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M Azuma

Publications and source records attributed to M Azuma.

At least 181 records · Page 10Linked to original sources

Effect of taurine on angiotensin II-induced hypertrophy of neonatal rat cardiac cells.

The effect of taurine on angiotensin II-induced hypertrophy of cultured neonatal rat heart cells (myocytes and nonmyocytes) was examined. Angiotensin II (1-100 nM) alone caused an increase in the rate of protein synthesis of myocytes without changing the rate of DNA synthesis and cell number. It mediated increases in DNA synthesis and cell number in nonmyocytes. Furthermore, at the lower concentration of 1 nM, it induced c-fos and c-jun expression in both cultured myocytes and nonmyocytes. Exposure of the cells to taurine (20 mM) in the absence of angiotensin II had no effect on either hyperplastic growth or immediate early response gene expression by the two types of cultured cardiac cells. However, myocytes pretreated for 24 h with 20 mM taurine exhibited reduced responsiveness to angiotensin II (1 nM), resulting in lower levels of angiotensin II-mediated stimulation in protein synthesis, and immediate early response gene expression was attenuated. Similarly, taurine treatment of nonmyocytes reduced the degree of hyperplastic growth (DNA synthesis and cell number) and immediate early response gene expression stimulated by angiotensin II. Finally, taurine partially prevented the increase in intracellular free calcium [Ca2+]i mediated by angiotensin II in cardiac cells. Our results indicate that taurine is an effective inhibitor of angiotensin II action.

Angiotensin II↗

Role of gamma delta TCR+ lymphocytes in the augmented resistance of trehalose 6,6'-dimycolate-treated mice to influenza virus infection.

Trehalose 6,6'-dimycolate (TDM), an immunomodulator, potentiates non-specific resistance in mice to influenza virus infection. When mice were injected intravenously with TDM, the striking proliferation of a minority of T-lymphocytes bearing gamma/delta T-cell receptors (gamma delta T-cells) that accumulated in granulomatous lungs was thought to be associated with the maintenance of acquired resistance to lethal influenza virus infection. To clarify the cellular basis of the defence against influenza virus, mice were depleted of gamma delta T-cells, alpha/beta (alpha beta) T-cells, or natural killer (NK) cells by in vivo administration of corresponding antibodies prior to influenza virus infection. The depletion of gamma delta T-cells significantly abrogated the augmented resistance of TDM-treated mice to infection, as did depletion of either alpha beta T-cells or NK cells. To gain insight into the functional ability of gamma delta T-cells, we evaluated the cytotoxic activity of this T-cell subset against a panel of target cell lines that were stably transfected with the influenza virus haemagglutinin (HA) gene from A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) strains. The gamma delta T-cells from TDM-treated mice showed profound cytotoxicity against the target cells expressing HA of either the H1 or H3 subtype, in a non-major histocompatibility complex-restricted manner. Taken together, these results indicate that gamma delta T-cells play a non-specific role, in conjunction with alpha beta T-cells and NK cells, in protecting mice against influenza virus infection, and that the recognition and destruction of HA-expressing target cells by the activated gamma delta T-cells is one of the steps involved in this anti-influenza virus immunosurveillance.

Adjuvants, Immunologic↗

Acyl amino acid derivatives as novel inhibitors of influenza neuraminidase.

We searched our chemical collection to identify non-N-acetylneuraminate (NeuAC) inhibitors of influenza neuraminidase (NA). Of the 62 acyl derivatives tested, several acyl amino acids, but not acyl alkanolamine derivatives, were effective and inhibited the NA activity in a dose-dependent manner. N-3-Hydroxymyristoyl D-cysteine and N-myristoyl-O-caproyl-D-serine were the more potent compounds and inhibited the enzyme in a noncompetitive manner (Ki = 102 and 125 microM, respectively) without respect to the substrate. An important consideration for the choice of inhibitor is the selectivity of the inhibition. These compounds were selective inhibitors of viral NA and effective for any variant enzyme, but the enzymes from V. cholerae and human placenta were insensitive. Accordingly, the acyl amino acid derivatives may be expected to be inhibitors without cellular toxicity and may serve as lead compounds for anti-influenza agents.

Acylation↗

Inhibition of octapeptide N-myristoylation by acyl amino acids and acyl alkanolamines.

Several acyl amino acids and acyl alkanolamines were prepared and screened for their inhibition of octapeptide N-myristoylation and HIV-1 replication in MT-4 cells. Of the 62 acyl derivatives tested, N-myristoyl-O-caproyl-L-serine, N-myristoyl-O-caproyl-D-serine and N-decanoyl-O-myristoyl-L-serine were found to be uncompetitive inhibitors of N-myristoylation, but did not prevent HIV-induced cytopathicity in MT-4 cells. However, other acyl derivatives such as N-3-hydroxymyristoyl ethanolamine, N-3-hydroxymyristoyl-D-serine and N-myristoyl-L-cysteine, which did not inhibit N-myristoylation, suppressed the cytopathicity in the infected cells. The acyl derivatives described here may serve as lead compounds for antiviral agents.

Acylation↗

[Costimulatory molecules in autoimmunity: role of CD28/CTLA4-CD80/CD86].

Optimal T cell activation requires at least two signals. One is provided by the interaction between antigen-specific T cell receptor and antigen-MHC complexes. The other is provided by costimulatory signals. One best characterized costimulation is mediated by CD28/CTLA4-CD80/CD86 (CD28/B7). In the past decade, costimulatory signal mediated by CD28 has been shown to be critical to augment T cell proliferative response and effector function such as cytokine production. Recent intensive analysis of the CD28/B7 pathway have revealed unexpected means in which this pathway may be involved in the maintenance and breakdown of self-tolerance. In vivo studies using antagonists or transgenes of this pathway reveals that inappropriate expression of CD80, ligand for CD28/CTLA4, could induce autoimmunity and blockade of CD28/B7 pathway could ameliolate several autoimmune disease models. Furthermore, CD80 and CD86 plays differential roles for the development of autoimmune disease since helper T cell development is differentially affected by the blockade of either. CD80 or CD86 in certain conditions. More recent studies demonstrated the crucial role of CTLA4 in negatively regulating T cell activation and autoreactivity. Taken together, this pathway play pivotal roles for autoimmunity, and manipulation of this pathway raises the possibility for controlling autoimmune diseases.

Abatacept↗

[Manipulation of costimulatory pathways in autoimmune disease].

The past year has seen significant advances in our understanding of the role of costimulatory pathways in antigen-specific T cell activation and maintenance of self-tolerance. It has been suggested that the absence of costimulators on normal tissue cells could serve to induce self-tolerance and that inappropriate expression of costimulators on antigen-presenting cells (APC) could activate self-reactive T cells, resulting in autoimmunity. CD28 on T cells and CD80 and CD86 on APC are key molecules in the maintenance and breakdown of anergy. CTLA-4Ig fusion protein, that binds to both CD80 and CD86 with high affinity and thereby prevents interaction of CD80/CD86 with CD28/CTLA-4, prevents or ameliorates several autoimmune disease in experimental animal models, supporting an importance of this pathway in the development of autoimmune diseases. However, the studies using specific monoclonal antibodies against CD80 and CD86 have shown different outcomes in individual autoimmune models. This suggests that the actual regulatory mechanisms of this pathway in autoimmunity is much more complex, because of the existence of two receptors (CD28 and CTLA-4) and two ligands (CD80 and CD86) and the opposite function of CD28 and CTLA-4 in T cell activation. Further investigation on physiological function of this pathway in vivo may help for developing rational therapeutic approaches manipulating this pathway.

Abatacept↗

Protective and therapeutic immunity against leukemia induced by irradiated B7-1 (CD80)-transduced leukemic cells.

B7 molecules provide an important costimulatory signal for T cell receptor/CD3-mediated T cell activation via binding to their cognate receptors, CD28 and CTLA-4. We have introduced B7-1 (CD80) into M1 cells, spontaneously-occurred mouse myelocytic leukemic cells and assessed its potential in the induction immunity to leukemia cells. Syngeneic, immunocompetent SL mice receiving polyclonal B7-1-transduced M1 cells showed prolonged survival than control mice. Two independent B7-1-transduced monoclonal sublines, M1-B7-1+ (F20) and M1-B7-1+ (F7), were rejected in 100% an 50% of SL mice, respectively. In vivo depletion of T cell subsets showed that both CD4+ and CD8+ T cells were indispensable for the B7-1-dependent anti-leukemic immunity. Although a single exposure to irradiated monoclonal M1-B7-1+ cells were not fully effective, multiple exposures induced protective immunity against subsequent challenge with M1 cells. Furthermore, hyperimmunization with irradiated monoclonal M1-B7-1+ (F7) cells could partly cure mice previously injected with a lethal number of M1 cells. Although other groups have demonstrated that live, proliferating B7-1-transduced leukemic cells can improve antitumor immunity, this is the first report which shows that irradiated B7-1-transduced myeloid leukemic cells can induce protective and therapeutic immunity against leukemia.

Animals↗

Interferon-gamma differentially regulates CD80 (B7-1) and CD86 (B7-2/B70) expression on human Langerhans cells.

CD80 (B7-1) and CD86 (B7-2/B70) have recently been identified in cultured human Langerhans cells (LCs), although their role and regulatory properties remain unclear. We present our comparison of the expression of the molecules, mRNAs and the function between CD80 and CD86 in human LCs treated by interferon gamma (IFN-gamma). We examined the regulatory properties of CD80 and CD86 expression in human LCs pretreated with IFN-gamma. Flow cytometric analysis indicated that the mean fluorescence intensity of CD86 but not CD80 was enhanced. However, the percentage modulation of both CD80 and CD86 positive cells were significantly up-regulated in a dose-dependent manner, after 48-h culturing with IFN-gamma. The regulatory properties of CD80 and CD86 mRNA expressions in human LC were studied using polymerase chain reaction methods. We found that both CD80 and CD86 mRNA of enriched LCs following IFN-gamma pretreatment for 12 h were higher than those without pretreatment. We have demonstrated that the primary allogeneic mixed epidermal cell-lymphocyte reaction induced by human LCs treated by IFN-gamma increased in a dose-dependent manner. There was a 61.5% inhibition by anti-CD86 monoclonal antibody and a 32.5% inhibition by anti-CD80 monoclonal antibody. These data indicate that the CD80 and CD86 expression of human LCs may be differently regulated by IFN-gamma.

Antigens, CD↗

Functional CD86 (B7-2/B70) is predominantly expressed on Langerhans cells in atopic dermatitis.

Recently, we reported the functional expression of CD86 on cultured human Langerhans cells derived from normal epidermis. In the present study, we investigated the expression and function of co-stimulatory molecules in the pathogenesis of atopic dermatitis. In immunohistochemical analysis, CD80 and/or CD86 were detected on dendritic-shaped cells not only in the epidermis but also in the dermis in the inflammatory lesions of atopic dermatitis (n = 12). CD80 was expressed in only five cases (42%), while CD86 was expressed in all cases (100%). These molecules were not detected in normal control subjects (n = 8). In non-lesional skin of atopic dermatitis (n = 4), CD86 but not CD80 was detected in one case. CD86 was preferentially induced on dendritic-shaped cells in positive patch test sites to Dermatophagoides pteronyssinus or house dust allergen in atopic dermatitis (n = 4). The CD80- or CD86-positive cells were confirmed as Langerhans cells by double immunostaining using anti-CD1a monoclonal antibody. Neither CD86 nor CD80 was detected on keratinocytes. Similar results of the stronger expression of CD86 over that of CD80 were obtained from psoriasis vulgaris (n = 11) and from contact dermatitis (n = 7), although CD86 was expressed only in 57% of the contact dermatitis cases. The percentage of Langerhans cells positive for CD86 was higher than for CD80, i.e. 48% compared with 9%, respectively, in the epidermis of lesional skin of atopic dermatitis (n = 8). The expression rate of these molecules on Langerhans cells increased in the dermis. To investigate the function of co-stimulatory molecules on Langerhans cells in atopic dermatitis, we conducted an inhibition test with antibodies. Anti-CD86 monoclonal antibody almost completely inhibited T-cell proliferation stimulated with crude extract of D. pteronyssinus in the presence of epidermal cells as antigen-presenting cells, whereas anti-CD80 monoclonal antibody produced less of an inhibitory effect. These data indicate that CD86 expressed on Langerhans cells may play an important part in the pathogenesis of atopic dermatitis.

Animals↗

Role of cytokines in the destruction of acinar structure in Sjögren's syndrome salivary glands.

Our aim in the present study was to understand the mechanism whereby specific destruction of acinar but not ductal structure occurs in salivary glands in Sjögren's syndrome (SS). Thus, we examined the effects of cytokines including TNF-alpha and IL-1 beta on the proteolytic activity of cultured normal human salivary gland cell clones, because degradation of the basement membrane by proteolytic enzymes leads to the disruption of acinar or ductal structure in salivary glands. Simian virus 40 (SV40)-immortalized normal human salivary gland cell clones with ductal (NS-SV-DC) or acinar (NS-SV-AC) phenotype were treated either with TNF-alpha or IL-1 beta alone or with a combination of both, and then proteolytic activity was examined. Although cytokine-treated NS-SV-AC demonstrated high matrix metalloproteinase-2 (MMP-2) activity at both protein and mRNA levels, no remarkable increase in MMP-2 activity was detected in NS-SV-DC. Expression of tissue inhibitor of metalloproteinase-2 (TIMP-2), a specific inhibitor of MMP-2, was similarly inhibited by cytokines in these cell clones. Thus, the net balance estimated by MMP-2/TIMP-2 suggested enhanced proteolysis in cytokine-treated NS-SV-AC and, to a much lesser extent, in NS-SV-DC. To examine whether enhanced expression of MMP-2 occurred in acinar cells of SS salivary glands, we carried out an immunohistochemical study using SS salivary gland tissues. This study indicated that acinar cells adjacent to the lymphocytic infiltrate exhibited enhanced expression of MMP-2 compared with those distant from infiltrated lymphocytes or with those in normal salivary glands. By Northern blot analysis, NS-SV-DC and NS-SV-AC expressed receptor mRNA for both cytokines. The signal-dependent activation of the transcription factor NF-kappa B was observed only in NS-SV-AC. I kappa B-alpha, a specific inhibitor of NF-kappa B, was down-regulated by treatment with cytokines in NS-SV-AC. However, the expression of I kappa B-alpha protein was not detected in NS-SV-DC at the basal level. Treatment of NS-SV-AC with calpain inhibitor-I restored the expression of I kappa B-alpha protein in cytokine-treated cells, thus leading to the inhibition of NF-kappa B activation. Reverse transcriptase-PCR analysis confirmed that there was a marked reduction in I kappa B-alpha mRNA expression in NS-SV-DC as compared to that in NS-SV-AC. As such, it seems likely that there is a relationship between the activation of MMP-2 and the activity of NF-kappa B, and that the NF-kappa B/I kappa B-alpha complex is not a signal mediator involved in cytokine-induced suppression of TIMP-2. These observations, therefore, indicate that the divergent response to cytokines in each constituent cell of salivary gland may result in the histopathologic manifestation of SS.

Clone Cells↗

Expression of integrin subunits in normal and malignant human salivary gland cell clones and its regulation by transforming growth factor-beta 1.

In this study, we have examined the expression of integrin subunits in normal and malignant human salivary gland cell clones as well as its regulation by transforming growth factor-beta 1 (TGF-beta 1). By the analysis using immunofluorescence staining, an SV40 immortalized normal human salivary gland duct cell clone (NS-SVDC) with no tumorigenic ability by s.c. implantation into nude mice was identified to express the integrin beta 1, alpha 2, alpha 3 and alpha 6 subunits on the cell surface, while the expression of these subunits, except for beta 1 subunit, was reduced or completely diminished in a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic but not metastatic potential in nude mice and metastatic cell clones derived after in vitro exposure of HSGc to N-methyl-N-nitrosourea. In addition, immunoblot analysis also exhibited the same results as those obtained with immunofluorescence staining. The alpha 1 subunit was not demonstrable in any of the cell clones by both techniques. TGF-beta 1 augmented the expression of the beta 1 subunit in NS-SV-DC, while HSGc and metastatic cell clones demonstrated no changes in the expression of the beta 1 subunit in response to TGF-beta 1. These findings, therefore, suggest that there is an inverse relationship between the malignancy and the expression mode of integrin subunits, especially alpha 2 subunit, in human salivary gland cell clones with varying degrees of malignant potential, and that TGF-beta 1 is a positive regulatory factor in the expression of the beta 1 subunit in normal but not malignant cell clones.

Animals↗

Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes.

We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN-gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.

Acid Phosphatase↗

Effects of eye position on saccades evoked by stimulation of the monkey superior colliculus.

Effects of different initial eye positions on saccades evoked by electrical stimulation of the superior colliculus (SC) were investigated in alert monkeys with the head restrained. The dependence of saccade vector on initial eye position was studied quantitatively by multiple regression analysis. Following stimulation at 240 of 367 sites in the SC, saccade vector was influenced by initial eye position. The magnitude of eye position effects on horizontal saccade component was similar to that for vertical component. These effects were mostly found with stimulation in the caudal SC. Results suggest that eye position signals may control excitability of neurones in the SC.

Animals↗

Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae.

The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.

Amino Acid Sequence↗

Lack of expression of transforming growth factor-beta type II receptor associated with malignant progression in human salivary gland cell clones.

To understand the molecular mechanisms whereby normal human salivary gland cells become malignant and escape growth-inhibitory control by transforming growth factor (TGF)-betaI, we examined the effect of TGF-betaI on the proliferation and expression of TGF-beta receptors in cells and the expression of TGF-beta type II receptor (TbetaR-II) mRNA. An SV40-immortalized normal human salivary gland duct cell clone (NS-SV-DC) with no tumorigenic ability, originally obtained via s.c. implantation into nude mice, was partially resistant to the growth-inhibitory effect of TGF-betaI, while a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic, but not metastatic, potential in nude mice was more resistant to the growth-suppressive effect of TGF-betaI than NS-SV-DC. Metastatic cell clones derived from carcinogen-treated HSGc were completely refractory to the anti-proliferative effect of TGF-betaI. Affinity cross-linking revealed that NS-SV-DC possesses the types I, II (TbetaR-II) and III receptors. However, HSGc and metastatic cell clones lacked expression of detectable levels of the TbetaR-II protein. Moreover, we evaluated TbetaR-II mRNA expression in these cell clones by Northern blot analysis and observed that, although NS-SV-DC expressed a large amount of TbetaR-II mRNA, a small amount of TbetaR-II mRNA was detectable in HSGc. In contrast, no significant bands were detected in metastatic cell clones. Our results, therefore, suggest that one of the possible mechanisms of escape from autocrine or paracrine growth inhibition by TGF-betaI during human salivary gland carcinogenesis involves reduced expression or lack of TbetaR-II.

Adenocarcinoma↗

The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G.

A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit. [14C]N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid. A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening. Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned. In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels. The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to the Vma10p protein, which behaved like a V0 subunit, the M. sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase. We designate this new V1 component subunit G.

Amino Acid Sequence↗