Search PubMed⌕ Search

Biomedical subjects

K Kikuchi

Publications and source records attributed to K Kikuchi.

At least 325 records · Page 18Linked to original sources
PubMed1998

Molecular cloning and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp.

cDNA clones encoding the myogenic regulatory factors (MRFs) myogenin, MyoD and myf-5 were isolated by reverse-transcription polymerase chain reaction from larvae and embryos of the common carp (Cyprinus carpio L.). Myocyte-specific enhancer factor 2 (MEF2) cDNAs were identified from a cDNA library from adult carp. Northern blot analysis showed that MyoD, myf-5 and MEF2C transcripts were present in three-somite embryos, whereas myogenin and MEF2A transcripts were not detected until the 15-somite stage. Intense signals of myogenin and MyoD transcripts were observed even in 1-month-old juveniles. Levels of MyoD, myogenin and MEF2A transcripts declined between 1 and 7 months after hatching, and myf-5 gave only a weak signal in the oldest fish. In contrast, levels of MEF2C transcripts were considerably higher in 7-month-old juveniles than in 1-month-old larvae. mRNAs encoding carp myosin heavy chain and -actin were first detected at approximately the time of the first heartbeat, and levels were maximal in juveniles 1 month post-hatching. The relatively high levels of MRF mRNA in juvenile fish probably reflect the recruitment of new muscle fibres from the satellite cell population. It was concluded that the relative importance of the different members of the MyoD and MEF2 families of transcription factors for muscle differentiation changes during ontogeny in the carp.

Journal Article↗
PubMed1998

Syntheses and structure-activity relationships of novel retinoid X receptor agonists.

As part of our studies to develop novel retinoids with increased affinity and selectivity for the retinoid X receptor (RXR) subfamily, we have designed and synthesized a series of (E,E,E)-7-(1,2,3, 4-tetrahydroquinolin-6-yl)-7-alkyl-6-fluoro-3-methylhepta-2, 4, 6-trienoic acid derivatives. These tetrahydroquinolines, generated by introducing a polar N atom into the hydrophobic part of the retinoid skeleton, showed high binding affinity to RXRs. Addition of fluorine at the 6-position of the 2,4,6-trienoic acid moiety afforded compounds which elicit potent and selective transactivation of the RXRs. Compound 14b (ER-35794), which possesses an ethyl substituent at the 7-position and fluorine at the 6-position of the triene moiety, is one of the most potent and selective RXR agonists reported to date.

Animals↗
PubMed1998

Telomerase activity in a specific cell subset co-expressing integrinbeta1/EGFR but not p75NGFR/bcl2/integrin beta4 in normal human epithelial cells.

Telomerase activity is correlated with the immortality of various cultured cells and cancer cells. The activity, however, is also demonstrated in various normal regenerating cells which normally have a finite life span in vivo and in vitro, though its biological implication remains unclear. Using cultured normal human epithelial cells, we show that telomerase activity is associated with epithelial cell subsets which actively proliferate in vitro. Unlike in most cancer cell lines, telomerase activity was evidently up-regulated when the cells entered into S phase in primary human epithelial cells. To characterize the cells which have telomerase activity, the primary human epithelial cell population of uterine cervix was dissociated into several distinctive cellular subsets by means of immunocytochemical cell fractionation. Telomerase activity was found to be closely associated with the subset which expressed predominantly integrin beta1 and epidermal growth factor receptor. We further identified the telomerase-negative subpopulation which contained a small subset that strongly coexpresses p75NGFR low affinity nerve growth factor receptor, integrin beta4 and bcl-2 protein. The location of the p75NGFR-expressing cells contrasts to that of the Ki-67 positive cells in vivo and is distinctive of telomerase positive cycling cells, indicating that these cells remain at the G0 phase. The present study supports the notion that telomerase activity is linked to cell cycle regulation, implying that telomerase is activated upon cell proliferation in regenerating normal human epithelial cells.

Antigens, CD↗
PubMed1998

Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins.

Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.

Animals↗
PubMed1998

Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins.

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green-fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.

Animals↗
PubMed1998

Different moieties of tautomycin involved in protein phosphatase inhibition and induction of apoptosis.

The effects of tautomycin and its derivatives on protein phosphatases PP1 and PP2A and their apoptosis-inducing activity toward human leukemia Jurkat cells were examined, and the relationship between chemical structure and function was discussed. Among the compounds we examined, tautomycin was the most potent inhibitor and the most effective inducer of apoptosis. It inhibited PP1 and PP2A enzymatic activity concentration-dependently with IC50 values of 20 and 75 pM, respectively, in the presence of 0.01% Brij-35, and an LC50 value of 1 microM. Esterification of the anhydride moiety of tautomycin markedly increased the IC50 for the protein phosphatases. The C1'-C7' fragment of tautomycin had no inhibitory effect, but the fragment containing the C22-C26 moiety was inhibitory. These results suggest that the C22-C26 moiety is essential for inhibition of protein phosphatase activity and that the anhydride moiety enhances the inhibition. However, the esterification of the anhydride did not decrease, nor did the inclusion of the C22-C26 moiety increase the apoptosis-inducing activity. On the other hand, the C1-C18 moiety of tautomycin was essential for induction of apoptosis, and the conformation and the arrangement of functionalities of the C18-C26 carbon chain affected the apoptosis activity. However, modification of C1-C18, C1-C21, or C1-C26 compounds had little effect on phosphatase inhibitory activity. Our results strongly suggest that different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis.

Antifungal Agents↗
PubMed1998

Interleukin-10 reduces natural killer sensitivity and downregulates MHC class I expression on H-ras-transformed cells.

We examined the effect of IL-10 on sensitivity to NK-cell-mediated cytotoxicity of the H-ras-induced transformants, W14 and W31. Incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of MHC class I antigens, but not in the ICAM-1 expression. However, prior incubation of W31 cells with rhIL-10 markedly decreased their susceptibility to cytolysis by rat splenic NK cells. This fact suggested that the IL-10-mediated decrease in MHC class I expression might not dominate the regulation of the NK sensitivity. This was true when rat IL-10 cDNA-introduced W31 cells were used as an endogenous IL-10 producer. The NK sensitivity in vitro of W31T-H, a high IL-10-producer clone, was suppressed downward to the equivalent level of W31 cells pretreated with exogenous rhIL-10. The decreased NK-sensitivity of W31T-H cells was further confirmed by in vivo Winn assay, in which nude mice challenged with W31T-H cells and rat NK cells together developed tumors, whereas nude mice challenged with W31T-L, a minimal-IL-10 producer clone, and NK cells did not. Since neither exogenous nor endogenous IL-10 affected the proliferation of W31 cells, the data indicated that W31T-H cells could evade the NK-cell-mediated immune response in vivo. Taken together, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune system by NK cells.

Animals↗
PubMed1998

p53 and Bcl-2 expression in pneumoconiosis-related pre-cancerous lesions and lung cancers: frequent and preferential p53 expression in pneumoconiotic bronchiolar dysplasias.

To explore the mechanism by which lung cancers excessively arise from pneumoconiosis, we determined the altered expression of p53 and Bcl-2 by immunohistochemistry (IHC) in lung cancers, dysplasias and non-cancerous pulmonary epithelia in pneumoconiotics in comparison with those from non-pneumoconiotic patients. We examined p53 expression in squamous cell carcinomas (SCCs) and dysplasias separately in the central and peripheral zones of bronchial trees, based on observations that SCCs from pneumoconiotic patients occurr more frequently in peripheral epithelia than those from non-pneumoconiotic patients (55 of 72 SCCs with pneumoconiosis vs. 33 of 72 SCCs without pneumoconiosis). Forty-one of 72 patients with pneumoconiosis-related lung cancers had altered p53 expression, which was comparable to the positivity of p53 expression in lung cancers without pneumoconiosis. p53 expression was observed significantly more frequently in bronchiolar dysplasias with pneumoconiosis than in those from non-pneumoconiotic patients (13 of 23 vs. 4 of 22), while p53 expression was found in bronchial dysplasias with pneumoconiosis as frequently as those without pneumoconiosis. Moreover, in patients with pneumoconiosis, bronchiolar dysplasias exhibited p53 expression more frequently than bronchial dysplasias (13 of 23 vs. 4 of 19). When comparison was restricted to bronchiolar dysplasias from patients without lung cancer, p53 expression had a strikingly higher frequency in the dysplasias with pneumoconiosis than in those from non-pneumoconiotic patients (8 of 15 vs. 0 of 14). Bcl-2 occasionally was expressed in squamous metaplasias and basal cell hyperplasias, in contrast to p53, for which immunostaining was negative in these lesions. Altogether, our results show that pre-cancerous and/or cancerous targets in pneumoconiosis may be distributed over a more peripheral zone than those in patients without pneumoconiosis.

Bronchi↗
PubMed1998

Structural organization of rat CD1 typifies evolutionarily conserved CD1D class genes.

The non-major histocompatibility complex (MHC)-encoded CD1 family has recently emerged as a new antigen-presenting system that is distinct from either MHC class I or class II molecules. In the present study, we determined the genomic structure of the rat CD1 locus. It was extremely similar to mouse CD1 genes, especially to CD1D1. The 5' flanking region of the CD1 gene contained the binding motifs for two cytokine-inducible transcription factors, NF-IL2-A and NF-IL6. Some regulatory elements found in MHC class I genes (enhancer A, enhancer B, and the IFN response element) were absent. It is of interest that a tyrosine-based motif for endosomal localization found in the human CD1b cytoplasmic tail was encoded by a single short exon which was conserved in all CD1 molecules except for CD1a. Southern blot and direct sequencing analyses of inbred rat strains suggested very limited polymorphism in the 5' region where a hydrophobic ligand-binding groove is encoded; a single base substitution resulted in amino acid alteration of alanine (GCT) to valine (GTT) at codon 119. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the intron was also characteristic to each of the two classes of CD1 genes, classic CD1 and CD1D; such categorization has hitherto been made according to the sequence similarity of the coding region. This finding provides further support for the hypothesis that the two classes have different evolutionary histories. In contrast to the complete absence of the classic CD1 in rats and mice, the entire region of nonpolymorphic CD1D has been conserved through mammalian evolution. Similar functional properties of rodent CD1 and human CD1d are implied.

Amino Acid Sequence↗
PubMed1998

Serum concentrations of carboxyterminal propeptide of type 1 procollogen and tissue inhibitor of metalloproteinase 1 in patients with dermatomyositis.

Serum levels of carboxyterminal propeptide of type I procollagen (PICP) and aminoterminal propeptide of type I procollagen (PINP) have been used as indices of collagen synthesis in patients with various fibrotic diseases during the active stages. One of the suggested contributory factors to the development of tissue fibrosis is a decrease in collagenase activity, which may be related to levels of serum tissue inhibitors of metalloproteinases-1 (TIMP-1). In this study, the serum levels of PICP and PINP in 20 patients with dermatomyositis and 29 control subjects and of TIMP-1 in 29 patients with dermatomyositis and 29 control subjects were measured using an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). We found that the mean PICP level in patients with dermatomyositis was significantly higher than that in normal controls (mean +/- SD 326+/-76 ng/ml vs 135+/-88 ng/ml; P < 0.001). In 60% of dermatomyositis patients, the serum PICP level was elevated (more than 311 ng/ml, i.e. 2 x SD above the mean control value). Elevated serum PICP levels were correlated with the incidence of elevated serum creatine kinase levels in patients with dermatomyositis. The serum concentration of PINP was not elevated in comparison with that of the normal control subjects (mean +/- SD 36+/-30 ng/ml vs 63+/-34 ng/ ml). The mean TIMP-1 level in the patients with dermatomyositis was also significantly higher than in the normal control subjects (mean +/- SD 438+/-328 ng/ml vs 163+/-63 ng/ml; P < 0.001). In 59% of dermatomyositis patients, the mean serum TIMP-1 level was elevated (more than 289 ng/ml, i.e. 2 x SD above the mean control value). Serum PICP and TIMP-1 levels might be useful for detecting disease activity or severity in dermatomyositis.

Adolescent↗
PubMed1998

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome.

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships.

DNA Mutational Analysis↗
PubMed1998

Differences in antigenic sites, recognized by anti-liver-kidney microsome-1 (LKM-1) autoantibody, between HCV-positive and HCV-negative sera in Japanese patients.

Anti-liver-kidney microsome-1 (LKM-1), which reacts with cytochrome P450 IID6 (CYP2D6), is an autoantibody present in autoimmune hepatitis type II, which affects primarily young patients. Recently, it has been shown some adult patients with chronic hepatitis C are also positive for anti-LKM-1. Thus, anti-LKM-1-positive patients can be classified into two subgroups: (1) those with autoimmune hepatitis type II and (2) those with chronic hepatitis C. We investigated the antigenic epitopes of CYP2D6 with which each of these two anti-LKM-1-positive subgroups reacted. Multiple deletion mutants of CYP2D6 were constructed from a human liver cDNA library and five recombinant fusion proteins expressed. Antigenic epitopes were determined by immunoblot analysis using these proteins. Anti-LKM-1 present in HCV-negative sera recognized at least two peptide regions of aa213-280 and aa341-477 of human CYP2D6. In contrast, anti-LKM-1 present in HCV-positive sera recognized only a single region of aa341-477. Thus, the sera of patients with autoimmune hepatitis type II and patients with chronic hepatitis C recognize different antigenic epitopes of the CYP2D6 molecule. To our knowledge, this is the first time LKM-1 autoantigens have been analyzed at the molecular level in Japanese patients.

Asian People↗
PubMed1998

Enhanced expression of heparin-binding EGF-like growth factor and its receptor in hypertrophied left ventricle of spontaneously hypertensive rats.

OBJECTIVES: Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) is thought to produce hypertrophy in isolated cardiomyocytes via an autocrine mechanism, the pathophysiological role of HB-EGF, in myocardial hypertrophy in vivo, is not yet known. To investigate the involvement of HB-EGF in cardiac remodeling associated with hypertension in vivo, we assayed the expression of HB-EGF mRNA and protein in the left ventricle (LV) during the development of left ventricular hypertrophy in spontaneously hypertensive rats (SHR). METHODS: Prior to sacrifice and assay of HB-EGF and EGF-receptor (EGF-R) mRNA, morphologic and hemodynamic variables were measured in SHR and in age-matched Wistar Kyoto rats (WKY). At 5, 9 and 12 weeks of age, rats were killed, their hearts were removed, and the expression of HB-EGF and EGF-R mRNA and protein were measured. In addition, SHR and WKY were treated with enalapril, atenolol, or both for 4 weeks. RESULTS: In untreated SHR, double products (i.e. systolic blood pressure (sBP) multiplied by heart rate (HR), an index of mechanical load, peaked at 9 weeks. Expression of HB-EGF mRNA was also observed to peak in these animals at 9 weeks, while expression of EGF-R mRNA increased from 5 to 9 weeks, but remained constant thereafter. In untreated WKY, double products and EGF-R mRNA expression did not change over time, whereas the level of HB-EGF message increased gradually. Antibody to HB-EGF reacted primarily with myocyte membranes in SHR, whereas antibody to EGF-R reacted mainly with interstitial cells in these animals. The angiotensin-converting enzyme inhibitor, enalapril, markedly decreased sBP in SHR, whereas the beta 1-adrenoreceptor antagonist, atenolol, significantly decreased HR. While neither alone affected the expression of HB-EGF mRNA, their combination significantly reduced the expression of HB-EGF mRNA, as well as double products, in these rats, but had no effect on expression of EGF-R mRNA. CONCLUSIONS: The enhanced expression of HB-EGF mRNA and protein in LV of SHR suggest that this growth factor may play an important role during the early development of LV hypertrophy and cardiac fibrosis in SHR. The association between double products and HB-EGF expression suggest that the latter may be induced by increased mechanical load and may contribute, in turn, to cardiac remodeling.

Adrenergic beta-Antagonists↗