• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.48-50
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• 2000

The energetics at the succinate:quinone oxidoreductase segment of V. alginolyticus was studied using a fluorescence quenching technique with inside-out membrane vesicles. A transient generation of the membrane potential (inside-positive) and ${\Delta}pH$ (inside-acidic) occurred in the presence of KCN and succinate when ubiquinone-1 (Q1) was added. The membrane potential (\Delta\psi$) generated by the succinate; quinone oxidoreductase segment was completely collapsed by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the membrane permeable anion $SCN^{-}$, whereas the ${\Delta}pH$ was completely collapsed by CCCP and $(NH_4)_2SO_4$. From these results, it was concluded that the succinate: quinone oxidoreductase segment as well as quinol oxidase [1] in the respiratory chain of V. alginolyticus generated $H^{+}$ electrochemical potential.

• Proceedings of the PSK Conference
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• 2002.04a
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• pp.108.2-109
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• 2002

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.55-66
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• 1990

In both resting and opsonized zymosan activated neutrophils, ATP stimulated superoxide generation, whereas adenosine inhibited it slightly. The superoxide generation in activated neutrophils to ATP was greater than that of resting neutrophils. In $Ca^{++}$ free medium, inhibitory effect of adenosine on superoxide generation was detectable, whereas ATP did not have any effect. The stimulatory effect of ATP on superoxide generation was inhibited by adenosine in a dose dependent manner. Neither ATP nor adenosine had any effect on NADPH oxidase acitivity. Effects of ATP or adenosine on superoxide generation were more prominent than that by other triphosphate nucleotides or nucleosides. ATP and ADP further stimulated $Ca^{++}$ uptake and increased cytosolic free $Ca^{++}$ level in neutrophils activated by opsonized zymosan, but adenosine inhibited a $Ca^{++}$ mobilization. Verapamil effectively and tetrodotoxin slightly inhibited an increase of cytosolic free $Ca^{++}$ level induced by ATP. Inhibitory effect of either verapamil or tetrodotoxin on superoxide generation in the ATP plus opsonized zymosan-activated neutrophils was greater than in the cells activated by opsonized zymosan alone. Tetraethylammonium chloride had no apparent effect on superoxide generation. CCCP, 2,4-dinitrophenol, diphenylhydantoin and procaine all inhibited superoxide generation in neutrophils activated by opsonized zymosan. Among these, CCCP only inhibited a stimulatory effect of ATP. ATP further stimulated a loss of sulfhydryl groups in activated neutrophils, whereas adenosine had no effect on it. These results suggest that functional responses of neutrophils may be regulated at least partly by purines. ATP and adenosine may further after functional responses of activated neutrophils through their effect on $Ca^{++}$ uptake, membrane phosphorylation and oxidation of soluble sulfhydryl groups.

• Molecules and Cells
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• v.19 no.2
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• pp.232-238
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• 2005

Corneal endothelial cells play an important role in maintaining the transparency and ionic balance of the cornea. Inflammation causes many changes in the intracellular and extracellular environment of the cornea, including acidosis. We examined the relationship between changes in extracellular pH and expression of cyclooxygenase-2 in cultured bovine corneal endothelial cells. When extracellular pH ($[pH]_o$) was reduced to pH 6.4, COX-2 mRNA increased, with a peak at 2 h. This was blocked by pretreatment with actinomycin D and incubation with spermine NONOate (SPER/NO, a nitric oxide donor). Exposure to the $H^+$ ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), also raised COX-2 mRNA levels. CCCP-induced COX-2 mRNA expression was also reduced by SPER/NO. These results were confirmed immuno-cytochemically. These data demonstrate that COX-2 expression is stimulated by the lowering of extracellular pH that could result from bacterial infection, and that this is countered by over-production of nitric oxide, which could also result from bacterial infection.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.103-106
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• 2000

Bacillus cereus KCTC 3674 excretes at least two kinds of extracellular proteases into the growth medium. Two major bands of the protease activity with molecular weights of approximately 100 and 38 kDa were obtained after gelatin-SDS-PAGE. The protease with a molecular weight of 38kDa was identified as an extracellular neutral (metallo-) protease. The neutral protease was quite thermostabile but labile to alkaline pH. On the contrary, the 100-kDa protease was thermolabile but stable to alkaline pH. The production of 38-kDa neutral protease was strongly affected by temperature, oxygen, carbonylcyanied m-chlorophenylhydrazone(CCCP) that was defined as a protonophofre, and cerulenin which inhibited lipid synthesis and caused changes in the membrane composition. On the other hand, the production of the 100-kDa protease was strongly affected by only temperature and cerulenin. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing proteases of Bacillus licheniformis, had no effect, whatsoever, on the production of extracellular proteases in B.cereus KCTC 3674.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.341-346
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• 2005

The mechanism underlying oxidant-induced intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular $Ca^{2+}$, the application of DTBNP ($20{\mu}M$), a membrane-permeable oxidant, caused an increase in $[Ca^{2+}]_i$, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The $[Ca^{2+}]_i$ increase induced by DTBNP was also observed in an extracellular $Ca^{2+}$-free/2 mM EGTA solution, indicating the release of $Ca^{2+}$ from intracellular store(s). After endoplasmic reticulum was depleted by an $IP_3$-generating agonist, ATP ($30{\mu}M$) or an ER $Ca^{2+}$ pump inhibitor, thapsigargin ($1{\mu}M$), DTBNP-stressed BAECs showed an increase of $[Ca^{2+}]_i$ in $Ca^{2+}$-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were $0.42{\pm}0.15$ and $0.49{\pm}0.07$, respectively (n=7), which are significantly reduced, compared to the control value of $0.72{\pm}0.07$ in a $Ca^{2+}$-free/2 mM EGTA solution. After the protonophore CCCP ($10{\mu}M$) challenge to release mitochondrial $Ca^{2+}$, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was $0.46{\pm}0.09$ (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced $[Ca^{2+}]_i$ increase. The above results together indicate that the increase of $[Ca^{2+}]_i$ by DTBNP resulted from the release of $Ca^{2+}$ from both endoplasmic reticulum and mitochondria.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1080-1086
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• 2005

Membranes prepared from Vibrio natriegens oxidized both NADH and deamino-NADH as substrates. The maximum activity of the membrane-bound NADH oxidase was obtained at about pH 8.5 in the presence of 0.2 M NaCl, whereas that of the NADH:ubiquinone oxidoreductase was obtained at about pH 7.5 in the presence of 0.2 M NaCl. Electron transfer from NADH or deamino-NADH to ubiquinone-l or oxygen generated a considerable membrane potential (${\Delta}{\psi}$), which occurred even in the presence of $20{\mu}M$ carbonylcyanide m-chlorophenylhydrazone (CCCP). However, the ${\Delta}{\psi}$ was completely collapsed by the combined addition of $10{\mu}M$ CCCP and $20{\mu}M$ monensin. On the other hand, the activity of the NADH oxidase and the ${\Delta}{\psi}$ generated by the NADH oxidase system were inhibited by about $90\%$ with $10{\mu}M$ HQNO, whereas the activity of the NADH:ubiquinone oxidoreductase and the ${\Delta}{\psi}$ generated at the NADH:ubiquinone oxidoreductase segment were inhibited by about $60\%$. Interestingly, the activity of the NADH:ubiquinone oxidoreductase and the ${\Delta}{\psi}$ generated at the NADH:ubiquinone oxidoreductase segment were resistant to the respiratory chain inhibitors such as rotenone, capsaicin, and $AgNO_3$, and the activity of the NADH oxidase and the ${\Delta}{\psi}$ generated by the NADH oxidase system were very sensitive only to $AgNO_3$. It was concluded, therefore, that V. natriegens cells possess a $AgNO_3$-resistant respiratory $Na^+$ pump that is different from the $AgNO_3$-sensitive respiratory $Na^+$ pump of a marine bacterium, Vibrio alginolyticus.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.982-995
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• 2020

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.391-396
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• 1996

The Gram-negative marine bacterium Marinomonas vaga, which requires 0.5 M NaCl concentration for optimal growth, is slightly halophilic. The growth of M vaga was highly resistant to the proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) under alkaline pH conditions (pH 8.5) but very sensitive to CCCP under acidic pH conditions (pH 6.5). These results suggest that the respiratory chain-linked NADH oxidase system of M. vaga may lead to generation of a $Na^{+}$ electrochemical gradient. In order to examine the existence of $Na^{+}$-stimulated NADH oxidase in M. vaga, membrane fractions were prepared by the osmotic lysis method. The membrane-bound NADH oxidase oxidized both NADH and deamino-NADH as substrates and required $Na^{+}$ for maximum activity. The maximum activity of NADH oxidase was obtained at about pH 8.5 in the presence of 0.2 M NaCl. The site of $Na^{+}$-dependent activation in the NADH oxidase system was at the NADH:quinone oxidoreductase segment. The NADH oxidase and NADH:quinone oxidoreductase were very sensitive to the respiratory chain inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) in the presence of 0.2 M NaCl but highly resistant to another respiratory inhibitor, rotenone. Based on these findings, we conclude that M. vaga possesses the $Na^{+}$-dependent NADH:quinone oxidoreductase that may function as an electrogenic $Na^{+}$ pump.

• Molecules and Cells
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• v.40 no.11
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• pp.864-870
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• 2017

The uncoupling protein 4 (ucp-4) gene is involved in age-dependent neurodegeneration in C. elegans. Therefore, we aimed to investigate the mechanism underlying the association between mitochondrial uncoupling and neurodegeneration by examining the effects of uncoupling agents and ucp-4 overexpression in C. elegans. Treatment with either DNP or CCCP improved neuronal defects in wild type during aging. Uncoupling agents also restored neuronal phenotypes of ucp-4 mutants to those exhibited by wild type, while ucp-4 overexpression attenuated the severity of age-dependent neurodegeneration. Neuronal improvements were further associated with reductions in mitochondrial membrane potentials. However, these age-dependent neuroprotective effects were limited in mitophagy-deficient mutant, pink-1, background. These results suggest that membrane uncoupling can attenuate age-dependent neurodegeneration by stimulating mitophagy.