-
[show abstract]
[hide abstract]
ABSTRACT: Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.
Cell Death and Differentiation 10/2004; 11(9):1009-16. · 8.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We cloned a gene which enabled Escherichia coli mutant host cells lacking all of the major Na(+)/H(+) antiporters to grow in the presence of 0.2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372. An Na(+)/H(+) antiport activity was observed with membrane vesicles prepared from E. coli cells possessing the cloned gene, but not with vesicles from the host cells. Lithium ion was also a substrate for the antiporter. We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence. The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58.1 kDa. None of the bacterial Na(+)/H(+) antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynNhaP (NhaS1) of Synechocystis sp., showed significant sequence similarity with the NhaG. However, the NhaP, the SynNhaP, animal NHEs (Na(+)/H(+) exchangers), and some hypothetical Na(+)/H(+) antiporters of several organisms showed significant sequence similarities with the NhaG. Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B. subtilis strain 168. We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B. subtilis ATCC9372 but not with that from strain 168. The missing DNA region (1,774 base pairs) is sandwiched by two identical sequences, TTTTCTT.
Journal of Biochemistry 12/2001; 130(5):711-7. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We found that an extract of Arctostaphylos uva-ursi markedly reduced the MICs of beta-lactam antibiotics, such as oxacillin and cefmetazole, against methicillin-resistant Staphylococcus aureus. We isolated the effective compound and identified it as corilagin. Corilagin reduced the MICs of various beta-lactams by 100- to 2,000-fold but not the MICs of other antimicrobial agents tested. The effect of corilagin and oxacillin was synergistic. Corilagin showed a bactericidal action when added to the growth medium in combination with oxacillin.
Antimicrobial Agents and Chemotherapy 12/2001; 45(11):3198-201. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.
AJP Gastrointestinal and Liver Physiology 11/2001; 281(4):G1092-100. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A chromosomal DNA fragment from Vibrio cholerae non-O1 containing a drug resistance determinant was cloned and sequenced. The deduced amino acid sequence suggested that the determinant gene encodes a multidrug efflux pump. We designated the pump VcmA. Escherichia coli cells transformed with a plasmid carrying the vcmA gene showed increased resistance against norfloxacin, ciprofloxacin, ofloxacin, daunomycin, doxorubicin, streptomycin, kanamycin, ethidium bromide, 4',6-diamidino-2-phenylindole dihydrochloride, Hoechst 33342 and acriflavine. Na+ (or Li+)-dependent efflux of ethidium bromide was detected in transformant cells. Efflux of Na+, elicited by ethidium bromide, was observed from transformant cells. Thus, we concluded that the VcmA is a Na+/drug antiporter.
FEMS Microbiology Letters 10/2001; 203(2):235-9. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We constructed a series of deletion mutants lacking all of the four major mex operons for Mex multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1. The drug specificity of MexAB-OprM, MexXY-OprM and MexCD-OprJ was investigated. Surprisingly, we found that the MexCD-OprJ was an inducible pump, inducers of which were tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G and acriflavine. Fluoroquinolones, chloramphenicol, erythromycin and tetracycline were not inducers although they were substrates of MexCD-OprJ.
FEMS Microbiology Letters 09/2001; 202(1):139-43. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Gastric mucosal cell death due to various gastric stressors can cause several types of gastric diseases, such as gastric ulcers. In this study, we examined cell death following the short-term treatment of guinea pig gastric mucosal cells in primary culture with various gastric stressors. The short-term treatment of cells with ethanol, hydrogen peroxide or hydrochloric acid caused, in a dose-dependent manner, cell death in the absence of apoptotic DNA fragmentation and chromatin condensation. Cells lost membrane integrity following the treatment with each of these gastric stressors, suggesting that necrosis was induced in gastric mucosal cells by short-term treatment of the cells with gastric stressors. Geranylgeranylacetone, an anti-ulcer drug with heat-shock protein inducing properties, protected gastric mucosal cells from the necrotic cell death caused by each of these gastric stressors. Pretreatment of cells with low concentrations of ethanol (3%), which also induced heat-shock protein, made cells resistant to the necrotic cell death caused by the gastric stressors. These results suggest that heat-shock proteins is involved in the cytoprotective effect of geranylgeranylacetone against necrotic cell death.
Biological & Pharmaceutical Bulletin 09/2001; 24(8):887-91. · 1.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIA(Glc) inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIA(Glc). It is concluded that there are multiple interaction sites for IIA(Glc) in these transporters.
Journal of Molecular Microbiology and Biotechnology 08/2001; 3(3):381-4. · 1.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.
Journal of Biochemistry 05/2001; 129(4):607-13. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be reactivated from the ADP-bound form to its ATP-bound form through stimulation of ADP release by acidic phospholipids such as cardiolipin. We previously reported that two potential amphipathic helices (Lys-327 to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved in the functional interaction between DnaA and cardiolipin. In relation to one of these helices (Asp-357 to Val-374), we demonstrated that basic amino acids in the helix, especially Lys-372, are vital for this interaction. In this study, we have identified an amino acid in the second potential amphipathic helix (Lys-327 to Ile-344), which would also appear to be involved in the interaction. We constructed three mutant dnaA genes with a single mutation (dnaAR328E, dnaAR334E, and dnaAR342E) and examined the function of the mutant proteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be more resistant to inhibition of its ATP binding activity by cardiolipin than the wild-type protein. The stimulation of ADP release from DnaAR328E by cardiolipin was also weaker than that observed with the other mutants and the wild-type protein. These results suggest that Arg-328 of DnaA protein is involved in the functional interaction of this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its ATP-binding pocket, which in turn stimulates the release of ADP from the protein.
Journal of Biological Chemistry 04/2001; 276(10):7450-6. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na(+)-driven Na(+)/drug antiporter. (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na(+) but not of K(+). (ii) An artificially imposed, inwardly directed Na(+) gradient elicited ethidium efflux from cells. (iii) The addition of ethidium to cells loaded with Na(+) elicited Na(+) efflux. Thus, NorM is an Na(+)/drug antiporting multidrug efflux pump, the first to be found in the biological world. Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na(+)/drug antiporters are present not only in V. parahaemolyticus but also in a wide range of other organisms.
Journal of Bacteriology 01/2001; 182(23):6694-7. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two new phenolic compounds, glicophenone (1) and glicoisoflavanone (2), were isolated from commercial licorice, and their structures were elucidated on the basis of spectroscopic data. Antibacterial assays of licorice phenolics for Staphylococcus aureus, including four strains of methicillin-resistant S. aureus (MRSA), and also for Escherichia coli K12 and Pseudomonas aeruginosa PAO1, were then examined. Two compounds among them, 8-(gamma,gamma-dimethylallyl)-wighteone (21) and 3'-(gamma,gamma-dimethylallyl)-kievitone (28), showed remarkable antibacterial effects [minimum inhibitory concentrations (MICs), 8 microg/ml on the MRSA strains and methicillin-sensitive S. aureus. Licochalcone A (14), gancaonin G (20), isoangustone A (24), glyasperins C (30) and D (31), glabridin, (32), licoricidin (33), glycycoumarin (34) and licocoumarone (40) showed antibacterial effects on the MRSA strains with MIC values of 16 microg/ml. Effects on the beta-lactam resistance of the MRSA strains were also examined, and licoricidin (33) noticeably decreased the resistance of the MRSA strains against oxacillin, as shown by the reduction in the MICs of oxacillin (lower than 1/128-1/1000 in the presence of 8 microg/ml of 33, and 1/8-1/32 in the presence of 4 microg/ml of 33). Mechanistic study suggested that 33 does not inhibit the formation of penicillin-binding protein 2' (PBP2'), but affects the enzymatic function of PBP2'.
CHEMICAL & PHARMACEUTICAL BULLETIN 10/2000; 48(9):1286-92. · 1.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: One of the major side effects of nonsteroidal antiinflammatory drugs, such as indomethacin, is gastropathy. The purpose of this study was to search for a therapeutic drug to prevent this side effect in vitro. We found that geranylgeranylacetone, a unique antiulcer drug with a heat-shock protein-inducing ability, protected cultured guinea pig gastric mucosal cells from cell damage caused by indomethacin. This cytoprotective effect of geranylgeranylacetone required concentrations of more than 10(-6) M and incubation periods of longer than 2 hr. Pretreatment of cells with an inhibitor of protein synthesis completely abolished the cytoprotective effect of geranylgeranylacetone, suggesting that some proteins induced by the drug are responsible for the cytoprotection. Since pretreatment of cells with low concentrations of ethanol, which also induced the heat-shock proteins, made cells resistant to indomethacin, heat-shock proteins are candidates for the proteins that are involved in the cytoprotective effect of geranylgeranylacetone against indomethacin.
Digestive Diseases and Sciences 09/2000; 45(8):1674-9. · 2.12 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture medium. This suppression was also observed with other high-temperature-sensitive dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations in the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hypothesized that the increase in DNA supercoiling is involved in the suppression of the temperature-sensitivity of the dnaA46 mutant by high concentrations of NaCl. This hypothesis was supported by in vitro and in vivo results as follows. A low DNA replication activity of purified DnaA46 protein at high temperatures was increased in line with an increase in DNA supercoiling of template DNA. The dnaA46 mutant showed higher sensitivity to nalidixic acid, a DNA-relaxing drug, than did the wild-type cells under the conditions of high temperatures and high concentrations of NaCl.
Biochemical Journal 07/2000; 348 Pt 2:375-9. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator.
Journal of Biological Chemistry 07/2000; 275(22):17058-63. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We found that extract from petals of Rosa canina L. (rose red) strikingly reduced the minimum inhibitory concentration of beta-lactams in methicillin-resistant Staphylococcus aureus. We isolated two compounds that reduced the minimum inhibitory concentrations of beta-lactams from the extract, tellimagrandin I and rugosin B. Tellimagrandin I was very effective regarding the reduction of the minimum inhibitory concentration, and rugosin B showed some effect. Tellimagrandin I showed a weak bactericidal action when added together with oxacillin. Judging from the fractional inhibitory concentration index, the effect of tellimagrandin I and oxacillin was synergistic. Tellimagrandin I also significantly reduced the minimum inhibitory concentration of tetracycline in some strains of methicillin-resistant Staphylococcus aureus.
FEMS Microbiology Letters 05/2000; 185(2):135-8. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The purpose of this study was to elucidate the mechanism of spontaneous and rapid cell death of cultured gastric pit cells. Gastric pit cells have a rapid cell turnover rate in vivo. We here show that guinea pig gastric pit cells in culture undergo spontaneous and rapid apoptotic DNA fragmentation, which may represent the rapid cell turnover cycle of gastric pit cells in vivo. This spontaneous apoptotic DNA fragmentation required the presence of fetal calf serum in the culture media. Furthermore, the spontaneous apoptotic DNA fragmentation was prevented by protein synthesis and caspase inhibitors.
Digestive Diseases and Sciences 03/2000; 45(2):291-7. · 2.12 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin. In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T. (1998) J. Biol. Chem. 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(364), and Lys(372)) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo. All mutant proteins maintained activities for DNA replication and ATP binding. A mutant protein in which Lys(372) was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins. Thus, Lys(372) seems to play an important role in the interaction between DnaA protein and acidic phospholipids. Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.
Journal of Biological Chemistry 03/2000; 275(6):4513-8. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have investigated protein-protein interaction between distinct domains of the human CD45 cytoplasmic region using a yeast two-hybrid system. Consequently, we have found that the spacer region between two tandem PTP domains specifically interacts with the membrane-distal PTP domain (D2). This interaction is mediated by a stretch of amino acid residues in the carboxyl-terminal half of the spacer region. Although the membrane proximal region does not directly interact with either of the two PTP domains, it appears to function in stabilizing the interaction between the spacer region and D2. We also demonstrate that the interaction between the spacer region and D2 might take place intramolecularly.
FEBS Letters 03/2000; 468(1):68-72. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We found that epicatechin gallate, a constituent of an extract of tea leaves (green tea) markedly lowered the minimum inhibitory concentration (MIC) of oxacillin and other beta-lactams, but not of other antibacterial agents tested, in strains of methicillin-resistant Staphylococcus aureus. The antibacterial action of epicatechin gallate plus oxacillin was a bactericidal one.
Biological & Pharmaceutical Bulletin 01/2000; 22(12):1388-90. · 1.66 Impact Factor
