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T Sawa

Publications and source records attributed to T Sawa.

At least 73 records · Page 4Linked to original sources
PubMed1998

In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections.

The role of quorum sensing by Pseudomonas aeruginosa in producing cytotoxicity has not been fully investigated. Strains of P. aeruginosa have been characterized as having an invasive or a cytotoxic phenotype (S. M. J. Fleiszig et al., Infect. Immun. 65:579-586, 1997). We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells. To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing. Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals. Nonetheless, only 6294 was cytotoxic. Cytotoxicity induced by 6294 correlated with increased elastase production. These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo. However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios. It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI [N-(3-oxododecanoyl) homoserine lactone] also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly. In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells.

4-Butyrolactone↗
PubMed1998

Diperamycin, a new antimicrobial antibiotic produced by Streptomyces griseoaurantiacus MK393-AF2. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

Antibacterial antibiotics, diperamycin (1) was produced in the culture broth of Streptomyces griseoaurantiacus MK393-AF2. Various spectroscopic analyses of 1 suggested that 1 belonged to a member of cyclic hexadepsipeptide antibiotic. Antibiotic 1 had potent inhibitory activity against various Gram-positive bacteria including Enterococcus seriolicida and methicillin-resistant Staphylococcus aureus.

Animals↗
PubMed1998

Cremimycin, a novel 19-membered macrocyclic lactam antibiotic, from Streptomyces sp.

A novel 19-membered macrocyclic lactam antibiotic, cremimycin, was isolated from the culture broth of an actinomycete strain. The producing organism, designated MJ635-86F5, was identified as a member of Streptomyces. Cremimycin was isolated from the mycelial cake by extraction with CHCl3-MeOH and precipitation with hexane-MeOH. The structure of cremimycin was determined by spectroscopic study. Cremimycin showed broad antimicrobial activities against Gram-positive bacteria including MRSA.

Animals↗
PubMed1998

Effect of a novel antibiotic, heliquinomycin, on DNA helicase and cell growth.

Heliquinomycin, a novel microbial product, was found to inhibit a human DNA helicase enzyme isolated from HeLa S3 cells at concentrations of 5 to 10 micrograms/ml. In contrast, adriamycin, etoposide and cisplatin did not inhibit this enzyme at the concentrations tested. Furthermore, the replication and repair of SV40 chromosome were not affected at heliquinomycin concentration of 50 micrograms/ml. The topoisomerase II and I enzymes were inhibited at 30 micrograms/ml and 100 micrograms/ml of heliquinomycin, respectively. Heliquinomycin inhibited the growth of HeLa S3, KB, LS180, K562 and HL60 human tumor cell lines at IC50 values of 0.96 to 2.8 micrograms/ml. In addition, the growth of adriamycin and cisplatin resistant P388 cell lines were inhibited at similar concentrations. Heliquinomycin inhibited both DNA and RNA synthesis in cell culture but did not inhibit protein synthesis. HeLa S3 cells were arrested at the G2/M phase by heliquinomycin. These studies suggest that heliquinomycin is a selective inhibitor of a cellular DNA helicase and in turn, inhibits growth of tumor cell lines.

Antibiotics, Antineoplastic↗
PubMed1998

Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis.

A diet high in fat and iron is known as a risk factor in cancer epidemiology. However, the details of the molecular mechanism remains to be elucidated. We examined the possible implication of lipid peroxyl radicals generated from fatty acids and heme-iron in DNA damage, and hence in the possibility of colon cancer. F344 female rats were given N-nitroso-N-methylurea six times during a 2-week period and then fed diets containing different amounts of safflower oil and hemoglobin (rich in iron) for 36 weeks; the occurrence of colon cancer was determined by H&E staining. In this animal model, simultaneous feeding of a fat diet and heme-iron produced a significant increase (P < 0.05) in the incidence of colon cancer compared with a diet without hemoglobin. Electron paramagnetic resonance and chemiluminescence studies revealed that oxidized refined vegetable oils, particularly safflower oil, readily generated lipid peroxyl radicals in the presence of various heme compounds, and the peroxyl radicals did effectively cleave DNA. Unpurified native vegetable oils contain a high amount of peroxyl radical scavengers, whereas conventional refining processes seem to reduce the levels of many valuable anti-peroxyl radical compounds abundant in plant seeds. In conclusion, lipid peroxides and heme components generate peroxyl radical species that exert DNA-cleaving activity. A plausible explanation is that lipid peroxyl radicals thus generated, which originated from routine dietary components such as fat and red meat, may contribute, at least in part, to the high incidence of colon cancer.

Animals↗
PubMed1998

[Current topics in the regulation of prostanoids-1. Inducible cyclooxygenase COX-2 and selective blockers].

Cyclooxygenases (COX) are prostaglandin synthases and are the main therapeutic targets for non-steroidal anti-inflammatory drugs. Recently it has been established that apart from the constitutive isoform, an inducible isoform of this enzyme is upregulated after injurious stimuli. The structures of the genes encoding these enzymes and protein structures have been determined, and with the utilization of knockout mice, the physiological properties of each isozyme are being revealed. COX-1 is responsible for the production of physiological levels of prostanoids, whereas COX-2 is upregulated during inflammatory states and produces prostanoids responsible for the generation of fever, pain or other inflammatory responses. Selective COX-2 blockers may be a safe and useful alternative in the treatment of inflammatory disorders, pain, and fever.

Animals↗
PubMed1998

[Current topics in the regulation of prostanoids--2. The interaction with cytokines and nitric oxide].

Cyclooxygenase (COX)-2 is induced by proinflammatory cytokines such as interleukin (IL)-1 beta, cytokines produced from helper T cell subpopulation Th 1, such as interferon-gamma and tumor necrosis factor-beta. Cytokines produced by the T cell such as IL-4, IL-10, and IL-13 down-regulate induction of COX-2. The novel MAP kinase pathway, JNK and/or p 38, are important intracellular signaling pathways for induction of COX-2. The increased production of prostaglandin E2 by upregulation of COX-2 increases IL-6 production. By utilizing a COX-2 blocker, it is possible to decrease IL-6 production via reduction of prostanoid production, thereby attenuating the systemic inflammatory response. Nitric oxide (NO) and prostanoids are also known to interact and regulate each other. It is important to note the interactions between prostanoids and cytokines or other inflammatory mediators such as NO in understanding the mechanism of the anti-inflammatory effects of prostanoid regulation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

IL-10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa is the most frequent Gram-negative pathogen causing nosocomial pneumonia. Four different strains of P. aeruginosa (including three isogenic transposon mutants) were utilized in experiments in mice to characterize the specific patterns of cytokine generation in response to bacterial products and cytotoxicity. Intratracheal instillation of any of the strains led to the up-regulation of IL-1beta, IL-6, and TNF-alpha mRNA. Instillation of the cytotoxic strains (PA103, PA103tox::omega) led to IL-10 mRNA up-regulation in the lungs and increased concentrations of IL-10 in the blood. In contrast, the instillation of the noncytotoxic strains (PA01, PA103exsA::omega) did not lead to an increase in IL-10 mRNA in the lungs or to an increase of IL-10 concentration in blood. IL-10 production appears to be a response to either cellular injury or to specific cytotoxic exoproducts produced by the bacteria. The systemic administration of rIL-10 significantly decreased the lung injury and the mortality in mice who had received the cytotoxic strains. The improvement in survival induced by administration of rIL-10 required the concomitant presence of IFN-gamma, as blockade of IFN-gamma with a neutralizing Ab led to 100% mortality, despite the administration of rIL-10. These results suggest that IL-10 is produced in response to specific bacterial products and that there is a potential role for IL-10 in the treatment of cytotoxic P. aeruginosa pneumonia.

Animals↗
PubMed1997

Functional importance of Shc tyrosine 317 on insulin signaling in Rat1 fibroblasts expressing insulin receptors.

Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity.

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC.

We compared the efficacy of gene transfer in vitro and in vivo using various formulations of DNA-lipid complexes based on the novel cationic lipid EDMPC (1,2-dimyristoylsn-glycero-3-ethylphosphocholine, chloride salt). In vitro studies analyzed delivery of marker genes to four established cell lines, including two of pulmonary origin. The in vivo analysis used intralobar delivery of marker genes and CFTR to mice and rats. We observed a lack of positive correlation between those DNA-EDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo. Intralobar DNA delivery to rodents mediated by EDMPC was efficient. The high level of gene delivery by DNA-EDMPC formulations demonstrates that efficient lipid-mediated gene transfer to the lung is possible.

Animals↗
PubMed1997

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::omega and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::omega. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

ADP Ribose Transferases↗
PubMed1997

Nitric oxide generation from hydroxyurea via copper-catalyzed peroxidation and implications for pharmacological actions of hydroxyurea.

We investigated the generation of nitric oxide (NO) by H2O2-dependent peroxidation of hydroxyurea in the presence of copper-containing proteins such as Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or ceruloplasmin as a catalyst. In the reaction mixture of hydroxyurea, CuZn-SOD, and H2O2, NO generation was identified by measuring the specific electron spin resonance (ESR) signal of 2-phenyl-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). The ESR signal of the NO-hemoglobin adduct was also detected in human red blood cells during copper-catalyzed peroxidation of hydroxyurea. The NO production during peroxidation of hydroxyurea was quantified as NO2- formation, measured by using the Griess assay, the amount of NO2- was dependent on the concentrating of hydroxyurea of the reaction mixture. ESR spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed hydroxy radical (OH) generation in the reaction of H2O2 with either Cu,Zn-SOD or ceruloplasmin. Several OH scavengers, such as ethanol, thiourea, DMPO, and dimethylsulfoxide, and the metalchelating agent diethylenetriaminepentaacetic acid significantly inhibited NO generation from hydroxyurea. This indicates that NO release from hydroxyurea may be mediated by OH derived from the copper-catalyzed Fenton-like reaction. Incubation of hydroxyurea and Cu,Zn-SOD with xanthine oxidase and hypoxanthine in a system forming O2- -->H2O2 also resulted in appreciable NO production. These results suggest that NO production from hydroxyurea catalyzed by copper-containing proteins may be the molecular basis of the pharmacological and antitumor action of hydroxyurea.

Ceruloplasmin↗
PubMed1997

Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S.

Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts. Previous studies indicated that P. aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells. In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P. aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype. In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology. Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy. Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain. In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay. The expression of cytotoxicity requires a functional exsA allele. In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression. Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53). Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT. These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants. In the absence of a functional cytotoxicity pathway (exsA::omega strains), invasion of eukaryotic cells is detectable.

ADP Ribose Transferases↗
PubMed1997

Nitric oxide attenuates lung endothelial injury caused by sublethal hyperoxia in rats.

Inhaled nitric oxide (NO) has been shown to prevent oxidant-induced lung injury in isolated-perfused lung models, whereas NO-derived oxidants may contribute to acute lung injury secondary to hyperoxia. Whether inhaled NO improves or contributes to oxidant-mediated lung injury may depend on the timing of NO administration or how lung injury is assessed. The objective of these studies was to determine whether inhaled NO (20 ppm) was protective or harmful to the different lung barriers when it was administered with 95% O2 for 60 h in Sprague-Dawley rats by measuring fluid transport and permeability to protein across the lung endothelium and the alveolar epithelium. Inhaled NO significantly attenuated the O2-mediated lung endothelial injury and abolished the increase in the bronchoalveolar lavage fluid content of rTI40, a specific and sensitive marker of alveolar epithelial type I cell injury, that occurs secondary to hyperoxia. In conclusion, inhaled NO administered with high concentrations of O2 may protect the lung endothelium and the alveolar epithelium against O2-mediated injury.

Administration, Inhalation↗
PubMed1997

Mechanism of posturally induced crackles as predictor of latent congestive heart failure.

We investigated the role of changes in pulmonary function in posturally induced crackles (PIC) in 76 patients with various heart diseases. Regional ventilation was evaluated by spirometric gated ventilation scanning using 133Xe in 23 of these patients and its relationship to PIC was analyzed. A change from the sitting to the supine position was associated with a significant decrease in the percent functional residual capacity (FRC, p < 0.01) and significant increases in closing volume (CV), CV/vital capacity (VC) and closing capacity (CC)/FRC (p < 0.01) in the PIC-positive subjects. CV, CV/VC and CC/FRC did not differ significantly between PIC-positive (n = 37) and PIC-negative (n = 39) subjects in the sitting position, but in the supine position, these values were significantly higher in the PIC-positive group than in the PIC-negative group (CV: p < 0.05, CV/VC and CC/FRC: p < 0.01). These results suggest that airway closure was markedly increased in PIC-positive subjects in the supine position compared with PIC-negative subjects. Regional ventilation (V) was assessed in the sitting and the supine position from right lateral images divided into 9 segments from the base to the apex of the lung using spirometric gated ventilation scanning. There was no significant difference in regional ventilation in the sitting position between PIC-negative (n = 11) and PIC-positive (n = 12) subjects; in the supine position, regional ventilation decreased significantly at the base in the PIC-positive group. Findings suggest that PIC at the base of the lungs may be related to airway closure at the base of the lungs in the supine position in PIC-positive subjects.

Adult↗
PubMed1997

Pyralomicins, novel antibiotics from Microtetraspora spiralis. IV. Absolute configuration.

The absolute configurations of pyralomicin la and pyralomicin 2a were determined by X-ray crystallographic analyses of crystalline 7'-O-p-bromobenzoylpyralomicin 1a and 2'-O-p-bromobenzoylpyralomicin 2a derived from pyralomicin 1a and pyralomicin 2a using anomalous scattering of the bromine atom. The absolute configurations of pyralomicins 1b approximately 1d and 2b approximately 2e were suggested to be the same as pyralomicin 1a and pyralomicin 2a, respectively, by comparing the circular dichroism spectra.

Actinomycetales↗