promoter activity and the GDP/GTP exchange of Ral.

Animals↗

PubMed1997

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage.

Basic helix-loop-helix (bHLH)/PAS proteins, such as Sim, act as transcriptional factors, playing a critical role in the control of central nervous system (CNS) development. To isolate novel bHLH/PAS factors in the CNS an iterative search of a database for expressed sequence tags (ESTs) resulted in the location of several bHLH/PAS protein-like sequences. The rapid amplification of cDNA end (RACE) method was applied to isolate full-length cDNAs of these ESTs. Several 5' and 3' terminal sequences were isolated using primers derived from an EST from the human brain cDNA library. The predicted novel factor polypeptide had bHLH and PAS domains that were highly homologous with those of Ah receptor nuclear translocator (Arnt) and Arnt2. Combination of the isolated cDNA fragments revealed the existence of several alternatively spliced variants. The distribution of the novel bHLH/PAS factor message was analyzed by Northern blot hybridization. This detected only one transcript, which was 2.9 kb in size. Strong hybridization was found in the brain, skeletal muscle and heart. Expression of the novel bHLH/PAS factor, brain and muscle Arnt-like protein 1 (BMAL1), was different from that of Arnt and Arnt2, suggesting that BMAL1 has a different function in the CNS and muscle than Arnt and Arnt2.

ARNTL Transcription Factors↗

PubMed1997

Atlantoaxial dislocation. A follow-up study of surgical results.

STUDY DESIGN: The cases of 47 patients with atlantoaxial dislocation exclusive of rheumatoid arthritis, cerebral palsy, and tumors as causative pathologies were reviewed after surgical treatment, which was performed between 1979 and 1993. OBJECTIVES: To investigate the surgical results of atlantoaxial dislocation itself without any systemic factors affected by rheumatoid arthritis, cerebral palsy, and tumors. METHODS: Neck pain (or occipitalgia) and extent of myelopathy at follow-up evaluation were compared with that present before surgery. The results were classified into four groups: excellent (no pain or recovery rate in myelopathy of more than 50%), good (decreased pain or recovery rate of 25% to 50%), fair (no improvement of pain or recovery rate of zero to 25%), and poor (aggravation of pain or recovery rate less than zero). The average follow-up period was 4 years and 2 months. RESULTS: Of the patients evaluated, 51% were assessed as excellent, 23% as good, 7% as fair, and 19% as poor. Pain relief was achieved in 95% of patients with non myelopathy. Extent of myelopathy, pathology of atlantoaxial dislocation (ligamentous or osseous instability), loss of reduction after surgery, and surgical procedures were recognized as the major factors affecting surgical results. Better surgical results were obtained in mild myelopathy cases (> 10 points in Japan Orthopaedic Association scoring), ligamentous instability, and cases without loss of reduction. The incidence of pseudarthrosis and loss of reduction was low in Brooks' method for atlantoaxial fusion and in Luque's segmental sublaminal wiring method for occipitocervical fusion. CONCLUSION: The best results occurred in patients with no myelopathy, and the worst results occurred in patients with severe myelopathy; therefore, surgery is best indicated for atlantoaxial dislocation with intractable pain or with mild myelopathy. To avoid pseudarthrosis and loss of reduction, a strong fixation method, such as Brooks' or Luque's segmental sublaminal wiring method, should be selected.

Adult↗

PubMed1997

Predominant expression of Brn-2 in the postmitotic neurons of the developing mouse neocortex.

The expression of Brn-2, a central nervous systems (CNS)-specific POU domain transcription factor, in the developing mouse neocortex was examined with an anti-Brn-2 antibody. Brn-2 protein was first detected in CNS on embryonic day (E) 11.5, and remained strong until E15.5. From E11.5 to postnatal day (P) 0, a high level of Brn-2 expression was observed in the subventricular zone, the intermediate zone, and the outer layer of the neocortex, but not in the ventricular zone. In the double-staining experiments, most of the Brn-2 positive cells were also positive for NCAM-H, an adhesion molecule specific to post-mitotic neurons. Furthermore, BrdU-labeling experiments demonstrated the presence of Brn-2 protein exclusively in postmitotic cells. These results indicated that, in the developing neocortex, Brn-2 expression is up-regulated after the final cell division. Therefore, this transcription factor may be involved in the migration and/or maturation process of the immature neuronal cells.

Animals↗

PubMed1997

Differential induction of constitutive and inducible nitric oxide synthases by distinct inflammatory stimuli in bovine aortic endothelial cells.

Exposure to various combinations of cytokines and lipopolysaccharide (LPS) has been reported to increase NO production in vascular endothelial cells. The molecular entity of the newly expressed nitric oxide synthase (NOS) in endothelial cells, however, has not yet been examined in detail. In this report, we carried out biochemical characterizations and molecular identification of NOS isoform(s) expressed in cytokine/LPS-treated bovine aortic endothelial cells (BAEC). The increased NOS activity in tumor necrosis factor-alpha (TNF-alpha)/LPS-treated BAEC was localized mainly in the cytosolic fraction and Ca2+-independent, whereas that in interferon-alpha,beta(IFN-alpha,beta)/LPS-treated BAEC was preferentially in the membrane fraction and Ca2+-dependent, suggesting that TNF-alpha/LPS increased an inducible NOS (iNOS)-like activity, and IFN-alpha,beta/LPS increased an endothelial constitutive NOS (ecNOS)-like activity. Correspondingly, the different responses to the cytokine/LPS pretreatment were demonstrated in semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for iNOS or ecNOS, that is, TNF-alpha/LPS elicited the expression of iNOS mRNA whereas IFN-alpha,beta/LPS increased that of ecNOS mRNA. A nuclear run-on transcription assay and an inhibition experiment by actinomycin D indicated that the apparent increase of ecNOS in the IFN-alpha,beta/LPS-treated BAEC was at least in part ascribed to the transcriptional activation. The nucleotide sequences of the amplified PCR products in TNF-alpha/LPS- and IFN-alpha,beta/LPS-treated BAEC were 93% and 99% identical to the corresponding regions of human hepatocyte iNOS and bovine ecNOS, respectively. These findings indicated that, in cytokine/LPS-treated BAEC, two NOS isoforms whose molecular natures were closely homologous to the conventional isoforms of iNOS and ecNOS were differently induced in response to distinct inflammatory stimuli.

Amino Acid Sequence↗

PubMed1997

Analysis of the 5' sequence, genomic structure, and alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease.

Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.

Alternative Splicing↗

PubMed1997

The primary structure of the Cl(-)-translocating ATPase, b subunit of Acetabularia acetabulum, which belongs to the F-type ATPase family.

The genes possibly encoding the b subunit (50 kDa) of the Cl(-)-translocating ATPase of Acetabularia acetabulum were cloned from total RNA and from poly(A)+ RNA and sequenced. The deduced amino acid sequence of the open reading frame consisted of 478 amino acids and showed high similarity to the beta subunit of chloroplast F1-ATPase. Gene fragments encoding the putative beta subunit of chloroplast F1- (273 bp) and mitochondrial F1-ATPases (332 bp) were also cloned from A. acetabulum and sequenced, respectively. The deduced amino acid sequence of the chloroplast F1-ATPase showed 92.5% identity to be primary structure of the b subunit of the Cl(-)-translocating ATPase, while the nucleotide sequences were 79.9% identical. The deduced amino acid sequence of the latter was 77.3% identical to that of the b subunit of the Cl(-)-translocating ATPase and the nucleotide sequences were 67.5% identical. By Northern analysis, these three beta-like genes were demonstrated to be transcribed with different sizes of RNA species. A putative chloroplast F1-beta fragment also hybridized with chloroplast DNA isolated from the organism.

Acetabularia↗

PubMed1997

[The effect of brain-derived neurotrophic factor on elongation of axons after transection with suture-morphometric evaluation].

Brain-derived neurotrophic factor (BDNF) is considered to play an important role in survival, maintenance, development and repair of the peripheral neuron. In this study, the effect of human recombinant BDNF on sprouting and elongation of axons, the early phase of regeneration of nerve fibers, was morphometrically evaluated 7 days following the sciatic nerve transection and juxtaposition of proximal and distal stumps with suture in Sprague-Dawley rats. In the experimental group (test), 20 mg/kg of BDNF was injected subcutaneously every day for seven days in eight rats, starting 30 minutes after the transection. In the control group (control), phosphate buffered-saline alone was injected in nine rats as in the experimental group. The various morphometric parameters were evaluated in the sciatic nerve of each rat of the control and test, 3 mm distal to the site of the transection on light and electron microscopy of Epon-embedded sections. On both light and electron microscopy only a few myelinated fibers with a very thin myelin sheath were found only in one nerve in each of the control and test. However, significant numbers of unmyelinated axons were found in each nerve of the control and test. There were no statistically significant differences in the total fascicular area per nerve, the numbers of myelinated and unmyelinated axons per mm2 of fascicular area and per nerve, their maximum and median diameters and their size distribution histograms between control and test. In addition, there were no statistically significant differences in the numbers of myelin ovoids per mm2 of the fascicular area and per nerve, their maximum and median diameters and their size distribution histograms between control and test. Therefore, we concluded that there was no definite evidence that BDNF promoted the sprouting and elongation of axons, the early phase of the regeneration of nerve fibers, at least under this experimental conditions.

Animals↗

PubMed1997

IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12.

We have previously reported the cloning of a novel cytokine, IFN-gamma-inducing factor (IGIF), which shared some biologic activities with IL-12. In this study, we analyzed the effects of murine IGIF on the activation of T cells, and compared the effects with those of IL-12. IGIF alone had no effect on the activation of T cell lines or Th1 clones, while IGIF increased the IFN-gamma production by antigen-stimulated T cell lines, but had no effect on IL-4 or IL-10 production. As reported with IL-12, IGIF served as a costimulatory factor for Th1 clones stimulated with Ag on B cell APC, immobilized anti-CD3, Con A, or IL-2 to augment IFN-gamma production and to induce IL-2R alpha-chain expression and proliferation of the Th1 clones, whereas IGIF had little or no effect on the IL-4 production and proliferation of Th2 clones stimulated with anti-CD3 or Ag. However, IGIF synergized with IL-12 to further augment the IFN-gamma production of the Th1 clones. Even in the presence of saturated amounts of IL-12, IGIF still augmented the IFN-gamma production and proliferation and enhanced the IL-2R alpha-chain expression of the Th1 clones. In contrast with IL-12, IGIF induced IL-2 production by Ag- or anti-CD3-stimulated Th1 clones. These two findings indicate that IGIF and IL-12 are utilizing different signal transduction pathways. We also found that IGIF as well as IL-12 was endogenously released through interaction between Th1 cells and spleen cell APC in the presence of specific Ag, and that it regulated IFN-gamma production. These results further suggest that IGIF may act as an immunoregulatory factor in the immune response.

Animals↗

PubMed1997

DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.

We have isolated and characterized the cDNA encoding a novel protein designated DJ-1. DJ-1, sharing no significant homology with the sequences so far reported, did not show transactivation activity in the Gal4 recombinant system, but transformed mouse NIH3T3 cells by itself. Furthermore, DJ-1 showed a cooperative transforming activity with H-Ras, more than 3 times as strong as the activity of ras/myc combination. DJ-1 was ubiquitously expressed in various human tissues, and the expression was induced by growth stimuli. Moreover, DJ-1 translocated from cytoplasm to nuclei in the S phase of the cell cycle. DJ-1 is thus suggested to be a novel mitogen-dependent oncogene product involved in a Ras-related signal transduction pathway.

3T3 Cells↗

PubMed1997

GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain.

We have cloned a cDNA encoding a novel association partner of cyclin G by West-Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase-like catalytic domain at the N-terminal. Hence, we named it GAK (cyclin G-associated kinase). The long C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co-immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co-precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK-cyclin G interaction was direct.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

Signal transduction system in epinephrine stimulated platelets; comparison between epinephrine sensitive and insensitive platelets.

We recently reported the high prevalence of impaired platelet responsiveness only to epinephrine in healthy Japanese. This abnormality was associated with a 50% decrease in the number of alpha 2-adrenergic receptors. Platelets from non-responders (NR) do not undergo secondary platelet aggregation even after exposure to 100 microM epinephrine, but they can potentiate the effect of ADP to provoke platelet aggregation. To further define the nature of the defect and to delineate controversial steps of epinephrine stimulated signal transduction, a signaling pathway of epinephrine was investigated in platelets from NR and R(normal responder to epinephrine). In a unique particle counting apparatus, epinephrine initially triggered the formation of small platelet aggregates composing of 10-1000 cells from both R and NR, but the aggregates became larger (4600 > cells) only in platelets from R. Thus, platelets from NR lack the ability to form larger aggregates. A similar defect was reproduced by treating normal platelets with aspirin. In the presence of fibrinogen, platelets from NR lacked phospholipase A2 activation, determined by arachidonic acid liberation in the presence of inhibitors to cyclooxygenase and lipoxygenase. In the absence of fibrinogen, aggregation and phospholipase A2 activation were not evident in R and NR. The surface expression of GPIIb/IIIa was markedly decreased in platelets from NR after stimulation by epinephrine, in comparison with those from R. The resting level and epinephrine stimulated increase in cAMP were not significantly different between NR and R. Incubating R platelets with a half saturating dose of yohimbine rendered them insensitive to epinephrine. These results indicated that the impaired platelet aggregation induced by epinephrine was due to the impaired surface exposure of glycoproteins GPIIbIIIa integral to the activation of phospholipase A2, which requires the full and normal occupancy of the alpha 2-adrenergic receptor by epinephrine.

Adenosine Triphosphate↗

PubMed1997

Expression of Src-like adapter protein mRNA is induced by all-trans retinoic acid.

By using a differential display method, specific bands were selected from ladder PCR products derived from ATRA-dependent differentiated U937 cells, in comparison with those of untreated U937. By screening the cDNA library of ATRA-dependent differentiated U937 cells with one of the PCR products, we cloned the src-like adapter protein (SLAP). Northern blot analysis of U937 cells with or without ATRA treatment indicated that the SLAP mRNA was clearly induced by ATRA. The induction was inhibited by the addition of cycloheximide, indicating that ATRA acted indirectly through synthesis of other proteins. The SLAP mRNA was induced in HL60 and NB-4 but not in K562 or THP-1. Interestingly, these cells in which SLAP mRNA was induced by ATRA all showed ATRA-dependent cell differentiation. The relationship between SLAP and cell differentiation is unclear, but SLAP may transduce a signal for cell differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Plasma nitrite/nitrate level is inversely correlated with plasma low-density lipoprotein cholesterol level.

BACKGROUND: Plasma nitrite/nitrate (NOx) is a stable end product of the vasodilator nitric oxide (NO). However, there are few reports about plasma NOx levels in humans. HYPOTHESIS: The purpose of this study was to assess the availability of plasma NOx for evaluating basal endogenously-synthesized or endothelium-derived NO, and to examine whether NOx levels are lowered in patients with coronary artery disease (CAD) or its risk factors. METHODS: Plasma NOx levels were measured using an automated system based on the Griess reaction. NOx levels for a 24-h period reproducibly became lowest at 6 A.M. in restricted healthy volunteers, and became stable in inpatient volunteers at 6 A.M. within 4 days after admission. RESULTS: Based on these findings, NOx levels at 6 A.M. in inpatients can be considered as the basal levels. In 40 inpatients suspected of CAD (28 men, 12 women; mean age 60 +/- 11 years), the basal levels of NOx were not related to CAD and its risk factors, except for hypercholesterolemia. The NOx level of patients with hypercholesterolemia was significantly lower than that of patients with normal cholesterol (n = 16,34 +/- 16 mumol/l vs. n = 24, 49 +/- 23 mumol/l, p < 0.03). Furthermore, the NOx levels correlated negatively with the total cholesterol and low-density lipoprotein cholesterol levels (r = -0.40, p < 0.01; r = -0.47, p < 0.003, respectively), but not with other lipid fraction levels. CONCLUSION: The results suggest that the quantity of basal endothelium-derived NO synthesis may be decreased in the presence of hypercholesterolemia.

Case-Control Studies↗

PubMed1997

Fourier Transform Microwave Spectroscopy of 13C-Substituted CCS Radicals

The rotational spectral lines of the 13C isotopic species of CCS (13CCS, C13CS, and 13C13CS) have been observed using a Fourier transform microwave spectrometer in combination with a pulsed-discharge nozzle. The hyperfine-resolved JN = 10-01, JN = 21-10, and JN = 32-21 transitions have been observed in the 11-, 22-, and 33-GHz regions, respectively, with an accuracy of about 5 kHz. The observed transition frequencies for 13CCS and C13CS are analyzed simultaneously with millimeter-wave data, and the hyperfine interaction constants for both species are determined accurately. Astronomical implications for these radicals are discussed. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗

PubMed1997

Predominant expression of the src homology 2-containing tyrosine phosphatase protein SHP2 in vascular smooth muscle cells.

src homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) beta and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues: it is lowest in the liver and kidney. Immunohistochemical staining and in situ hybridization showed SHP2 was expressed ubiquitously but predominantly in vascular smooth muscle cells (SMC). During the development of granulations. SHP2 was expressed predominantly in vascular SMC and also highly expressed in capillary cells. The functional associations of SHP2 with PDGFR beta, which transduces major growth signals in vascular SMC, identify a crucial function of SHP2 in blood vessels in consert with PDGFR beta.

Actins↗

PubMed1997

Sclerotherapy for varicose veins of the lower legs in patients with dysplasminogenemia.

Sclerotherapy combined with ligation has become a widely accepted treatment for varicose veins; however, it is associated with some risk of the serious complications of deep vein thrombosis (DVT). We investigated the incidence of thrombophilia in 164 consecutive patients undergoing treatment for varicose veins and determined the activities of antithrombin-III, protein C, and plasminogen. Of the 164 patients, 10 were diagnosed as having dysplasminogenemia (DPG), showing an incidence of 6.1%, in accordance with previous reports. DVT was not found to be caused by DPG in any patient, and no difference was found between patients with and those without DPG, suggesting that DPG is not a risk factor for varicose veins. We also investigated the activation of coagulation by measuring the thrombin-antithrombin III complex (TAT). The activation of coagulation after sclerotherapy was inhibited when ligation was performed 1 month prior to sclerotherapy, whereas it was increased when sclerotherapy and ligation were performed simultaneously. Of the 10 patients with DPG, 5 were treated uneventfully, and their TAT level increased to 4.0 micrograms/l, which was comparable to the level after sclerotherapy and ligation. These findings indicate that sclerotherapy can be performed safely in the majority of patients with DPG, and that the temporal separation of sclerotherapy and surgery is an alternative for these patients to prevent the activation of coagulation.

Adult↗