• Toxicological Research
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• v.27 no.1
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• pp.37-41
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• 2011

This study investigated the antioxidative action of Corni Fructus aqueous extract on kidneys of diabetic mice. The electron donating abilities of Corni Fructus aqueous extract and its antioxidant activities (XO, SOD, CAT, GST, eNOS) in kidneys of C57BL/6 or db/db mice were evaluated. For in vivo study, seven week-old male mice were divided into normal control group (NC, C57BL/6 mice), diabetic control group (DC, db/db mice) and Corni Fructus (500 mg/kg/day for 8 weeks) treated diabetic group (DCF, db/db mice). The electron donating abilities of Corni Fructus aqueous extract exhibited 7%, 24.4%, and 42.7% at concentrations of 100, 500, and $1000\;{\mu}g/ml$, respectively. The activity of XO in the DCF group was significantly lower than the DC group by 35% (p < 0.05). The SOD activity was significantly higher in the DCF group than the DC group by 26% (p < 0.05). The activities of CAT and GST were lowered in the DCF group than the DC group by 26% (p < 0.05) and 7.6%, respectively. The mRNA expression of eNOS in kidneys was lower in the DCF group than the DC group by 24%. These results indicate that Corni Fructus reduced oxidation stress as evidenced by the restoration of the enzymatic antioxidative defense system in renal tissues of db/db mice. It is suggested that these antioxidative actions of Corni Fructus on renal tissues in db/db mice could contribute to its renoprotective effects on diabetic nephropathy.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.99-103
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• 2016

In perennial ginseng plantations, the effective control of various diseases is one of the most critical factors for increasing yields. Enhancing the resistance to disease through induced systemic resistance (ISR) and anti-microbial activity of beneficial soil bacteria, is currently considered to be a potential promising approach to integrate pathogen management for sustainable agriculture. However, the effective in vitro culture systems for testing ISR in ginseng plants have been rarely reported. In this study, I have successfully developed an in vitro germ-free culture system of Panax ginseng seedling for diverse purposes. With this useful system, we also tested BTH-induced priming effects against Botrytis cinerea and Colletotrichum panacicola. Compared to the drain method for enhancing ISR effects to ginseng seedlings, the direct method of spraying leaves somewhat increased the defense activity to these major fungal pathogens. Consistently, the expression of pathogen related PgPR10 and PgCAT were greatly and rapidly enhanced in the BTH-treated ginseng seedlings by treatment with C. panacicola. Our results revealed that the in vitro culture system can be used for developing eco-friendly and versatile bio-control agents for harmful diseases in ginseng cultivation.

• Applied Chemistry for Engineering
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• v.10 no.8
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• pp.1210-1215
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• 1999

Optimum conditions of the hydrogenation of PNA to pure PPD were determined in a three-phase slurry reactor with suspended Pd/C catalyst particles. Minimization of mass transfer resistances at the interfaces of both gas-liquid and liquid-catalyst particles and control of overall reaction rate on catalyst surface leaded to decrease the hydrogen starvation on reaction active sites and to reduce the side reactions during hydrogenation. The optimum temperature, pressure, and catalysst concentration were confirmed to be in the range of $60^{\circ}C$, 60~70 psig, and 1~2 g-cat/L, respectively. Reaction rate was zero order with respect to the concentration of PNA and 1st order with respect to the pressure of hydrogen(P). Overall rate expression of the reaction was $R_A=6.44{\times}10^6{\cdot}H{\cdot}P{\cdot}m{\cdot}$exp(-4659/T) where H is constant, m is concentration of catalyst, and T is temperature.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.82-88
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• 2023

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni²⁺-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type I spectral changes, with K_d values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k_cat value of 0.038 ± 0.002 min^-1 and a K_m value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k_cat value of 0.135 ± 0.007 min^-1 and a K_m value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.2
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• pp.105-113
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• 2024

Background: Aflatoxin B1 (AFB1) is a toxic metabolite generated by Aspergillus species and is commonly detected during the processing and storage of food; it is considered a group I carcinogen. The hepatotoxic effects, diseases, and mechanisms induced by AFB1 owing to chronic or acute exposure are well documented; however, there is a lack of research on its effects on the intestine, which is a crucial organ in the digestive process. Dogs are often susceptible to chronic AFB1 exposure owing to lack of variation in their diet, unlike humans, thereby rendering them prone to its effects. Therefore, we investigated the effects of AFB1 on canine small intestinal epithelial primary cells (CSIc). Methods: We treated CSIc with various concentrations of AFB1 (0, 1.25, 2.5, 5, 10, 20, 40, and 80 μM) for 24 h and analyzed cell viability and transepithelial-transendothelial electrical resistance (TEER) value. Additionally, we analyzed the mRNA expression of tight junction-related genes (OCLN, CLDN3, TJP1, and MUC2), antioxidant-related genes (CAT and GPX1), and apoptosis-related genes (BCL2, Bax, and TP53). Results: We found a significant decrease in CSIc viability and TEER values after treatment with AFB1 at concentrations of 20 μM or higher. Quantitative polymerase chain reaction analysis indicated a downregulation of OCLN, CLDN3, and TJP1 in CSIc treated with 20 μM or higher concentrations of AFB1. Additionally, AFB1 treatment downregulated CAT, GPX1, and BCL2. Conclusions: Acute exposure of CSIc to AFB1 induces toxicity, and exposure to AFB1 above a certain threshold compromises the barrier integrity of CSIc.

• Journal of fish pathology
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• v.22 no.3
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• pp.293-303
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• 2009

Effects of stress on the low salinity stress were examined in the pacific abalone Haliotis discus discus. Changes in survival rate, hemolymph count, antioxidant enzyme activities (catalase: CAT and superoxide dismutase: SOD), respiratory burst activity, phenoloxidase activity, lysozyme activity and expression of heat shock protein 70 (HSP70) mRNA were measured 0, 3, 6, 12, 24 or 48hours after low salinity treatment with 25, 30, 33 and 35 psu. Survival rates of pacific abalone were 100% at 33 and 35 psu, but 93 and 97% at 25 and 30 psu for 48 hours, respectively. Hemolymph counts decreased in the time elapsed-dependent way at all of the experimental groups. At low salinity, 25 and 30 psu, SOD and CAT activity increased compared to the experimental group of 33 psu. Moreover, respiratory burst activities of the pacific abalone seemed to have no effect on low salinity stress at any experimental group. However, phenoloxidase activity is an important component of the defence against pathogen that was decreased in a reduction of salinity dependent way. Lysozyme activity also immediately reduced at 25 psu experimental group for 48 h. The HSP70 mRNA was weakly expressed at 33 psu, but strongly detectable at 25 psu experimental group. The HSP 70 mRNA expression in gill increased in the time elapsed-dependent way at 25 psu experimental group and then recovered at 48 h. These results suggest that low salinity stress give rise to inhibitory action of immune system as a result of the decrease of phenoloxidase and lysozyme activity in the pacific abalone, especially.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Molecular & Cellular Toxicology
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• v.5 no.4
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• pp.273-282
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• 2009

Cadmium (Cd) and tributyltin (TBT) are common contaminants of marine and freshwater ecosystems, and can induce the formation of reactive oxygen species (ROS). These ROS can, in turn, cause oxidative stress. In the present study, we investigated time-related effects of Cd (0.05 and 0.1 ppm) and TBT (5 and 10 ppb) treatment on antioxidant enzyme activity, i.e., the activity of superoxide dismutase (SOD) and catalase (CAT) in the gills and digestive glands of the ark shell, Scapharca broughtonii. In addition, hydrogen peroxide ($H_2O_2$) concentrations, lysozyme activity, and glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels were measured in the hemolymph. We found that Cd and TBT treatment significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. In response to the Cd and TBT treatments, antioxidant enzymes mRNA expression and activity increased up to day 5 in the digestive glands and then decreased by day 7. In the gills, antioxidant enzymes mRNA expression and activity increased up to day 3 and then decreased by day 5. Likewise, $H_2O_2$ concentrations significantly increased up to day 5 and then decreased by day 7. Finally, lysozyme activity decreased during the experimental period, whereas GOT and GPT levels were significantly increased in a time-dependent manner. These results suggest that antioxidant enzymes play an important role in decreasing ROS levels and oxidative stress in ark shells exposed to Cd and TBT.

• BMB Reports
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• v.37 no.4
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• pp.408-415
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• 2004

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and $40^{\circ}C$. The catalytic efficiency ($k_{cat}/K_m$) values for $\alpha$-, $\beta$-, and $\gamma$-CD were $3.0{\times}10^5$, $8.8{\times}10^5$, and $5.5{\times}10^5\;M^{-1}\;min^{-1}$, respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.