• Journal of Acupuncture Research
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• v.18 no.3
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• pp.114-122
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• 2001

Objective : The present study was carried out to determine if Juglandis Semen Aquacupuncture(JSA) exerts beneficial effect against the urine concentrating defect induced by glycerol injection in rabbits. Methods : In order to test the effect of JSA, rabbits were acupunctured with $0.5m{\ell}$ of 1%JSA for 7days at both sides of $Sh{\grave{e}}ns{\bar{u}}$(BL23) before the glycerol injection. The other animals were pretreated with an equal volume of saline instead of JSA. Results : The urine flow was reduced, but the urine osmolality was significantly lower than the basal period in glycerol-injected animals, indicating that glycerol causes the urine concentrating defect. The fractional excretion of Na and K was increased in glycerol-treated animals. The Free-water clearance and fractional water excretion(V/GFR) were increased in animals treated with glycerol. Conclusion : These results indicate that glycerol injection resulted an impairment in the urine concentrating ability in rabbits. Such changes were prevented by JSA. JSA may be used as a method to treat and prevent glycerol-induced acute renal failure.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1077-1083
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• 2012

Previously, an extracellular ${\alpha}$-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [$BKA{\Delta}$(N44) and $BKA{\Delta}$(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The $K_m$ and $V_{max}$ values for $BKA{\Delta}$(N44) were lower than $BKA{\Delta}$(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.612-619
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• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1070-1083
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• 2015

A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His⁴⁰⁰-Glu⁴⁰¹-X-XHis⁴⁰⁴). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepF_Ba from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the K_m, V_max, and k_cat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10^-6 M, 2.65 ± 0.03 × 10^-3 µM/min, and 5.99 ± 0.07 s^-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.659-665
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• 2016

The oligosaccharides in human milk constitute a major innate immunological mechanism by which breastfed infants gain protection against infectious diarrhea. Clostridium difficile is the most important cause of nosocomial diarrhea, and the C-terminus of toxin A with its carbohydrate binding site, TcdA-f2, demonstrates specific abolishment of cytotoxicity and receptor binding activity upon diethylpyrocarbonate modification of the histidine residues in TcdA. TcdA-f2 was cloned and expressed in E. coli BL21 (DE3). A human milk oligosaccharide (HMO) mixture displayed binding with TcdA-f2 at 38.2 respond units (RU) at the concentration of 20 μg/ml, whereas the eight purified HMOs showed binding with the carbohydrate binding site of TcdA-f2 at 3.3 to 14 RU depending on their structures via a surface plasma resonance biosensor. Among them, Lacto-N-fucopentaose V (LNFPV) and Lacto-N-neohexaose (LNnH) demonstrated tight binding to TcdA-f2 with docking energy of −9.48 kcal/mol and −12.81 kcal/mol, respectively. It displayed numerous hydrogen bonding and hydrophobic interactions with amino acid residues of TcdA-f2.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.873-880
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• 2009

A novel xylanase gene, Kxyn, was cloned from Kocuria sp. Mn22, a bacteria isolated from the deep sea of the east Pacific. Kxyn consists of 1,170 bp and encodes a protein of 390 amino acids that shows the highest identity (63%) with a xylanase from Thermohifida fusca YX. The mature protein with a molecular mass of approximately 40 kDa was expressed in Escherichia coli BL21 (DE3). The recombinant Kxyn displayed its maximum activity at $55^{\circ}C$ and at pH 8.5. The $K_m,\;V_{max}$, and $k_{cat}$ values of Kxyn for birchwood xylan were 5.4 mg/ml, $272{\mu}mol/min{\cdot}mg$, and 185.1/s, respectively. Kxyn hydrolyzed birchwood xylan to produce xylobiose and xylotriose as the predominant products. The activity of Kxyn was not affected by $Ca^{2+},\;Mg^{2+},\;Na^+,\;K^+$, ${\beta}$-mercaptoethanol, DTT, or SDS, but was strongly inhibited by $Hg^{2+},\;Cu^{2+},Zn^{2+}$, and $Pb^{2+}$. It was stable over a wide pH range, retaining more than 80% activity after overnight incubation at pH 7.5-12. Kxyn is a cellulase-free xylanase. Therefore, these properties make it a candidate for various industrial applications.

• Korean Journal of Pharmacognosy
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• v.40 no.3
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• pp.205-212
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• 2009

Antitumor activity of Korean mistletoe extract (KM-110) and European commercial mistletoe preparation (Helixor) was investigated. KM-110 showed the cytotoxic effect that it is high for various tumor cell lines and normal splenocytes in comparison with Helixor. Administration of two mistletoe extracts ($100{\mu}g$) to mice did not show any significant changes on the level of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvate transaminase (GTP), blood creatinine (CRE) and blood urea nitrogen (BUN) in sera. The culture supernatant of macrophages stimulated with KM-110 inhibited effectively tumor growth whereas Helixor had little effect. Administration of KM-110 or Helixor resulted in a effective inhibition of lung metastasis after the i.v. inoculation of colon 26-M3.1 lung carcinoma, B16-BL6 melanoma and L5178Y-ML25 lymphomas. In all cases, the mice treated with KM-110 showed more effective anti-tumor metastatic activity than the mice of Helixor. These results suggest that Korean mistletoe extracts, KM-110 might be used as an alternative methods having antitumor activity like European mistletoe preparation, Helixor.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3065-3071
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• 2012

Aim: The present investigation was to evaluate the effects of essential oils of Wedelia chinensis (Osbeck) on free radicals and in vivo antioxidant properties. Methods: Essential oils were extracted using hydro-distillation and compound analysis was performed by GC-MS analysis. Screening for inhibitory activity was conducted by DPPH and OH-scavenging assays. In addition an in vivo study was carried out in cell line implanted cancer bearing mice with assessment of levels of catalase, superoxide dismutase, glutathione peroxidase, lipid peroxidation, nitric oxide and reduced glutathione. Finally, lungs were dissected out for histopathology study of metastasis. Results: GC-MS analysis revealed the presence of carvocrol and trans-caryophyllene as the major compounds with 96% comparison with the Wilily and NBS libraries. The essential oil exhibited significant inhibition in DPPH free radical formation. Whereas reducing power and hydroxyl radical scavenging activity are dose dependent. When compared with the standard, it was found that the essential oil has more or less equal activity in scavenging free radicals produced. In the animal studies, the level of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase, as well as glutathione, were found to be increased in treated groups whereas lipid peroxidation and nitric oxide were reduced. Histopathology report also shows that the essential oil has a significant combating effect against cancer development. Conclusion: In all the in vitro assays, a significant correlation existed between the concentrations of the essential oil and percentage inhibition of free radicals. The in vivo studies also has shown a very good antioxidant property for the essential oil during cancer development. From, these results the essential oil can be recommended for treating disease related to free radicals and to prevent cancer development.

• The Korean Journal of Food And Nutrition
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• v.26 no.3
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• pp.375-382
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• 2013

The present study is designed to explore an anti-tumor activity on crude extracts of Oplopanax elatus. Water extractions of Oplopanax elatus were performed at $100^{\circ}C$(OeE-100). OeE-100 doses up to $62.5{\mu}g/m{\ell}$ had no cytotoxicity on the tumor cell lines in vitro. In experimental lung metastasis of colon26-M3.1 carcinoma or B16-BL6 melanoma, the prophylactic intravenous ($4{\sim}100{\mu}g/mouse$) or oral (2 mg/mouse) administration of OeE-100 significantly inhibited tumor metastasis as compared with tumor controls. Peritoneal macrophages stimulated with OeE-100 produced various cytokines such as TNF-${\alpha}$, IL-6 and IL-12. In an analysis of NK-cell activities, i.v. administration of OeE-100 ($10{\sim}100{\mu}g/mouse$) significantly augmented the cytotoxicity to YAC-1 tumor cells. Vaccination of mice with boiling-treated tumor cells (BT-vaccine) in combination with OeE-100 ($100{\mu}g/mouse$) showed higher inhibitions in tumor metastasis when compared with the mice of BT-vaccine treatment. In addition, the splenocytes from OeE-100 admixed BT-vaccine immunized mice secreted a higher concentration of Th1 type cytokine such as IFN-${\gamma}$. These results suggested that the OeE-100 stimulated immune system and was a good candidate adjuvant of anti-tumor immune responses.