Biomedical subjects

J Sawada

Publications and source records attributed to J Sawada.

At least 37 records · Page 2Linked to original sources

PubMed2000

Effect of Ca(2+) ATPase inhibitors on MCP-1 release from bone marrow-derived mast cells and the involvement of p38 MAP kinase activation.

The effect of two Ca(2+) ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), on the release of MCP-1 from bone marrow-derived mast cells (BMMCs) were investigated. CPA and DTBHQ increased the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and induced MCP-1 release in a dose-dependent manner. These Ca(2+) ATPase inhibitors induced MCP-1 release in the absence of phorbol ester, in contrast to their induction of TNF-alpha. MCP-1 release reached a maximum at 6-9 h. It was inhibited by treatment with actinomycin D, the immunosuppressant cyclosporin A, and the cytosolic Ca(2+) chelator BAPTA-AM. Furthermore, RT-PCR showed a time-dependent increase of MCP-1 mRNA. Thus MCP-1 release seems to depend on Ca(2+)-dependent transcriptional activation. MCP-1 release was dose-dependently inhibited by the p38 MAP kinase inhibitor SB202190, but not by the p44/42 MAP kinase inhibitor PD98059. Therefore, transcriptional activation of MCP-1 production and its release seem to be dependent on the nuclear factor of activated T cells and p38 MAP kinase activation. This is the first report to show the regulation of MCP-1 production in BMMCs.

Animals↗

PubMed1999

Synergistic transcriptional activation by hGABP and select members of the activation transcription factor/cAMP response element-binding protein family.

The Ets-related DNA-binding protein human GA-binding protein (hGABP) alpha interacts with the four ankyrin-type repeats of hGABPbeta to form an hGABP tetrameric complex that stimulates transcription through the adenovirus early 4 (E4) promoter. Using co-transfection assays, this study demonstrated that the hGABP complex mediated efficient activation of transcription from E4 promoter synergistically with activating transcription factor (ATF) 1 or cAMP response element-binding protein (CREB), but not ATF2/CRE-BP1. This synergy also partially occurred when hGABPalpha was used alone in place of the combination of hGABPalpha and hGABPbeta. hGABP activated an artificial promoter containing only ATF/CREB-binding sites under coexistence of ATF1 or CREB. Consistent with these results, physical interactions of hGABPalpha with ATF1 or CREB were observed in vitro. Functional domain analyses of the physical interactions revealed that the amino-terminal region of hGABPalpha bound to the DNA-binding domain of ATF1, which resulted in the formation of ternary complexes composed of ATF1, hGABPalpha, and hGABPbeta. In contrast to hGABPalpha, hGABPbeta did not significantly interact with ATF1 and CREB. Taken together, these results indicate that hGABP functionally interacts with selective members of the ATF/CREB family, and also suggest that synergy results from multiple interactions which mediate stabilization of large complexes within the regulatory elements of the promoter region, including DNA-binding and non-DNA-binding factors.

Activating Transcription Factor 1↗

PubMed1999

Localization of opioid-binding cell adhesion molecule (OBCAM) in adult rat brain.

We investigated the tissue distribution and brain localization of opioid-binding cell adhesion molecule (OBCAM) in the adult rats by immunoblotting and immunohistochemistry using a monoclonal anti-OBCAM peptide antibody that is specific for OBCAM. OBCAM was preferentially expressed in the central nervous system (CNS) and at a very low level in the spleen. Within the brain, OBCAM was distributed in almost all the gray matter, but little or no immunoreactive OBCAM was found in the white matter. Morphologically, the distribution pattern of OBCAM immunoreactivity was very similar to that of synaptophysin, suggesting a role in the synaptic machinery.

Animals↗

PubMed1999

Role of ecto-kinase in phorbol ester-enhanced growth hormone-binding protein release from human IM-9 cells.

Previously we reported that a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), increased the release of human growth hormone-binding protein (hGH-BP) in IM-9 cells, and that this phorbol ester-enhanced release was mediated by protein kinase Ca (PKCalpha). In the present study, the mechanisms of the phorbol ester-enhanced hGH-BP release were further investigated. Treatment of IM-9 cells with PDBu did not increase hGH-BPs (55-60 kDa) in the intracellular soluble fraction. When the cells were treated with trypsin to remove human growth hormone receptors (hGHRs) on the cell surface after stimulation, no hGH-BPs were detected in the culture supernatants, nor did treatment with bafilomycin A1 or chloroquine affect the PDBu-enhanced hGH-BP release. These results suggest that hGH-BPs released by PDBu stimulation are derived from cell surface hGHRs and not generated within the cells. Protein kinase inhibitors with broad specificities, K-252a and K-252b, inhibited the PDBu-enhanced release with almost the same dose-dependency, although only a trace amount of K-252b was incorporated into IM-9 cells than K-252a, suggesting that K-252b probably inhibits an ecto-kinase extracellularly. PDBu actually enhanced the phosphorylation of several extracellular proteins, and this enhanced phosphorylation was completely inhibited by K-252b treatment. Moreover, the PKCalpha-specific inhibitor bisindolylmaleimide III which inhibits PDBu enhanced hGH-BP release inhibited the PDBu-enhanced phosphorylation of extracellular proteins. On the other hand, the impermeable PKC inhibitor PKC inhibitor peptide 19-31 did not inhibit PDBu-enhanced release, suggesting that the target PKCalpha for PDBu is not present on the extracellular surface. Taken together, these results suggest that, in addition to intracellular PKCalpha, activation of an undefined ecto-kinase may also be involved in the PDBu-enhanced hGH-BP release.

Carbazoles↗

PubMed1999

Casein kinase II-like ectokinase activity on RBL-2H3 cells.

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.

Animals↗

PubMed1999

IgE hyperproduction through enhanced tyrosine phosphorylation of Janus kinase 3 in NC/Nga mice, a model for human atopic dermatitis.

IgE hyperproduction frequently observed in patients with atopic dermatitis (AD) may greatly contribute to the pathogenesis of AD, but its mechanisms are still unclear. NC/Nga mice raised in nonsterile circumstances spontaneously suffered from AD-like skin lesions with elevation of plasma IgE levels. We investigated mechanisms of the IgE hyperproduction in NC/Nga mice. Splenic T cells from SPF NC/Nga mice had a level of CD40 ligand (CD40L) expression comparable to that of BALB/c mice. Although there was no difference in the expression of CD40 on B cells between NC/Nga and BALB/c mice, B cells of NC/Nga mice produced much more IgE in the presence of soluble CD40L and IL-4. The stimulation with CD40L and/or IL-4 resulted in tyrosine phosphorylation of Janus kinase 3 (JAK3) in B cells, which was more strongly inducible in NC/Nga mice than in BALB/c mice. In B cells isolated from PBMC of AD patients with high serum IgE levels, JAK3 was constitutively phosphorylated at the tyrosine residue, and its phosphorylation was enhanced by the treatment with CD40L and/or IL-4 as was that in splenic B cells of NC/Nga mice with dermatitis and high IgE levels. Thus, it is suggested that constitutive and enhanced JAK3 phosphorylation in B cells highly sensitive to CD40L and IL-4 may be attributable to IgE hyperproduction in NC/Nga mice and patients with AD.

Aluminum Hydroxide↗

PubMed1999

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.

Amino Acid Sequence↗

PubMed1999

The effect of feeding carrots on immunoglobulin E production and anaphylactic response in mice.

Carrot juice was administered orally to BALB/c mice immunized intraperitoneally with dinitrophenylated (DNP)-OVA for about 1 month. The titers of DNP-specific IgE, DNP-specific IgG, and the levels of total IgE in mouse sera were determined. The DNP-specific IgE production by mice fed carrot juice was significantly inhibited. On the other hand, the DNP-specific IgG production and the level of total IgE in mice fed carrot juice were not significantly different from those in control mice. We also examined the effect of feeding carrots on immediate-type hypersensitivity. One hour after antigen stimulation, the ears of mice fed carrots swelled less than those of control mice. Furthermore, the rise in serum histamine in the mice fed carrots under active systemic anaphylaxis was lower than in controls. We then examined the pattern of cytokine production by spleen cells from mice followed by restimulation with DNP-OVA in vitro. The spleen cells from the mice fed carrots produced more interferon-gamma than those from the control group. In contrast, the spleen cells from the mice fed carrots produced less interleukin-4 than those from the control group. Furthermore, the interleukin-12 production of the spleen cells from mice fed carrots was also higher than that of the control group. These findings suggest that feeding carrots improves the helper T cell (Th)1/Th2 balance, inhibiting specific IgE production and antigen-induced anaphylactic response.

Anaphylaxis↗

PubMed1999

Characterization of an antagonist monoclonal antibody, GHBP116, specific for human growth hormone receptors.

To obtain an antagonist antibody against human growth hormone receptors (hGHRs), we prepared monoclonal antibodies against the recombinant hGHR extracellular domain. One of the clones, GHBP116, exhibited binding activity to intact human IM-9 cells and effectively immunoprecipitated the receptors in cell lysate. GHBP116 competitively inhibited 125I-human growth hormone (hGH) binding to the cells. The antagonist activity of GHBP116 was assessed in terms of ligand-induced receptor internalization, degradation, and phosphorylation of signal transducer and activator of transcription (STAT) 5. The antibody alone did not cause internalization or degradation of hGHRs, but a 1:25000 dilution of ascitic fluid almost completely inhibited ligand (1 nM hGH)-induced internalization and degradation of surface hGHRs. Moreover, GHBP116 alone did not stimulate the phosphorylation of STAT5, used as an indicator of Janus kinase (JAK)-STAT signaling, but almost completely inhibited hGH-induced phosphorylation of STAT5. These results suggest that GHBP116 acts as a specific antagonist of hGH.

Animals↗

PubMed1998

Activation of protein kinase C alpha enhances human growth hormone-binding protein release.

The effect of phorbol ester on human growth hormone-binding protein (hGH-BP) release was investigated. The hGH-BP release from human IM-9 cells measured by immunoblotting was dose-dependently enhanced by a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and reached plateau at 100 nM. The increased hGH-BP release was shown after 10 min incubation with PDBu and reached a plateau at 60 min after stimulation. Similarly, a diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, enhanced hGH-BP release. The enhancement was not inhibited by cycloheximide pretreatment, suggesting that the enhanced hGH-BP release does not require de novo protein synthesis. The PDBu-enhanced hGH-BP release was strongly inhibited by extracellular EDTA, and was dose-dependently inhibited by protein kinase C (PKC)-specific inhibitor, Ro 31-8220. These results suggest that activation of PKC mediates the PDBu-enhanced hGH-BP release. Of the 11 known PKC isoforms in human cells, PKCalpha, delta, mu and iota were detected in IM-9 cells by immunoblotting. Of these isoforms, PKCalpha, delta and mu were present in the membrane fraction, which is a known activation marker of PKC. Furthermore, when several PKC-specific inhibitors (Gö 6976, GF 109203X or bisindolylmaleimide III) with different specificities for each isoform were used, there was a good correlation between inhibition of the enhancement of hGH-BP release and inhibition of the phosphorylation of PKC isoforms, another activation marker of PKC, in PKCalpha but not in PKCdelta and mu. These results suggest that activation of PKCalpha is involved in PDBu-enhanced hGH-BP release.

Carrier Proteins↗

PubMed1998

Functional interactions of transcription factor human GA-binding protein subunits.

The transcription factor human GA-binding protein (hGABP) is composed of two subunits, the Ets-related hGABPalpha, which binds to a specific DNA sequence, and either one of two hGABPalpha-associated subunits, hGABPbeta or hGABPgamma. The DNA-binding protein hGABPalpha cannot affect transcription by itself, but can modify hGABP-dependent transcription in vitro and in vivo in the presence of its associated subunits. In this study, co-transfection assays showed that the ratio of hGABPbeta to hGABPgamma affected transcription from a promoter containing hGABP binding sites. Biochemical analysis showed that they bind to hGABPalpha competitively, indicating that the ratio of hGABPbeta to hGABPgamma is important for hGABP complex formation. Kinetic analysis of the protein-protein interaction using the surface plasmon resonance system showed that hGABPalpha binds to hGABPbeta or hGABPgamma with similar equilibrium constants. Kinetic analysis of the DNA-hGABP interaction showed that the binding of hGABPgamma to hGABPalpha stabilized the interaction of hGABPalpha with its DNA binding site. In addition, the kinetic analysis revealed that this was due to a slower dissociation of the protein complex from the DNA. These results suggest that hGABPalpha-associated subunits influence the DNA binding stability of hGABPalpha and regulate hGABP-mediated transcription by competing with each other.

Base Sequence↗

PubMed1998

Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.

Homologous passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) to ovalbumin and DNP-hapten were studied in the ears of female BALB/c mice by means of assessing Evans blue dye leakage. For the quantitative evaluation of PCA and ACA, a hand-held spectrophotometer and the conventional colorimetric method were used to detect the amount of extravasated dye. The value of deltaE*ab (a numerical expression of color) obtained with the hand-held spectrophotometer and the amount of extravasated dye showed a good correlation. In the mouse ear, the sensitivity of PCA reaction was comparable to that of PCA in the rat, and deltaE*ab in the PCA and ACA reactions correlated well with the dilutions of sera and of the antigen, respectively. Thus, using a hand-held spectrophotometer is a simple, quantitative and sensitive method for ascertaining the extent of immediate-type hypersensitivity in the mouse.

Anaphylaxis↗

PubMed1998

Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice.

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

Animals↗

PubMed1998

Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-alpha production in RBL-2H3 cells.

OBJECTIVE AND DESIGN: To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells. MATERIAL: Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used. TREATMENT: Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4). RESULTS: All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01). CONCLUSIONS: These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.

Animals↗

PubMed1998

Production of antiserum for sensitive enzyme-linked immunosorbent assay of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid by chemiluminescence.

To obtain a specific antiserum for use in enzyme-linked immunosorbent assay (ELISA) of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), we prepared a hapten-carrier conjugate in which the CMPF hapten was linked to a carrier protein through the 5-(1-hydrazopropyl) group. The antisera raised against this antigen in guinea pigs had excellent specificity for CMPF, showing little cross-reactivity with closely related compounds and no significant cross-reactivities with other furan compounds. The results indicated that a specific antiserum to CMPF could be produced by an antigen whose CMPF moiety is linked to a carrier protein through a position remote from the inherent functional groups. A standard curve of CMPF by ELISA using a chemiluminescence system showed a high sensitivity and a linearity in the range of 5-100 ng/mL.

Animals↗

PubMed1998

Ca2+-ATPase inhibitor induces IL-4 and MCP-1 production in RBL-2H3 cells.

The effects of a Ca2(+)-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-alpha required both a Ca2(+)-ATPase inhibitor and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the Ca2(+)-ATPase inhibitors induced IL-4 and MCP-1 production without TPA. The release of IL-4 and MCP-1 reached a maximum at 9 and 6 h, respectively. IL-4 and MCP-I release was inhibited by treatment with the immunosuppressant FK-506 and actinomycin D. Therefore, in our system IL-4 and MCP-1 release involves Ca2(+)-dependent and FK-506-sensitive signaling pathways. This is the first report about Th-2 type cytokine and chemokine production in RBL-2H3 cells.

Animals↗

PubMed1998

Changes in the specificity of antibodies against steroid antigens by introduction of mutations into complementarity-determining regions of the V(H) domain.

An attempt was made to change the specificity of antibodies (Abs) by introduction of mutations. A monoclonal Ab specific for 17alpha-hydroxyprogesterone (17-OHP) was used as the starting Ab. On the basis of a model that was generated by a computer-driven model-building system, we constructed a phage-display library of Abs in which 16 residues were mutated in three complementarity-determining regions of the heavy chain that appeared to form the steroid-binding pocket. We screened the library with 17-OHP and cortisol that had been conjugated with bovine serum albumin, and we isolated many clones that had retained 17-OHP-binding ability as well as clones with the newly developed ability to bind cortisol in addition to 17-OHP. We compared the amino acid sequences between 17-OHP-specific and cortisol-binding Abs, and then constructed several additional Abs. Our results indicated that a change in specificity could be achieved by changing only a single, critical amino acid residue. Models of the 17-OHP-and cortisol-binding pockets formed by the mutated Abs could explain these observations.

17-alpha-Hydroxyprogesterone↗

PubMed1998

Soluble factors from rat basophilic leukemia (RBL-2H3) cells stimulated cooperatively the neurite outgrowth of PC12 cells.

Culture media from rat basophilic leukemia cells (RBL-2H3) induced the neurite outgrowth of rat pheochromocytoma PC12 cells, a model system for neuronal differentiation. The extension of the neurite outgrowth was dependent on the culture time of RBL-2H3 cells in the DMEM medium. The DMEM medium conditioned by RBL-2H3 cells for 48 h induced neurite outgrowth of PC12 cells significantly. The neurite extension was much higher than that by medium containing 1 ng/ml nerve growth factor (NGF) but was rather lower than that by medium containing 10 or 50 ng/ml NGF. The neurite extension by 50 ng/ml NGF was completely suppressed by excess anti-NGF antibody (1-1.5 microg/ml), while the extension by culture medium conditioned by RBL-2H3 cells for 48 h was not completely suppressed in the presence of the same amount of anti-NGF antibody. The neurite extension by the culture medium of RBL-2H3 cells was also suppressed by anti-interleukin (IL)-6 antibody (1 microg/ml), although IL-6 itself (20 units) could scarcely induce the neurite outgrowth of PC12 cells. This suggests that IL-6 in the culture medium of RBL-2H3 cells could be effective in inducing the neurite extension in cooperation with NGF. In the presence of an excess of both anti-NGF and anti-IL-6 antibodies, the culture medium of RBL-2H3 cells induced the neurite extension of PC12 cells. This suggests that the action of the various factors from RBL-2H3 cells may be synergistic as far as the neurite outgrowth of PC12 cells is concerned.

Animals↗