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Biomedical subjects

Tohru Mizushima

Publications and source records attributed to Tohru Mizushima.

At least 19 recordsLinked to original sources
PubMed2007

Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides.

Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.

Activating Transcription Factor 4↗
PubMed2007

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells.

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.

Adenosine Triphosphate↗
PubMed2006

Genetic evidence for a protective role of heat shock factor 1 against irritant-induced gastric lesions.

Gastric lesions result from an imbalance between aggressive and defensive factors. Indirect lines of evidence suggest that heat shock proteins (HSPs) induced by various aggressive factors provide a major protective mechanism. In this study, we compared gastric ulcerogenic response in wild-type mice and in those lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes. The severity of gastric lesions induced by ethanol or hydrochloric acid was worsened in HSF1-null mice. Immunoblotting, real-time reverse transcription-polymerase chain reaction, immunohistochemical analysis, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed that the ethanol administration up-regulated gastric mucosal HSPs, in particular HSP70, in an HSF1-dependent manner, and more apoptotic cells were observed in the gastric mucosa of HSF1-null mice than in wild-type mice. In contrast, other parameters governing the gastric ulcerogenic response, including gastric acid secretion, gastric mucosal blood flow, and prostaglandin E(2) levels, were not significantly affected by the absence of the hsf1 gene. Geranylgeranylacetone (GGA), a clinically used antiulcer drug with HSP-inducing activity, suppressed ethanol-induced gastric lesions in wild-type mice but not in heat shock factor 1 (HSF1)-null mice. The results suggest that the aggravation of irritant-induced gastric lesions in HSF1-null mice is due to their inability to up-regulate HSPs, leading to apoptosis. It is also suggested that the HSP-inducing activity of GGA contributes to the drug's antiulcer activity. This study provides direct genetic evidence that HSPs, after their HSF1-dependent up-regulation, confer gastric protection against the irritant-induced lesions.

Animals↗
PubMed2006

Up-regulation of 150-kDa oxygen-regulated protein by celecoxib in human gastric carcinoma cells.

Induction of apoptosis by nonsteroidal anti-inflammatory drugs, such as celecoxib, is involved in their antitumor activity. An endoplasmic reticulum chaperone, 150-kDa oxygen-regulated protein (ORP150) is essential for the maintenance of cellular viability under hypoxia and is reported to be overexpressed in clinically isolated tumors. We here found that ORP150 was up-regulated by celecoxib in human gastric carcinoma cells. In conjunction with the suppression of tumor growth, orally administered celecoxib up-regulated ORP150 in xenograft tumors. Both the ATF4 and ATF6 pathways were activated by celecoxib, and suppression of ATF4 and ATF6 mRNA expression by small interfering RNA (siRNA) inhibited the celecoxib-dependent up-regulation of ORP150. Celecoxib administration led to an increase in the intracellular concentration of Ca2+, whereas 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca2+ chelator, inhibited the up-regulation of ORP150 and the activation of the ATF4 and ATF6 pathways. These results suggest that these Ca2+-activated pathways are involved in the celecoxib-mediated up-regulation of ORP150. Clones overexpressing ORP150 were less susceptible to celecoxib-induced, but not staurosporine-induced, apoptosis and displayed less up-regulation of C/EBP homologous transcription factor (CHOP), a transcription factor with apoptosis-inducing activity. In contrast, siRNA for ORP150 stimulated apoptosis and expression of CHOP in the presence of celecoxib but not staurosporine. These results suggest that up-regulation of ORP150 in cancer cells inhibits celecoxib-induced apoptosis, thereby decreasing the potential antitumor activity of celecoxib.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2006

Heme oxygenase-1 protects gastric mucosal cells against non-steroidal anti-inflammatory drugs.

Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAIDs) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions.

Animals↗
PubMed2006

Functional gene cloning and characterization of MdeA, a multidrug efflux pump from Staphylococcus aureus.

A DNA fragment conferring drug resistance was cloned from the chromosomal DNA of Staphylococcus aureus N315 using a drug hypersensitive Escherichia coli KAM32 as the host. Although E. coli KAM32 cells were sensitive to many antimicrobial agents, transformed cells harboring a recombinant plasmid carrying the DNA region became resistant to several structurally unrelated antimicrobial agents, such as tetraphenylphosphonium chloride, Hoechst 33342 and norfloxacin. These results suggest that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. We partially determined the nucleotide sequence of the cloned DNA and found the mdeA gene within it. The E. coli cells transformed with the mdeA gene showed efflux activity of Hoechst 33342. On the other hand, S. aureus cells transformed with mdeA showed elevated resistance to doxorubicin, daunorubicin, tetraphenylphosphonium chloride, Hoechst 33342, ethidium bromide and rhodamine 6G. Elevated energy-dependent efflux of ethidium was observed with transformed S. aureus. We found that the mdeA gene was expressed under normal growth conditions in S. aureus N315.

Anti-Bacterial Agents↗
PubMed2005

Geranylgeranylacetone protects membranes against nonsteroidal anti-inflammatory drugs.

Direct gastric mucosal cell damage mediated by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. We recently suggested that this direct cytotoxicity of NSAIDs is caused by their membrane-permeabilization activity. Geranylgeranylacetone (GGA), a clinically used antiulcer drug, can protect gastric mucosa against lesion formation mediated by NSAIDs. However, the mechanism by which this occurs is not fully understood. In this study, we show that GGA acts to stabilize membranes against NSAIDs. GGA suppressed NSAID-induced permeabilization of calcein-loaded liposomes and NSAID-induced stimulation of K(+)-efflux across the cytoplasmic membrane in cells. GGA was effective even when coadministered with NSAIDs and was also able to restore membrane fluidity that had been compromised by NSAIDs. This mechanism seems to play an important role in the antiulcer activity of GGA.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Involvement of intracellular Ca2+ levels in nonsteroidal anti-inflammatory drug-induced apoptosis.

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Animals↗
PubMed2005

Inhibitory activities against topoisomerase I and II by isoaurostatin derivatives and their structure-activity relationships.

Isoaurostatin A (IAS-A) isolated from Thermomonospora alba showed weak inhibition against topoisomerase (topo) I (IC(50)=307microM). To get more strong inhibition, derivatives of IAS-A were prepared and their structure-activity relationships against topo I and II were investigated. The addition of hydroxyl group on aromatic rings increased the activities, 3-(3,4,5-trihydroxybenzylidene)-5-hydroxy-3H-benzofuran-2-one (IAS-9) showed strong inhibition (IC(50)=3microM) against topo I. And also, the increasing of hydroxyl group increased growth inhibition against a variety of cancer cells, and IAS-9 showed most potent inhibition. Unlike camptothecin and etoposide, IAS-9 neither stabilized DNA-topo cleavable complex nor intercalated into DNA, and it inhibited topo I and II noncompetitively. The inhibitory activities also increased by opening of lactone ring in the molecule of IAS-9.

Benzofurans↗
PubMed2005

Induction of claudin-4 by nonsteroidal anti-inflammatory drugs and its contribution to their chemopreventive effect.

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca2+ concentration. Other drugs that increased the intracellular Ca2+ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca2+ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction-related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Low direct cytotoxicity of nabumetone on gastric mucosal cells.

Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa.

Administration, Oral↗
PubMed2005

Low direct cytotoxicity and cytoprotective effects of nitric oxide releasing indomethacin.

Nitric oxide (NO) releasing non-steroidal anti-inflammatory drugs (NSAIDs) have shown a marked reduction of gastrointestinal side effects and we here examined the cytotoxicity of NCX 530 (NO-indomethacin). Under conditions where indomethacin clearly induced both necrosis and apoptosis, NCX 530 induced neither. NCX 530 protected cells from celecoxib-induced necrosis and apoptosis. NCX 530 partially suppressed celecoxib-dependent membrane permeabilization and an inhibitor for guanylate cyclase suppressed the cytoprotective effect of NCX 530 against celecoxib. In vivo, NCX 530 alone produced fewer gastric lesions in rats than did indomethacin. A combination of the oral administration of celecoxib together with the intraperitoneal administration of indomethacin, but not of NCX 530, clearly resulted in the production of gastric lesions. The low direct cytotoxicity and the cytoprotective effect of NCX 530 observed in vitro may also act in vivo, thus ensuring that NCX 530 is safe for use on the gastric mucosa.

Acetates↗
PubMed2005

Physiological roles of three Na+/H+ antiporters in the halophilic bacterium Vibrio parahaemolyticus.

Vibrio parahaemolyticus mutants lacking three Na+/H+ antiporters (NhaA, NhaB, NhaD) were constructed. The DeltanhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The DeltanhaA and DeltanhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na+/H+ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na+/H+ and Li+/H+ antiport activities were observed with everted membrane vesicles of DeltanhaB strains. However, Li+/H+ antiport activities of DeltanhaB strains were two times higher than those of DeltanhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2-heptyl-4-hydroxyquinoline N-oxide), which is a potent inhibitor of the respiratory Na+ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na+/H+ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na+/H+ antiporters cooperate with the respiratory Na+ pump in ionic homeostasis in V. parahaemolyticus.

Lithium↗
PubMed2005

Gene cloning and characterization of four MATE family multidrug efflux pumps from Vibrio cholerae non-O1.

There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V. cholerae non-O1 NCTC4716. Here we cloned four remaining genes for putative MATE family efflux pumps by the PCR method from this microorganism and designated them as vcmB, vcmD, vcmH and vcmN. Each one of the four genes was introduced and expressed in the drug hypersusceptible host Escherichia coli KAM32 cells. We observed elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium bromide and Hoechst 33342 in the transformants. Energydependent efflux of substrate was observed with the transformed cells. We found that efflux activities of VcmB, VcmD and VcmH were Na+-dependent, but that of VcmN was Na+-independent. Thus, all six of the MATE family multidrug efflux pumps of V. cholerae non-O1 have been characterized. We also found that all six genes were expressed in cells of V. cholerae non-O1.

Bacterial Proteins↗
PubMed2005

Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa.

We cloned two operons for putative RND-type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(-opmE) and in another operon mexMN. Introduction of the mexPQ-opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ-OpmE and MexMN-OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.

Bacterial Outer Membrane Proteins↗
PubMed2005

Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus.

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin-resistant Staphylococcus aureus(MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7-trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with beta-lactams. Thus, mechanisms of the synergies seem to be versatile.

Anti-Bacterial Agents↗
PubMed2005

Multidrug resistance in Klebsiella pneumoniae MGH78578 and cloning of genes responsible for the resistance.

Klebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniae MGH78578 by shotgun method using Escherichia coli KAM32, a drug hypersensitive strain, as host. We obtained 43 hybrid plasmids that made host cells resistant to several antimicrobial agents. We classified them into 17 groups based on growth properties in the presence of each one of 9 antimicrobial agents and on restriction patterns of each hybrid plasmid. Analysis of the cloned genes must be very useful for investigation of major parts of multidrug resistance systems including multidrug efflux pumps in K. pneumoniae MGH78578 in which genome sequence is available.

Biological Transport↗