• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.197-208
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• 1996

Endosomes lower their internal pH by an ATP-driven proton pump, which is critical to dissociation of many receptor-ligand complexes, the first step in the intracellular sorting of internalized receptors and ligands. Endosomes are known to exhibit n great range of pH values that can vary between 5.0 and 7.0 within a single cell although the factors that regulate endosomal pH remain uncertain. To evaluate the morphological and topological differences of endosomes in the different stages, confocal microscopy was used. The early endosomes labeled with fluorescein isothiocyanate-dextran for 10 min at $37^{\circ}C$ were identifiable at the peripheral and tubule-vesicular endosome compartment. In contrast, the late endosomes formed by 10 min pulse and 20 min trace were located deeper in the cytoplasm and showed more vesicular features than early endosomes. For the purpose of determining whether ATP-dependent acidification was heterogeneous and whether the differences in acidification were attributed to differences in the activity of $Na^{+}-K^{+}$-ATPase and/or $Cl^{-}$ channel, endocytic compartments were fractionated into subpopulation using percoll gradient and measured ATP-dependent acidification. While all fractions exhibited ATP-dependent acidification activity, both the initial rate of acidification and extent of proton translocation were lower in early endosomes and gradually increased in late endosomes. Phosphorylation by PKA and ATP enhanced ATP-dependent acidification in both early and late endosomes, hut there was no difference in the degree of enhancement by phosphorylation between two subpopulations. When ATP-dependent acidification was determined in the presence or absence of vanadate ($Na_{3}VO_{4}$) or ouabain, only early endosomes exhibited the vanadate or ouabain dependent stimulation of acidification activity, suggesting the inhibition of $Na^{+}-K^{+}$-ATPase. Therefore, it seems probable that the inhibition of early endosome acidification by $Na^{+}-K^{+}$-ATPase observed in vitro at least in part plays a physiological role in controlling the acidification of early endosomes in vivo.

• 한국환경과학회지
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• 제16권7호
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• pp.779-784
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• 2007

Dinitrophenol is preventing cells from making energy for growth and it has been suggested that pH may be important in mitigating effects of uncouplers. The effect of pH on toxicity of dinitrophenol at high concentration was investigated, over a pH range of 5.7 to 8.7. DNP inhibition was found to be strongly dependent on mixed liquor pH. The DNP degradation rate was highest in the pH range of 7.0 to 7.8; at pH 6.0 degradation of 0.41 mM dinitrophenol was significantly inhibited; at pH <5.7, dinitrophenol degradation was completely inhibited after approximately 25% of the dinitrophenol was degraded. However no significant effect of pH variation was seen on glucose uptake by the activated sludge mixed culture.

• Applied Biological Chemistry
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• 제50권4호
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• pp.257-261
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• 2007

Pseudomonas tolaasii에 의해서 분비되는 펩티드 독소인 tolaasin은 재배버섯에 세균성 갈반병을 유발한다. Tolaasin의 독성은 적혈구를 파괴하는 용혈활성으로 평가된다. Tolaasin 펩티드는 C-말단부위에 두 개의 amine기를 갖고있어, 이 펩티드의 세포막 결합은 amine기의 전하상태에 따라 다를 수 있다. 이것을 확인하기 위하여 tolaasin을 적정하였을 때, 적정곡선은 pH 7.0에서 9.6 사이에서 적정이 되는 amine기가 있음을 보였다. Tolaasin에 의한 용혈활성의 pH 의존성을 조사하였을 때, 용혈활성은 알칼리 조건에서 증가함을 확인하였다. 따라서, pH 변화에 따른 tolaasin의 막결합 특성을 조사하기 위하여 적혈구를 tolaasin과 사전배양한 후, tolaasin을 포함하지 않은 완충액으로 씻어내고, 사전배양중 적혈구에 결합한 tolaasin의 용혈활성을 측정하였을 때, 활성은 pH 8 이상에서 크게 증가하였다. 이러한 결과는 tolaasin이 전하가 없거나 양전하량이 적은 상태에서 세포막에 잘 결합하여 세포독성이 커진다는 것을 의미한다.

• Bulletin of the Korean Chemical Society
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• 제31권6호
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• pp.1479-1484
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• 2010

Photosynthesis uses light energy to drive the oxidation of water at an oxygen-evolving catalytic site within photosystem II (PSII). Chlorophyll binding by the photosystem II subunit S protein, PsbS, was found to be necessary for energy-dependent quenching (qE), the major energy-dependent component of non-photochemical quenching (NPQ) in Arabidopsis thaliana. It is proposed that PsbS acts as a trigger of the conformational change that leads to the establishment of nonphotochemical quenching. However, the exact structure and function of PsbS in PSII are still unknown. Here, we clone and express the recombinant PsbS gene from Arabidopsis thaliana in E. coli and purify the resulting homogeneous protein. We used various biochemical and biophysical techniques to elucidate PsbS structure and function, including circular dichroism (CD), fluorescence, and DSC. The protein shows optimal stability at $4^{\circ}C$ and pH 7.5. The CD spectra of PsbS show that the conformational changes of the protein were strongly dependent on pH conditions. The CD curve for PsbS at pH 10.5 curve had the deepest negative peak and the peak of PsbS at pH 4.5 was the least negative. The fluorescence emission spectrum of the purified PsbS protein was also measured, and the ${\lambda}_{max}$ was found to be at 328 nm. PsbS revealed some structural changes under varying temperature and oxygen gas condition.

• 상하수도학회지
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• 제12권1호
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• pp.96-101
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• 1998

Substrate inhibition of 2,4-dinitrophenol (DNP) degradation was investigated using activated sludge which had been adapted to mineralize DNP. DNP is a metabolic uncoupler, preventing cells from making energy for growth and it has been suggested that pH may be important in mitigating effects of uncouplers. After acclimation of the activated sludge, the effect of pH on toxicity of DNP at high concentration (75 mg/L) was investigated, over a pH range of 5 to 9. DNP inhibition was found to be strongly dependent on mixed liquor pH. The DNP degradation rate was highest in the pH range of 6.95 to 7.84; at pH 5.94 degradation of 75 mg/L DNP was significantly inhibited; at pH < 5.77, DNP degradation was completely inhibited after approximately 30% of the DNP was degraded. By comparison, no significant effect of pH variation in the same range was seen on glucose uptake by the activated sludge culture.

• 동의생리병리학회지
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• 제19권3호
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• pp.628-632
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• 2005

The aim of this study was to investigate the cytotoxic effect and its mechanism on Radix Aconiti(RA) extract in lung cancer cell lines. RA extract treatment decreased the cell viability in a dose-dependent fashions in lung cancer cells including A549, H460, H23 and H157 cells. Many investigators reported that A549 and H460 cells expressed wild-type p53, but H23 and H157 cells preserved mutated p53. After treatment with RA extract in A549 and H460 cells, we measured the expression of p53 protein levels using Western blot. analysis. In both cells treated with RA extracts, p53 protein expressions were increased in a dose-dependent manner. In our experiments, RA extracts also have cytotoxic effects in H23 and H157, which have mutated p53. Treatment with RA extract decreased bcl-2 protein expressions in both cells. These results suggest that RA extracts have cytotoxic effects via p53 expression increase and bcl-2 inhibitable pathways in A549, H460 cells and H23, H157 cells, respectively.

• 한국전통의학지
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• 제15권1호
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• pp.106-112
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• 2006

The aim of this study was to investigate the cytotoxic effect and its mechanism on Radix Aconiti(RA) extract in lung cancer cell lines. RA extract treatment decreased the cell viability in a dose-dependent fashions in lung cancer cells including A549, H460, H23 and H157 cells. Many investigators reported that A549 and H460 cells expressed wild-type p53, but H23 and H157 cells preserved mutated p53. After treatment with RA extract in A549 and H460 cells, we measured the expression of p53 protein levels using Western blot. analysis. In both cells treated with RA extracts, p53 protein expressions were increased in a dose-dependent manner. In our experiments, RA extracts also have cytotoxic effects in H23 and H157, which have mutated p53. Treatment with RA extract decreased bcl-2 protein expressions in both cells. These results suggest that RA extracts have cytotoxic effects via p53 expression increase and bcl-2 inhibitable pathways in A549, H460 cells and H23, H157 cells, respectively.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1790-1794
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• 2012

Recently, it has been suggested that $p27^{Kip1}$, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of $p27^{Kip1}$ in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs) grown for 10 ($EB_{10}$) and 20 days ($EB_{20}$). Our results demonstrate that the proteins $p27^{Kip1}$ and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of $p27^{Kip1}$ protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.

• 한국식품위생안전성학회지
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• 제9권4호
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• pp.185-189
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• 1994

This study was to investigate time-dependent degradation process under various pH condition for organophosphorus(org-p) insecticides, namely Demeton-s-methyl, diazinon, Parathion, Phenthoate, and EPN in several waters. They were analysed by GC-FTD according to standard methods for the examination of water and wastewater. In pH=4, diazinon showed disappearance after 14 days in chromatogram. In pH=11, org-p insecticides were almost degradable after 7 days. In this condition, effect of pH on degradation process was greater than of light. In pH=7, org-p insecticides persisted residues after 112 days except Demeton-s-methyl. In BOD5 120, 250 ppm and domestic water, org-p insecticides showed also rapid degradation process.