• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.44-47
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• 2000

Cell growth and DNA synthesis were studied from a cultured early- and late- pas- sage mouse aorta smooth muscle cell (MASMC) because the proliferation of vascular smooth muscle cell (VSMC) is a key factor in development of atherosclerosis. In this study, the cells were cultured in fetal bovine serum (FBS) and stimulated by growth factors such as thrombin and platelet-derived growth factor-BB (PDGF-BB). Compared to the number of early-passage MASMC (passage 3 to 9) the number of late-passage MASMC (passage 30 to 40) in a normal serum state was increased 2 fold at Day 1, 3 and 6 in culture, respectively. Incorporation of $[^3H]$ thymidine into DNA induced by serum, PDGF and thrombin in late-passage MASMC was greater than those in early-passage MASMC. We also examined whether intracellular zinc levels would be an aging factor or not. The intracellular zinc level in early- and late-passage MASMC was monitored by using the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide. It is interested that late-passage MASMC increased the intracellular fluorescence level of zinc, more than the early passage MASMC did. The alterations of intracellular zinc level occur concurrently with changes in MASMC proliferation rate during aging. This data suggest that the age-associated changes in zinc concentrations may provide a new in vitro model for the study of smooth muscle cell differentiation.

• 한국동물생명공학회지
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• 제34권2호
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Molecules and Cells
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• 제19권2호
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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.

• Preventive Nutrition and Food Science
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• 제7권4호
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• pp.421-426
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• 2002

In order to obtain basal data for industrial application of inulinase from Bacillus sphaeicus 188-1, its intracellular inulinase was purified by ammonium sulfate fractionation and column chromatography on DEAE-Sephadex A-50 and Sephadex G-100. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent molecular weight of 29 kDa. Inulinase activity was optimal at pH 6.5 and 4$0^{\circ}C$. The enzyme activity was significantly inhibited by Cu$^{2＋}$, Cd$^{2＋}$ and Hg$^{2＋}$. The inulinase exhibited an apparent Km value of 0.014% for inulin.

• 대한의생명과학회지
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• 제11권3호
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• pp.301-305
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• 2005

MR spectroscopy of intracellularly located $^{133}Cs$ has been used to monitor the uptake of Gd-EOB-DTPA by the isolated rat liver. As shown by ${31}P$ spectroscopy, accumulation of $^{133}Cs$ ions in hepatocytes does not produce detectable effects on the metabolism. The hepatic internalization of Gd-EOB-DTPA was followed by the paramagnetic relaxation enhancement of the intracellular $^{133}Cs$ ions, and confirmed by parallel quantitations of Gd and Cs run by inductively coupled plasma analysis of liver samples and aliquots of perfusate. Two peaks are observed at -22.0 and -23.5 ppm, with respect to the line of the external reference arbitarily set to 0 ppm. Upon rinsing of the extracellular compartment with regular K-H free of CsCl, the high-field resonance disappears within 20min. The intracellular concentration was confirmed by ICP, which gives a $Cs^+$ content of $22.0\pm3.5mM$. The relaxation data significantly underestimate the Gd content, suggesting a potential compartmentation of $Cs^+$ and the contrast agent.

• 미생물학회지
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• 제30권4호
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• pp.299-304
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• 1992

An intracellular protease form cells of Pseudomonas carboxydovorans DSM 1227 grown on carbon monoxide was purified 57-fold in six steps to homogeneity with a yield of 4.3% using azocoll as a substrate. The molecular weight of the enzyme was determined to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed the purified enzyme to be a dimer with two identical subunits of molecular weight 72,000. The enzyme was stimulated by $Mg^{2+}$ but was inhibited completely by $Cd^{2+}$ $Fe^{2+}$ $Hg^{2+}$, and $^Zn{2+}$ The enzyme activity was also inhibited by EDTA, EGTA, phenylmethylsulfonyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl)carbodiimide, iodoacetamide and dithiothereitol. The optimal pH and temperature for the enzyme reaction were found to be 7-8 and 50.deg.C, respectively. Casein and bovine serum albumin were hydrolyzed by the enzyme, but carbon monoxide dehydrogenase was not.

• 미생물학회지
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• 제29권3호
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• pp.167-171
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• 1991

An intracellular protease from cells of Pseudomonas carboxydohydrogena grown on nutrient broth was purified to better than 95% homogeneity in five steps using azocaseine as a substrate. The molecular weight of the native enzyme was determined to be 125, 000. Sodium dodecyl sulfate-gel electrophoresis revealedat least two non-identical subunits of molecular weight 70, 000 and 56, 000. The enzyme activity was completely ingibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The enzyme was also inhibited by $Mg^{2+}$ , $Zn^{2+}$ , $Cd^{2+}$, $Cu^{2+}$ , and $Fe^{2+}$ , but was stimulated by iodoacetamide. Maximal reaction rate of the enzyme was observed at pH8.0 and 30.deg.C. The isoelectric point of the enzyme was found to be 7.5. The enzyme was unable to hydrolyze carbon monoxide dehydrogenase.

• BMB Reports
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• 제35권4호
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• pp.384-388
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• 2002

A method for the ratiometric pH sensing using LysoSensor DND-192 is presented in this paper. It works in the physiological pH range. It is based on the use of two fluorophores which differ significantly in their lifetimes. As the discrimination of their emissions is performed through two different frequencies, this method can allow significant overlap of the emission spectra. A simple long-pass filter, or a combination of long-and short-pass filters, was used instead of narrow-bandpass devices. Importantly, the measurements were carried out under strong ambient light. The method could be used in a wide variety of applications, such as intracellular measurements, microscopy, bioprocess monitoring, etc.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1182-1189
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• 2004

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. The intracellular cytosine deaminase from Chromobacterium violaceum YK 391 was purified to apparent homogeneity with 272.9-fold purification with an overall yield of $13.8\%$. The enzyme consisted of dimeric polypeptides of 63 kDa, and the total molecular mass was calculated to be approximately 126 kDa. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 6-azacytosine, and 5-methylcytosine, but not 5-azacytosine. Optimum pH and temperature for the enzyme reaction were 7.5 and $30^{\circ}C$, respectively. The enzyme was stable at pH 6.0 to 8.0, and at 30T for a week. About $70\%$ of the enzyme activity was retained at $60^{\circ}C$ for 5 min. The apparent $K_{m}$ values for cytosine, 5-fluorocytosine, and 5-methylcytosine were calculated to be 0.38 mM, 0.87 mM, and 2.32 mM, respectively. The enzyme activity was strongly inhibited by 1 mM $Hg^{2+},\;Zn^{2+},\;Cu^{2+},\;Pb^{2+},\;and\;Fe^{3+}$, and by o-phenanthroline, $\alpha,\;{\alpha}'$-dipyridyl, p-choromercuribenzoate, N-bromosuccinimide, and cWoramine­T. In addition, the enzyme activity was strongly inhibited by I mM 2-thiouracil, and weakly inhibited by 2-thiocytosine, or 5-azacytosine. Finally, intracellular and extracellular cytosine deaminases from Chromobacterium violaceum YK 391 were found to have a different optimum temperature, apparent $K_{m}$ value, and molecular mass.