Biomedical subjects

J Sawada

Publications and source records attributed to J Sawada.

At least 55 records · Page 3Linked to original sources

PubMed1997

Expression of opioid-binding cell adhesion molecule (OBCAM) and neurotrimin (NTM) in E. coli and their reactivity with monoclonal anti-OBCAM antibody.

Opioid-binding cell adhesion molecule (OBCAM), neurotrimin (NTM) and limbic system-associated membrane protein (LAMP) are homologous and are the members of the IgLON family which is a subfamily within the immunoglobulin superfamily. We cloned the cDNAs for OBCAM and NTM, prepared recombinant proteins, and examined the reactivity of the previously prepared monocolonal anti-OBCAM antibody, OBC53, with the recombinant proteins by immunoblotting. These experiments revealed that OBC53 recognizes OBCAM about 1000 times as efficiently as NTM. Moreover, the NTM and LAMP peptides which have sequences homologous to the OBCAM peptide used for the preparation of OBC53 were 150 times less reactive to OBC53. Thus, the OBC53 antibody is a useful tool for specifically detecting OBCAM in immunochemical experiments.

Amino Acid Sequence↗

PubMed1997

Retinoblastoma binding factor 1 site in the core promoter region of the human RB gene is activated by hGABP/E4TF1.

We previously reported two oncogenic point mutations present in the RB (retinoblastoma) gene promoter region, found at consensus Sp1 and ATF sites, respectively, and in two separate hereditary RB families. However, Sp1 protein was shown not to bind to the Sp1 site; this indicated that the Sp1 consensus site mutation was blocking the action of an alternative transcription factor, which we called RBF-1 (retinoblastoma binding factor 1). Subsequent purification of RBF-1 revealed it to be hGABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. In this study, we directly examined the effects of hGABP/E4TF1 on transactivation of the RB gene promoter through the RBF-1 site. As expected, hGABP/E4TF1 enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site. We therefore conclude that the most essential transcription factor in the human RB gene is likely to be hGABP/E4TF1.

Animals↗

PubMed1997

Effect of an ectokinase inhibitor, K252b, on degranulation and Ca2+ signals of RBL-2H3 cells and human basophils.

We examined the effects of K252b, an ectoprotein kinase inhibitor of microbial origin, on the activation process of RBL-2H3 cells by cross-linking of IgE receptors by the endoplasmic reticulum Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone or by the Ca2+ ionophore A23187. Analysis of phosphorylation of ectoproteins following IgE receptor cross-linking revealed that K252b mainly inhibited the phosphorylation of a 130-kDa protein. The inhibitor simultaneously inhibited degranulation and the sustained increase in the cytosolic calcium ion concentration even after addition of Ag. In contrast, K252b did not inhibit the increase in degranulation and cytosolic calcium ion concentration caused by stimulation with 2,5-di(tert-butyl)-1,4-hydroquinone and A23187. Permeation of K252b into RBL-2H3 cells, assessed by fluorescence intensity, was very low. K252b also inhibited degranulation caused by IgE receptor cross-linking in human basophils, but did not inhibit the degranulation caused by A23187. Thus, our findings suggest that the effects of K252b may be mediated by outer surface-bound or -anchored K252b-sensitive molecules on RBL-2H3 cells and human basophils, and that the phosphorylation of ectoprotein may involve a transmembrane influx of Ca2+ by IgE receptor cross-linking.

Basophils↗

PubMed1997

The promoter of an androgen dependent gene in the hamster flank organ.

Hamster flank organs are useful for studying androgen-dependent growth of hair follicles and sebaceous glands. A cDNA clone (FAR-17a) was isolated from the hamster flank organ, whose expression was highly sensitive to androgen. The mRNA level of this gene was reduced after castration but reappeared after testosterone treatment. To elucidate the mechanism of expression of this gene regulated by androgen we isolated a genomic clone, from a hamster genomic library, that includes the promoter and upsteam region. The promoter region was used to drive a luciferase reporter gene in Cos 7 cells. This construct was activated five to six times higher over a control plasmid lacking the promoter region. We tested the effects of testosterone by transfection of the reporter plasmid into androgen dependent SC-3 cells. The results showed up to fivefold stimulation after the addition of androgen. The sequence of this promoter region was analyzed and the transcription factor binding sites were predicted. Since no obvious androgen responsive elements were included in the promoter region, we suggest that the stimulation of the reporter construct has to be mediated indirectly by androgen-dependent transcription factor(s).

Amino Acid Sequence↗

PubMed1997

Autoregulation of Pax6 transcriptional activation by two distinct DNA-binding subdomains of the paired domain.

BACKGROUND: Pax6 is a transcription factor that plays a central role in eye development. Pax6 contains a DNA-binding domain called paired domain, which consists of a highly conserved N-terminal subdomain and a variable C-terminal subdomain. Recent findings have suggested that both subdomains possess distinct DNA-binding activities. RESULTS: To understand the mechanism of DNA-binding and transcriptional activation by Pax6 via these subdomains, we employed Pax6 paired domain mutants previously isolated from patients with ocular disorders. Analysis of these mutants by gel shift assay revealed that the N-terminal and C-terminal subdomains can independently bind to their respective cognate sites, P6CON and 5aCON. Results from a luciferase assay, however, showed that the two functional subdomains negatively regulate their transactivation potentials each other. Wild-type Pax6 and its hyperactive variants show different patterns of DNA contact. CONCLUSION: These results support a new model for the regulation of Pax6 transactivation: When one DNA-binding subdomain binds to its cognate site, the other subdomain also interacts with the flanking sequences nonspecifically, and this interaction constrains its structure to give a reduced level of transactivation.

Animals↗

PubMed1997

[A family with facioscapulohumeral muscular dystrophy and hereditary long QT syndrome].

We describe a family with facioscapulohumeral muscular dystrophy (FSHD) and hereditary long QT syndrome (LQT) for three generations. The proband, a 50-year-old woman, had noticed difficulty in raising the upper extremities since the age of 40. At the age of 48, she was admitted to our hospital because of arrhythmia attack. She, her mother, and one of her three children were diagnosed as having LQT. These three individuals and the proband's two siblings were clinically diagnosed as having FSHD which was confirmed by genetic analysis using EcoR1. FSHD is an autosomal dominant disorder and the gene locus is mapped to chromosome 4q35-ter, but the gene has not been isolated. LQT is a group of disorders which cause syncope and sudden death from ventricular arrhythmia in an autosomal dominant fashion. Four loci for this syndrome (LQT1-4) have been known, and three of the genes have been shown to encode ion-channels. Genetic analysis of the proband failed to detect any of previously known mutations in the LQT1, LQT2, and LQT3 genes. The locus for LQT4 has been mapped to chromosome 4q25-7. There have so far been no report of FSHD associated with LQT. Although the pathogenesis is unclear, we speculate that these two diseases are linked each other on chromosome 4q.

Chromosomes, Human, Pair 4↗

PubMed1996

Functional domains of transcription factor hGABP beta1/E4TF1-53 required for nuclear localization and transcription activation.

Transcription factor E4TF1 is the human homolog of GABP and has been renamed hGABP (human GABP). hGABP is composed of two types of subunits; hGABP beta1/E4TF1-53 and the ets-related protein hGABP alpha/E4TF1-60. Both bind together to form an (alpha)2(beta1)2 heterotetrameric complex on DNA and activate transcription at specific promoters in vitro. Tetramer formation depends on two regions of hGABP beta1; the N-terminal region containing the Notch/ankyrin-type repeats is necessary for binding to hGABP alpha and the C-terminal region is necessary for homodimerization. In this report, we constructed various deletion mutants of hGABP beta1 in order to delimit the functional regions required for nuclear localization and transcription activity. We found that hGABP beta1 localization in the nucleus is dependent on a region located between amino acids 243 and 330 and that the presence of hGABP beta1 influences the efficiency of hGABP alpha transport into the nucleus. Next, we demonstrated that the hGABP complex composed of alpha and beta1 subunits activates transcription from the adenovirus early 4 promoter in vivo. This transcription activation needs the C-terminal region of hGABP beta1 and is consistent with results obtained with the in vitro assay. Furthermore, site-directed mutagenesis analysis of the C-terminal region reveals that the alpha-helix structure and the leucine residues are important for formation of a heterotetrameric complex with hGABP alpha in vitro and for transcription activation in vivo. These results suggest that hGABP beta1 stimulates transcription as part of a heterotetrameric complex with hGABP alpha in vivo.

Animals↗

PubMed1996

Confocal fluorescence microscopy for antibodies against a highly conserved sequence in SH2 domains.

We have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Mouse IgG1s (12E and 32D) prepared against a peptide-conjugated keyhole lympet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analysis showed that one IgG1 (12E) recognized mainly 62 kDa proteins (possibly src-family tyrosine kinases) from triton X-100 extracts of RBL-2H3 cells and that another (32D) recognized mainly 32 and 110 kDa proteins. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribution was observed in the cells stained with 12E and 32D IgGs. 12E IgGs strongly stained the plasma membranes while 32D IgGs stained small granules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggest that tyrosine kinases cluster along the plasma membranes and/or than conformational changes occur in the domains after antigen stimulation.

Amino Acid Sequence↗

PubMed1996

Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells.

We previously reported that a hydroquinone-type antioxidant, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), increases intracellular free Ca2+ concentration ([Ca2+]i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five-hydroquinone-type and phenolic antioxidants (2,5-di(tert-amyl)-1,4-hydroquinone [DTAHQ], 2-tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di(tert-butyl)-4-hydroxytoluene [BHT], 3,5-di(tert-butyl)-4-hydroxyanisole [DTBHA], and 3-tert-butyl-4-hydroxyanisole [MTBHA]) on [ca2+]i and degranulation (beta-hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (> or = 3 microM) showed effects similar to those of DTBHQ (10 microM) on [Ca2+]i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 microM) and DTBHA (50 microM) caused [Ca2+]i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca2+]i and degranulation, even at 50 microM. The degree of Ca2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca2+]i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca2+-ATPase.

Animals↗

PubMed1996

Activation of the retinoblastoma gene expression by Bcl-3: implication for muscle cell differentiation.

The retinoblastoma (Rb) protein is a master regulator of cell cycle. Accumulating evidence suggests that elevation of Rb expression is a key event in differentiation of various cell types. However the mechanism of regulation of Rb expression is poorly understood. Here we report that the candidate oncoprotein Bcl-3, previously characterized as a member of the IkappaB family, activates transcription of the Rb gene, whose promoter has no typical kappaB sites. A target element for Bcl-3 that matches the consensus for the E4TF1/GABP transcription factor was identified. Bcl-3 was shown to promote tetramer formation of E4TF1. During muscle cell differentiation, increased bcl-3 expression was observed before the induction of the Rb mRNA. Transient expression of Bcl-3 in myoblasts was shown to induce expression of the endogenous Rb. Furthermore, expression of the antisense bcl-3 RNA in myoblasts suppressed induction of Rb and myogenic differentiation. These results suggest that Bcl-3 is an upstream regulator of Rb expression during differentiation of muscle cells.

B-Cell Lymphoma 3 Protein↗

PubMed1996

Surface expression of CD63 antigen (AD1 antigen) in P815 mastocytoma cells by transfected IgE receptors.

The surface expression CD63 antigen in rat basophilic leukemia cells (RBL-2H3) was observed after antigen stimulation by confocal fluorescence microscopy. The surface expression of CD63 antigen reflected the degranulation in RBL-2H3 cells. Then, we did the same experiments in P815 mastocytoma cells with transfected IgE receptors. The expression was observed in P815 cells with normal IgE receptors, but not in P815 variant cells with IgE receptors which were missing a C-terminal cytoplasmic domain of beta or gamma subunit. In addition, the expression in P815 cells with normal IgE receptors was mostly blocked by the pretreatment of herbimycin A. The results suggested that tyrosine phosphorylation of the C-terminal cytoplasmic domains of beta and gamma subunits was essential for degranulation.

Animals↗

PubMed1996

Effects of three different Ca(2+)-ATPase inhibitors on Ca2+ response and leukotriene release in RBL-2H3 cells.

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

Animals↗

PubMed1996

Characterization and tissue distribution of opioid-binding cell adhesion molecule (OBCAM) using monoclonal antibodies.

Monoclonal antibodies to opioid-binding cell adhesion molecule (OBCAM) were produced against a synthetic OBCAM peptide. Immunoblotting analysis revealed that the antibodies reacted with 58 and/or 51 kDa proteins in P2 membranes from bovine, rat, mouse, guinea pig and rabbit brains. In bovine brain, the 58 and 51 kDa proteins were present in the striatum and cerebral cortex at high levels, but not in the pituitary. OBCAM was also detected in the cerebellum mainly in the 51 kDa form. In other tissues, the proteins were found in the spleen at very low levels, but not at all in the liver or kidney of the rat. OBCAM was effectively solubilized from bovine P2 membranes by bacterial phosphatidylinositol specific-phospholipase C (PI-PLC), indicating that OBCAM is a glycosylphosphatidylinositol (GPI)-anchored protein. PI-PLC treatment, however, had little effect on the opioid binding activity of the residual P2 membranes. The molecular weight of the proteins (58 and 51 kDa) was reduced to 36 kDa following treatment with N-glycanase but not further reduced after subsequent treatment with neuraminidase and O-glycanase, suggesting that OBCAM has N-glycosylated carbohydrate chains and that its two isoforms are different, at least, in the degree of N-glycosylation. Taken together, these results suggest that OBCAM consists of 58/51 kDa GPI-anchored glycoproteins which are highly N-glycosylated and are expressed mainly in the nervous system.

Amino Acid Sequence↗

PubMed1996

Quantitative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.

A hand-held spectrophotometer was used for the quantitative evaluation of PCA caused by anti-TNP-IgE and TNP-BSA. A good relationship existed between the dilution of sera and the value of delta E*ab (a numerical expression of color) obtained by a hand-held spectrophotometer. In addition, the value of delta E*ab and the amount of Evans blue measured by the conventional colorimetric method correlated well. Because the method using a hand-held spectrophotometer provides a simple and objective analysis, it appears that the method is suitable as a substitute for the conventional method, which is time-consuming and requires killing animals cruelly.

Animals↗

PubMed1996

[Transesophageal echocardiography as a early postoperative monitoring patients after cardiovascular surgery: analysis of 500 consecutive studies].

In cardiac surgery significant residual lesions increase postoperative morbidity and mortality. To evaluate the usefulness of transesophageal echocardiography (TEE) as a early postoperative monitor in patients immediately after cardiovascular surgery, 500 consecutive patients were studied from April 1990 to December 1994. TEE was performed at the bedside in the intensive-care unit in all of these patients. TEE detected findings unsuspected by other perioperative tests or clinical examination in 103 (20.6%) of 500 patients. Imaging revealed unsatisfactory operative results that necessitated further surgery in 18 (3.6%) of the 500 patients. These data indicate that early postoperative TEE is useful in assessing immediate operative results and identifying patients with unsatisfactory results who are at increased risk for postoperative complications.

Cardiac Surgical Procedures↗

PubMed1996

[Surgical pathology of infective endocarditis].

Underlying pathologic disorders of infected valves were analyzed in 36 consecutive patients with infective endocarditis (from April 1987 to May 1995) of 18 aortic, 11 mitral and 8 prosthetic valves. Among 29 cases of native valve endocarditis, 27 had known organic changes [aortic valve prolapse 8, bicuspid aortic valve 4, annuloaortic ectasia 1, mitral valve prolapse 9 (including 2 cases associated with hypertrophic cardiomyopathy), looping chordae 1, rheumatic heart disease 4]. However, the remaining two cases had unknown etiology. Histological examination of these two aortic valves revealed proliferation of small vessels and remnants of vascular smooth muscle cells, suggesting postinflammatory valve prolapse. Five cases with definite pathology had no clinical signs of infection. The preponderance of surgically resected valves with infective endocarditis in Japan is non-rheumatic in origin.

Adult↗

PubMed1995

Assignment of the E4TF1-60 gene to human chromosome 21q21.2-q21.3.

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Chromosomes, Artificial, Yeast↗

PubMed1995

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells.

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca2+ level ([Ca2+]i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1-10 mumol/l) alone induced rapid and sustained increases in [Ca2+]i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 mumol/l) further increased the antigen (dinitrophenylated BSA)-induced Ca2+ response. In the absence of external Ca2+ with addition of 1 mmol/l EGTA, both DTBHQ (10 mumol/l) and the antigen (10 microgram/ml) induced transient increase in [Ca2+]i. In sensitized cells, both DTBHQ (10 mumol/l) and antigen (10 micrograms/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca2+]i and enhances antigen-induced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.

Adenosine Triphosphatases↗