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J Sawada

Publications and source records attributed to J Sawada.

At least 73 records · Page 4Linked to original sources
PubMed1995

Release of a soluble form of growth hormone receptors (growth hormone-binding proteins) from human IM-9 cells by proteolytic cleavage.

Soluble forms of growth hormone receptors, growth hormone-binding proteins (GH-BPs), with molecular weights of 60 and 55kDa were found to be constitutively released from human IM-9 cells. The release of the GH-BPs was not inhibited by inhibitors of protein synthesis (cycloheximide) and transport (brefeldin A). Down-regulation by human growth hormone or trypsin pretreatment of surface growth hormone receptors abolished the GH-BP release, suggesting that cell-surface human growth hormone receptors are involved in the GH-BP release. Several inhibitors of serine-, thiol-, and acid-proteases did not affect the GH-BP release. EDTA efficiently blocked the GH-BP release. This inhibition by EDTA was restored by addition of Mg(2+) and Co(2+). These results suggest that human GH-BPs are constitutively released by proteolytic cleavage of cell-surface growth hormone receptors by a metalloprotease.

Brefeldin A↗
PubMed1995

[Immunotoxicity of chemicals].

Immunotoxicity is the detrimental effects of xenobiotics on immune functions of hosts, which result in enhanced susceptibility to infectious agents and tumours. A variety of environmental chemicals, such as pharmaceuticals, food additives, contaminating chemicals in food and drinking water, pesticides, and airborne chemicals, have been reported to exert immunotoxic effects on experimental animals and humans. In this paper, the mechanisms of immune responses, the effects of typical immunotoxic chemicals on the immune systems, and the methods for detecting immunotoxicity were described. In addition, the strategies and guidelines for immunotoxicity assessment were briefly outlined and discussed.

Animals↗
PubMed1994

Thapsigargin-induced nuclear calcium signals in rat basophilic leukaemia cells.

By a confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) we have studied thapsigargin-induced calcium signals in individual rat basophilic leukaemia (RBL-2H3) cells. In the presence or absence of external calcium ions, thapsigargin-induced calcium signals were transferred to the nucleus as well as to the cytoplasm of RBL-2H3 cells. The calcium signals were generally much stronger in the nucleus than in the cytoplasm. However, some of the RBL-2H3 cells had apparently reduced nuclear calcium signals. They had a basophil-like bilobed (multilobed) nucleus, although most RBL-2H3 cells had a mast-cell-like monolobed nucleus. In the cells with a bilobed nucleus, IgE-receptor-mediated calcium signals were neither transferred to the nucleus nor to the cytoplasm. The results gave a new insight into the understanding of the mechanism of the nuclear calcium signals in RBL-2H3 cells.

Animals↗
PubMed1994

Stimulatory effect of pervanadate on calcium signals and histamine secretion of RBL-2H3 cells.

We examined the effect of pervanadate on the activation of rat basophilic leukaemia (RBL-2H3) cells. The pervanadate, generated from a combination of H2O2 and vanadate (Vi), induced concomitantly protein tyrosine phosphorylation, formation of inositol 1,4,5-trisphosphate (IP3), an increase in [Ca2+]i, and histamine secretion in RBL-2H3 cells. These effects were clearly dependent on the ratio of H2O2/Vi. The secretion of histamine, IP3 formation, and sustained increase in [Ca2+]i were effectively induced by treatment of the cells with the pervanadate produced from 1 mM H2O2 and 1 mM Vi. These effects mimic the stimulatory effects of an antigen (dinitrophenylated BSA) on Ca2+ signals, histamine secretion and morphological changes. Protein tyrosine phosphorylation, formation of IP3 and transient increase in [Ca2+]i were markedly induced by the pervanadate produced from 3 mM H2O2 and 1 mM Vi. However, histamine secretion induced by the pervanadate was very low. After the pervanadate from 3 mM H2O2 and 1 mM Vi was treated with catalase, it was able to induce the [Ca2+]i increase and histamine secretion as much as the antigen did. This indicates that pervanadate from a lower H2O2 concentration (1 mM H2O2/1 mM Vi) and catalase-treated pervanadate from a higher H2O2 concentration (3 mM H2O2/1 mM Vi) are able to mimic the activity that was caused by cross-linking of IgE receptors with antigen. The present results also demonstrate that protein tyrosine phosphorylation seems to have a crucial role in Ca2+ entry from the external medium, and that a sustained [Ca2+]i increase is an important step for histamine secretion in RBL-2H3 cells.

Animals↗
PubMed1994

Transcriptional activation through the tetrameric complex formation of E4TF1 subunits.

Transcription factor E4TF1 is composed of two types of subunit, an ets-related DNA binding protein, E4TF1-60, and its associated proteins with four tandemly repeated Notch-ankyrin motifs, E4TF1-53 and E4TF1-47. To determine the functional domains, we constructed various mutants of the subunits. E4TF1-60 bound to DNA as a monomer. The ets domain and its N-terminal flanking region were necessary to recognize the specific DNA sequence. The 48 amino acids at the E4TF1-60 C-terminus were required for interaction with the other type of subunit. E4TF1-53 and E4TF1-47 share the N-terminal 332 amino acids but differ at the C-termini. They interacted with E4TF1-60 through the N-terminal flanking region to form a heterodimer. E4TF1-53 dimerized with itself, whereas E4TF1-47 did not. The C-terminal region specific for E4TF1-53 was required for the dimerization. Therefore, heterodimers composed of E4TF1-53 and E4TF1-60 were further dimerized, resulting in the formation of a tetrameric complex, which stimulated transcription in vitro. Heterodimers of E4TF1-47 and E4TF1-60 weakly stimulated transcription in vitro. The results indicated that the tetrameric complex formation of E4TF1 subunits was necessary to activate transcription efficiently in vitro.

Base Sequence↗
PubMed1994

Effects of herbimycin A and ST638 on Fc epsilon receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells.

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Animals↗
PubMed1994

Ligand-induced internalization and phosphorylation-dependent degradation of growth hormone receptor in human IM-9 cells.

The human growth hormone (hGH) induced a marked reduction in the number of human growth hormone receptors (hGHR) within 60 min, as assessed by immunoblotting of the crude membrane fraction from human IM-9 cells, without an increase in soluble forms of hGHR. The disappearance of hGH-induced hGHR was markedly inhibited by reagents that raise the internal pH of acidic organella and partially by protease inhibitors. These results suggest that hGH stimulation results in degradation of internalized hGHRs, where proteases in acidic compartments such as lysosomes may be involved. The relationship between the hGH concentration and the number of residual cell surface hGHRs 60 min after hGH stimulation yielded a curve with an inverted bell shape showing maximum internalization at 10 nM hGH. A similar relationship was shown in the hGHR degradation. The fact that the ligands in excess gave reduced internalization and degradation supports the idea that dimerization of hGHRs on the cell surface through the bivalent ligand hGH is required for their internalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. These phosphorylations were inhibited by pretreatment with a protein kinase inhibitor, staurosporine. The hGHR internalization, however, was not markedly affected by the inhibitor. In contrast, the staurosporine inhibited the degradation of hGHR in a dose-dependent manner. These results suggest that staurosporine-sensitive phosphorylation is not required for the hGHR internalization, but the phosphorylation is involved in the degradation of hGHR.

Alkaloids↗
PubMed1994

Effect of alpha-linolenic acid on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine release in RBL-2H3 cells.

We examined the effect of alpha-linolenic acid (18:3 (n-3)) pretreatment on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine content and release of RBL-2H3 cells. RBL-2H3 cells grew without reduction in number when incubated with subculture media for 3 d and then placed again in serum-free medium with bovine serum albumin (BSA) and epidermal growth factor (EGF). Cholesterol pullulan (10 micrograms/ml) emulsified alpha-linolenic acid (20 micrograms/ml) was recommended as an additional form serum free medium. We determined the fatty acid composition in all neutral lipids, free fatty acids and all phospholipids in alpha-linolenic acid-treated cells. In all cases the concentration of alpha-linolenic acid and docosahexenoic acid (DHA, 22:6 (n-3)) was increased, while linolenic acid (18:2 (n-6)) was slightly and arachidonic acid (20:4 (n-6)) was markedly decreased. Content of histamine in alpha-linolenic acid-treated cells was remarkably lower than that of untreated cells. Accordingly, net histamine release stimulated by antigen or A23187 was also markedly decreased in the alpha-linolenic acid-treated cells, as was the percent histamine release stimulated by antigen. Results from our in vitro experiment suggest that the anti-allergic effect of alpha-linolenic acid may be caused either by the decrease in histamine content or by inhibition of the release of chemical mediator resulting from changes in the fatty acid composition.

Animals↗
PubMed1994

Preparation of monoclonal antibodies for immunoblotting human growth hormone receptor and growth hormone-binding protein.

Monoclonal anti-peptide antibodies against the extracellular domain of human growth hormone receptor (hGHR) were prepared. Four monoclonal antibodies (mAbs) reacted with an extracellular domain protein produced by genetic engineering. Among them, GHRP2-88 was the most reactive against hGHRs from human IM-9 cells. The lower limit of detection for immunoblotting using this mAb was about 200 pg hGHR. The GHRP2-88 antibody also reacted with deglycosylated hGHRs from tunicamycin-treated IM-9 cells and with the growth hormone-binding protein (GH-BP) in human plasma.

Antibodies, Monoclonal↗
PubMed1994

[A case of lobar atrophy with stereotypic unusual laughing].

We report a case of a Pick's disease (frontal predominant type) with unusual laughing. The patient was a 54-year-old, right handed, female. The patient began to show low activity and inappropriate laughing at about 51 years. She did not speak at all and showed weird laughing with a voice "hi hi hi hi hi hi" frequently at first presentation. When asked some questions, she sometimes answered with a nod. Almost all of her laughing did not match with the situation and seemed to lack a relationship between affective change and observed expressions. The laughing could be elicited by non specific and varied stimuli. She often interrupt laughing voluntarily unlike pathological laughing. Neurological examination revealed visual rooting reflex and forced grasping reflex but there was no signs of pseudobulbar palsy often observed in patients with pathological laughing. Her electromyogram of facial expression during the laughing showed that electric discharge of M. corrugator supercilii was over 4 times as much as that in normal laughing. And it showed also that the duration of her laughing was several seconds as normal laughing. CT and MRI scans demonstrated atrophy in the fronto-temporo-parietal lobe bilaterally (L > R), most prominent in the frontal lobe. SPECT scan showed a significant diminution of blood flow in the atrophic region. An EEG was normal. We consider that the laughing of the present case is not due to an emotional disturbance but rather a disorder of affective expression as pathological laughing.

Dementia↗
PubMed1994

The retinoblastoma binding factor 1 (RBF-1) site in RB gene promoter binds preferentially E4TF1, a member of the Ets transcription factors family.

The tumor suppressor retinoblastoma gene product, pRB, is a well known regulator of G1/S cell cycle progression. Moreover, mutational inactivations within the retinoblastoma gene (RB) are found in many human malignant tumors, and thus, believed to be an essential step in tumor formation. The human RB gene is considered as a housekeeping gene with no characteristic TATA or CAAT elements in its promoter region, but the sequence between 206 and 185 bases upstream of the initiation codon, essential for RB promoter activity, contains putative Sp1 and ATF recognition sites. We have previously reported that point mutations in this region, causing low penetrance retinoblastomas, completely reduced RB promoter activity, and that a nuclear factor, named RBF-1 (retinoblastoma binding factor 1), could specifically bind to this sequence, overlapping Sp1 recognition sequence. We show here, that RBF-1 can recognize a specific DNA sequence, 5'-GGCGGAAGT-3', overlapping the Sp1 and ATF sites and corresponding to the consensus DNA binding site for members of Ets transcription factors family. When RBF-1 site was used for sequence specific DNA affinity purification from erythroleukemia cells, reconstitution assays, immunoblotting analysis and peptide mapping show that the two major co-purified proteins are identical to human E4TF1-60 and -53 proteins. This reveals that E4TF1 can bind to the RBF-1 site of RB gene promoter, which, thus, constitutes a new target for this member of Ets transcription factors family.

Base Sequence↗
PubMed1993

Induction of metallothionein in a human astrocytoma cell line by interleukin-1 and heavy metals.

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti-MT antibody (MT45). IL-1, CdCl2 (50 microM) or ZnCl2 (500 microM) remarkably augmented intracellular MT levels, whereas IL-6 or 10 microM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL-1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 microM) or IL-1 plus ZnCl2 (10 microM). However, this accumulation was not observed when only IL-1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Astrocytoma↗
PubMed1993

Significance of subclinical entrapment of nerves in lead neuropathy.

We examined a left-handed 25-year-old man engaged for 2 years at a vinyl chloride resin factory where he had been exposed to lead stearate, a stabilizer of resin. Neurological examination revealed atrophy of small hand muscles, especially in the left dorsal interossei. Sensation of all modalities was intact. A nerve conduction study showed conduction block at the elbow, indicating possible cubital tunnel syndrome. Following CaEDTA therapy, continued recovery of conduction velocities, amplitude of compound muscle action potential, and diminution of conduction block at elbow were observed within a few months. In lead intoxication, nerves may incur mechanical damage. Subclinical entrapment may thus be an important factor leading to vulnerability of nerves in lead neuropathy.

Action Potentials↗
PubMed1993

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus.

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

Amino Acid Sequence↗
PubMed1993

Molecular and biochemical analyses of combining sites of monoclonal anti-morphine antibodies.

V region nucleotide sequences were determined by mRNA sequencing for 11 monoclonal anti-morphine antibodies with slightly different specificities for morphine-related opiates. The VH region nucleotide sequences of the antibodies MOR8, MOR33, MOR35, MOR44, MOR83, and MOR76 were classified into the VH-5 (7183) family, while the antibodies MOR39, MOR115, MOR131, MOR158 and MOR180 used VH-1 (J558) family genes. MOR39, MOR115 and MOR131 used the V lambda-1 gene for their L chain V region. MOR158 and MOR180 used the Vk-10 gene. MOR8, MOR33, OR35, MOR44, MOR76 and MOR83 used VK-21D. The antibody sets MOR158 and MOR180; MOR39 and MOR131; and MOR8, MOR33, MOR35, MOR44, MOR76 and MOR83 appeared to be somatic mutants derived from the same clones since they showed the same VH/VL usage and V(D)J recombination pattern. The pH-reactivity profiles for these antibodies revealed that the binding of morphine to the antibodies is highly dependent on the pH value of the assay solution, suggesting the importance of the electrostatic interaction between the positive charge of morphine and the negative charges at or near the combining sites. Direct UV-photoaffinity labeling with 3H-morphine was carried out in order to estimate the orientation of morphine in the combining sites. The H chains were preferentially labeled in MOR8, MOR33, MOR35, MOR76, and MOR83, whereas most of the crosslinked hapten was found in the L chains in MOR39, MOR115, MOR131, MOR158 and MOR180. Thus, these 11 antibodies were classified into two types in terms of reactivity in the photoaffinity labeling.

Affinity Labels↗
PubMed1993

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

Adenoviridae↗
PubMed1992

Identification of metallothionein in cultured cells by immunoblotting and immunofluorescence using a new monoclonal antibody.

A hybridoma clone (MT45-5-3) producing an IgG-class monoclonal antibody specific for metallothioneins (MTs) was established. The monoclonal antibody (MT45) cross-reacted with mouse, rat and rabbit Cd(2+)-induced MTs 1 and 2 and Zn(2+)-induced MT 2 as assessed by enzyme-linked immunosorbent assay. When the antibody was used to detect the MTs transferred to nylon membranes after SDS-polyacrylamide gel electrophoresis, the antibody reacted with cultured human cell MTs as well as rat, mouse and rabbit MTs 1 and 2 even after carboxymethylation. The antibody could be used for the indirect immunofluorescence test for Cd(2+)-induced MTs in cultured human and mouse cells.

Animals↗