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Comparative Studies on the Enzymatic Properties of Trypsins from Cat-shark and Mackerel -2. Enzymatic Properties of the Trypsins- (복상어와 고등어의 Trypsin에 관한 비교효소학적 연구 -2. 정제 Trypsin의 효소적 성질-)

  • PYEUN Jae-Hyeung;CHO Deuk-Moon;HEU Min-Soo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.25 no.5
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    • pp.383-391
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    • 1992
  • A comparative study of enzymatic properties between the trypsin from the cat-shark Cephaloscyllium umbratile ( C-T) and the two trypsins from the mackerel Scomber japonicus $(M-T_A\;and\;M-T_B)$ was carried out following after the previous paper(Pyeun et al., 1991). Trypsin from cat-shark(C-T) showed the higher heat stability compared to the others $(M-T_A\;and\;M-T_B)$ and its denaturation constant$(K_D)$ was $10.68\times10^{-4}\;sec^{-1}\;at\;55^{\circ}C$ with BA-p-NA substrate. The activation energies(Ea) of the trypsins measured at a temperature range from $30^{\circ}C\;to\;50^{\circ}C$ were estimated to be 4.07 kcal/mole for C-T, 11.61 kcal/mole for $M-T_A$, and 8.43kcal/mole for $M-T_B$, respectively. The Km values were $24.9\times10^{-5}\;M\;for\;C-T,\;5.37\times10^{-5}\;M\;for\;M-T_A,\;and\;9.65\times10^{-5}\;M\;for\;M-T_B$. On the other hand, the Ki values for TLCK and DFP determined by Dixon plot were $1.50\times10^{-6}\;M\;and\;9.28\times10^{-6}\;M\;for\;C-T\;2.86\times10^{-6}\;M\;and\;2.11\times10^{-4}\;M\;for\;M-T_A\;and\;3.90\times10^{-6}\;M\;and\;1.60\times10^{-4}\;M\;for\;M-T_B$ Similar amino acid profiles were showed between three trypsins each other, with few exceptions of $M-T_B$ containing higher amount of arginine, and the smaller amount of tryptophan in C-T than the others.

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A Human Case of Zoonotic Dog Tapeworm, Dipylidium caninum (Eucestoda: Dilepidiidae), in China

  • Jiang, Peng;Zhang, Xi;Liu, Ruo Dan;Wang, Zhong Quan;Cui, Jing
    • Parasites, Hosts and Diseases
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    • v.55 no.1
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    • pp.61-64
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    • 2017
  • We described a human case of zoonotic dog tapeworm, Dipylidium caninum (Eucestoda: Dilepidiidae), rarely occurring in China. The mother of a 17 month-old boy noted the appearance of small white and active worms over a month period in her son's feces, but the boy was asymptomatic except mild diarrhea. We observed 3 tapeworm proglottids resembling cucumber seeds in his stool sample. Microscopically, each proglottid had 2 genital pores, 1 on each lateral edge, and numerous egg capsules in the uterus. The patient was successfully treated with a single oral dose of praziquantel. Adult worms were recovered in the diarrheic stool after praziquantel treatment and purgation. His family had household pet dogs for several years, and he might have acquired the infection by ingestion of infected fleas of his pet dogs. A history of dog or cat pets and flea bites may be important clues to diagnosis of D. caninum infection. The infected pets should also be treated.

Ozone Impacts on Soluble Carbohydrates, Antioxidant Activity and Macro-element Concentrations in Rice Seedling

  • Sung Jwa-Kyung;Park So-Hyeon;Lee Su-Yeon;Lee Ju-Young;Jang Byoung-Choon;Hwang Seon-Woong;Kim Tae-Wan;Song Beom-Heon
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.51 no.2
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    • pp.142-147
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    • 2006
  • The present study describes carbohydrate metabolism, macro-element utilization and antioxidant defenses in response to an ozone dose (100 ppb, 8d) in two rice varieties. Tolerant (cv. Jinpumbyeo) and sensitive (cv. Chucheongbyeo) varieties of rice were grown in growth chamber for 30 days after sowing. Concentrations of chloroplast pigments and non-structural carbohydrates as well as activity of antioxidant enzymes were determined to evaluate the resistance against ozone stress. Ozone caused the decrease in chlorophyll a and carotenoid contents, and also resulted in faster decomposition of non-structural carbohydrate in leaf blade and leaf sheath. The contents of nitrogen and potassium in leaves were visibly decreased in cv. Chucheongbyeo with an increase in ozone exposure, but not in cv. Jinpumbyeo. Enzymatic antioxidants against ROS in both varieties responded in the order of POD, SOD and CAT, and their capacity was stronger in cv. Jinpumbyeo.

Purification and Characterization of a Chitinase from Cytophaga sp. HJ Isolated from Sea Sand

  • Lee, Dong-Mi;Noh, Hee-Jung;Lee, Kang-Man
    • Journal of Microbiology and Biotechnology
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    • v.9 no.6
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    • pp.839-846
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    • 1999
  • An extracellular chitinase-producing bacterial strain induced by colloidal chitin was isolated from sea sand and was identified to be a member of the genus Cytophaga. The chitinase was purified successively by 30-60% ammonium sulfate fractionation, and DEAE-Bio gel A column, Octyl-Sepharose CL-4B column, and DEAE-Bio gel A column chromatographies. The enzyme had a molecular mass of 59.75 kDa, and the amino terminal amino acid sequence was ATPNAPVISW MPTDXXLQNXS. The enzyme acted better on colloidal chitin as a substrate than on chitosan. For colloidal chitin and chitosan (Degree of Acetylation, 15-25%), $K_{cat}$ values were 0.60U/mg and 0.08U/mg, respectively. HPLC analysis of the enzymatic reaction products showed that the chitinase produced mostly N-acetyl-D-glucosarnine and di-N-acetylchitobiose. The optimum temperature and pH for the enzyme were $50^{\circ}C$ and 4.0, respectively. N-Bromosuccinimide and $Hg^{2+}$ inhibited the chitinase activity as much as 90%, and $Sb^{3+}$, diethylpyrocarbonate, and $Ag^{+}$ inhibited it by 50-70%.

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Purification and Characterization of a Thermostable Cellobiohydrolase from Fomitopsis pinicola

  • Shin, Keum;Kim, Yoon-Hee;Jeya, Marimuthu;Lee, Jung-Kul;Kim, Yeong-Suk
    • Journal of Microbiology and Biotechnology
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    • v.20 no.12
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    • pp.1681-1688
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    • 2010
  • A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a $t_{1/2}$ value of 42 h at $70^{\circ}C$ and catalytic efficiency of $15.8mM^{-1}s^{-1}(k_{cat}/K_m)$ for p-nitrophenyl-${\beta}$-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.

Eliminatory Effect of Mixture including Hot Water Extract of Dendropanax morbifera Lev. on Alcohol-induced Blood Alcohol Concentration and Hangover in Rat (흰쥐에서 황칠나무 열수 추출물을 포함한 혼합물의 혈중 알코올 농도와 숙취 해소 효과)

  • Na, Ju-ryun;Kim, Eun;Park, Soyi;Lee, Kihoon;Jeong, Eui-seon;Kim, Jinseok;Kim, Youngjae;Kim, Sunoh
    • Journal of Chitin and Chitosan
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    • v.23 no.4
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    • pp.267-276
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    • 2018
  • This study was performed to investigate the ameliorating effect of a hangover beverage mixture (SBJ) that contains Dendropanax morbifera Lev. and several medicinal plant extracts, on hepatoprotection and alcohol-metabolizing enzymes in alcohol-induced hangover in both in vitro and in vivo models. In human hepatoma cell line, HepG2, 300 mM of ethanol-induced hepatotoxicity was significantly improved by pretreatment of SBJ by dose-dependent manner. In the in vivo study, administration of alcohol to rats raised to the concentration of blood alcohol and lactate dehydrogenase (LDH). Blood alcohol and LDH levels in SBJ-treated rats significantly decreased at 0.5 h and 8 h after acute ethanol administration (40%, 4.6 g/kg body weight) as compared to alcohol-treated rats. Hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity were significantly higher in SBJ-treated rats than in alcohol-treated rats. SBJ supplementation reduced formation of malondialdehyde (MDA), and inhibited reductions of hepatic superoxide dismutase (SOD), hepatic glutathione (GSH), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) levels, compared with rats administered alcohol. Plasma catalase (CAT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels showed unaltered resulted in all experimental groups compared with the control group. These results suggest that SBJ exhibit hepatoprotective properties by enhancing ADH, ALDH activity and stimulating the antioxidant defense system in alcohol-induced hangover.

Anti-inflammatory effects of the Rubus occidentalis seed extracts on UV-B induced inflammation in HaCat cells (각질형성세포에서 UV-B와 사이토카인에 의하여 유도되는 염증 반응에 대한 복분자 씨앗 추출물의 항염증 효과)

  • Kim, Dong-Hee;Park, Tae-Soon;Son, Jun-Ho
    • Journal of Applied Biological Chemistry
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    • v.59 no.3
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    • pp.247-253
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    • 2016
  • It confirmed the applicability as an anti-inflammatory material from Rubus occidentalis seed (RSE) extract. In HaCaT cells to evaluate the anti-inflammatory potential as a material RSE extract on the activity of the inflammatory factors caused by UVB and $IFN-{\gamma}/TNF-{\alpha}$. We measured the activity of ROS, interleukin-$1{\beta}$ ($IL-1{\beta}$), interleukin-6 (IL-6) and interleukin-8 (IL-8) by ROS-Glo $H_2O_2$ assay and ELISA kit. Our results showed that the RSE extracts inhibit the UVB and $IFN-{\gamma}/TNF-{\alpha}$-induced ROS activities and expression of $IL-1{\beta}$, IL-6 and IL-8 in a dose-dependent manner. Also it was found that inflammatory mediators of the expression of cyclooxygenase-2 (COX-2) inhibition were also brought, the expression of which is increased $PGE_2$ by COX-2 also inhibited. Finally RSE extracts measure the seed expression of filaggrin in the skin barrier, the main factor of the extract could be confirmed to increase the expression of the filaggrin damaged as a result of this concentration-dependent manner. Through this, it was able to confirm that the efficacy RSE extract to protect the inflammation by restoring the damaged layers of the epidermis. Results from more than RSE extract was able to confirm that the extract that has anti-inflammatory effects by improving the inflammation being produced from UVB.

Effects of Bombesin on Electrical and Mechanical Activities of Gastric Smooth Muscle Strips of Cats (적출한 고양이 위(胃) 평활근 절편의 전기적 및 기계적 활동에 미치는 Bombesin의 영향과 그 작용기전)

  • Park, Hyoung-Jin;Kwon, Hyeok-Yil;Suh, Sang-Won;Kim, Jeong-Mi;Lee, Tae-Hyung
    • The Korean Journal of Physiology
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    • v.24 no.1
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    • pp.39-49
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    • 1990
  • It has been reported that bombesin induces contraction of the smooth muscle of the gastrointestinal tract. Thus, the present investigation was undertaken to see an influence of bombesin on electrical activity of the gastric smooth muscle, since electrical activity is associated with contractile activity in the smooth muscle of the stomach. Smooth muscle strips $(5\;{\times}\;1.5\;cm)$ that included the corpus and antrum were prepared from the ventral and dorsal portion of the feline stomach along the greater curvature. Circular muscle strips $(1\;{\times}\;0.3\;cm)$ of the corpus were also obtained. Electrical activity of the corpus and antrum of the muscle strip was monophasically recorded by using Ag-AgCl capillary electrodes placed on the circular muscle layer. Contractile activity of the circular muscle strip was also recorded. The recordings were performed in Krebs-Ringer solution that was continuously aerated with $O_{2}$ containing 5% $Co_{2}$, and kept at $36^{\circ}C$. Dose-related responses of electrical activity and contractility to bombesin was studied after frequency of slow waves and contraction of each strip reached to a steady state. An action of $D-leu^{13}-{\psi}\;(CH_{2}NH)-D-leu^{14}-bombesin,\;D-pro^{2}-D-trp^{7,9}-substance\;P$, tetrodotoxin, hexamethonium, atropine, phentolamine or propranolol on the effect of bombesin was also observed. 1) Bombesin increased frequency of slow waves and contractions dose-dependently at concentrations from $10^{-9}\;M\;to\;3\;{\times}\;10^{-8}\;M$. 2) The bombesin analogue at a concentration of $3\;{\times}\;10^{-7}\;M$ antagonized the effect of bombesin on frequency of slow waves. 3) The effect of bombesin on frequency of slow waves was inhibited by tetrodotoxin $(10^{-6}\;M)$ and hexamethonium $(10^{-3}\;M)$ but unaffected by atropine $(10^{-6}\;M)$, phentolamine $(10^{-5}\;M)$ and propranolol $(10^{-5}\;M)$. 4) The effect of bombesin on frequency of slow waves was blocked by the substance P analogue at a concentration of $10^{-5}\;M$. 5) Substance P at a concentration of $10^{-5}\;M$ failed to change frequency of slow waves. It is concluded from the above results that bombesin increases the frequency of slow waves as well as contractions of the smooth muscle strip from the feline stomach, and the effect of bombesin might be mediated by non-cholinergic or non-adrenergic mechanism at neuromuscular junction. However, enteric nerves that have substance P as a neurotransmitter do not appear to participate in the action of bombesin on frequency of slow waves.

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P2X and P2Y Receptors Mediate Contraction Induced by Electrical Field Stimulation in Feline Esophageal Smooth Muscle

  • Cho, Young-Rae;Jang, Hyeon-Soon;Kim, Won;Park, Sun-Young;Sohn, Uy-Dong
    • The Korean Journal of Physiology and Pharmacology
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    • v.14 no.5
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    • pp.311-316
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    • 2010
  • It is well-known that electrical field stimulation (EFS)-induced contraction is mediated by a cholinergic mechanism and other neurotransmitters. NO, ATP, calcitonin gene-related peptide (CGRP), and substance P are released by EFS. To investigate the purinergic mechanism involved in the EFS-induced contraction, purinegic receptors antagonists were used. Suramine, a non-selective P2 receptor antagonist, reduced the contraction induced by EFS. NF023 ($10^{-7}{\sim}10^{-4}M$), a selective P2X antagonist, inhibited the contraction evoked by EFS. Reactive blue ($10^{-6}{\sim}10^{-4}M$), selective P2Y antagonist, also blocked the contraction in a dose-dependent manner. In addition, P2X agonist ${\alpha}$,${\beta}$-methylene 5'-adenosine triphosphate (${\alpha}{\beta}MeATP$, $10^{-7}{\sim}10^{-5}M$) potentiated EFS-induced contraction in a dose-dependent manner. P2Y agonist adenosine 5'-[${\beta}$-thio]diphosphate trilithium salt ($ADP{\beta}S$, $10^{-7}{\sim}10^{-5}M$) also potentiated EFS-induced contractions in a dose-dependent manner. Ecto-ATPase activator apyrase (5 and 10 U/ml) reduced EFS-induced contractions. Inversely, 6-N,$N$-diethyl-D-${\beta}$,${\gamma}$- dibromomethylene 5'-triphosphate triammonium (ARL 67156, $10^{-4}M$) increased EFS-induced contraction. These data suggest that endogenous ATP plays a role in EFS-induced contractions which are mediated through both P2X-receptors and P2Y-receptors stimulation in cat esophageal smooth muscle.

Characterization of polysaccharide A-1 from Opuntia ficus-indica and it's protection effect on alcoholic induced hepatic oxidative stress (Opuntia ficus-indica 다당 A-1의 특성 및 알코올유도 간 산화스트레스의 보호 효과)

  • Ryu, Il-Hwan;Kwon, Ji-Wung;Lee, Eoh-Jin;Yun, Young-Gab;Kwon, Tae-Oh
    • Herbal Formula Science
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    • v.17 no.2
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    • pp.163-174
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    • 2009
  • Reactive oxygen species(ROS) can induce hepatotoxicity and trigger apoptosis in the liver. In this study, we investigated the sulfated polysaccharide A-1 from Opuntia ficus-indica against alcoholic oxidative stress in human liver Hep G2 cell. An antioxidant substance A-1 obtained from the enzymatic extract of Opuntia ficus-indica fruit was purified by DEAE-cellulose ion exchange and sephadex G-100 gel permeation chromatography. The purification yield and molecular weight were 14.3% and 1.8 KDa, respectively. The A-1 predominately contained arabinose, galactose, rhamnose and also sulfate group. The structure of A-1 was investigated by periodate oxidation, FT-IR spectroscopy, $^1H$-NMR spectroscopy. The A-1 mainly composed of alternating unit of ${\rightarrow}4$)-$\alpha$-L- Rapp-2-$SO_3^-$-$\alpha$-L-Galp-($1{\rightarrow}$ and branched linkage of $\beta$-D-Arbp- ($5{\rightarrow}$. The antioxidative activity was measured using the SOD, CAT activity and GSH assay, respectively. The expression of Nrf2 protein was analyzed by western blotting. The viable cell count analyzed by autofluorescence. Oxidative stress induced by ethanol(1 M) were dramatically reduced by A-1 treatment. A-1 also prevented cell death induced by oxidative stress. It also increased expression Nrf2 protein level. We concluded that sulfated polysaccharide A-1 from Opuntia ficus-indica effectively protect Hep G2 liver cell from alcoholic oxidative stress.

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