• Title/Summary/Keyword: enzyme transport

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Influence of Temperature on the Bacterial Community in Substrate and Extracellular Enzyme Activity of Auricularia cornea

  • Zhang, Xiaoping;Zhang, Bo;Miao, Renyun;Zhou, Jie;Ye, Lei;Jia, Dinghong;Peng, Weihong;Yan, Lijuan;Zhang, Xiaoping;Tan, Wei;Li, Xiaolin
    • Mycobiology
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    • v.46 no.3
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    • pp.224-235
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    • 2018
  • Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures ($20^{\circ}C$, $25^{\circ}C$, $30^{\circ}C$, $35^{\circ}C$, and $40^{\circ}C$) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above $35^{\circ}C$. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at $35^{\circ}C$ and $40^{\circ}C$. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE GENE FOR THE HEMIN-BINDING PROTEIN FROM Prevotella intermedia (Prevotella intermedia에서의 Hemin 결합 단백질 유전자의 분리 및 염기서열 분석)

  • Kim, Shin;Kim, Sung-Jo
    • Journal of the korean academy of Pediatric Dentistry
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    • v.33 no.2
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    • pp.304-310
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    • 2006
  • Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.

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Characterization of a Mucolipidosis Type II Mouse Model and Therapeutic Implication of Lysosomal Enzyme Enriched Fraction Derived from Placenta (뮤코지방증 2형 마우스 모델의 특징과 태반에서 추출한 리소좀 효소 투여의 결과)

  • Cho, Sung Yoon;Kim, Ki-Yong;Kim, Su Jin;Sohn, Young Bae;Maeng, Se Hyun;Kim, Chi Hwa;Ko, Ah-Ra;Song, Junghan;Yeau, Sung-Hee;Kim, Kyung-Hyo;Jin, Dong-Kyu
    • Journal of The Korean Society of Inherited Metabolic disease
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    • v.12 no.1
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    • pp.5-13
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    • 2012
  • I-cell disease (mucolipidosis type II; MIM 252500) and pseudo-Hurler polydystrophy (mucolipidosis type III; MIM 252600) are disorders caused by abnormal lysosomal transport in cells. The presence of numerous inclusion bodies in the cytoplasm of fibroblasts, a lack of mucopolysacchariduria, increased lysosomal enzyme activity in serum, and decreased GlcNAc-phosphotransferase activity are hallmark. Here, we attempted to investigate phenotypical and biochemical characteristics of the knockoutmouse of GlcNAc-phosphotransferase ${\alpha}/{\beta}$ subunits; in addition, we also attempted to determine whether the lysosome enriched fraction derived from placenta can be beneficial to phenotype and biochemistry of the knockout mouse.We found that the knockout mouse failed to thrive and had low bone density, as is the case in human. In addition, skin fibroblasts from the animal had the same biochemical characteristics, including increased lysosomal enzyme activity in the culture media, in contrast to the relatively low enzyme activity within the cells. Intravenous injection of the lysosome rich fraction derived from placenta into the tail vein of the animal resulted in a gain of weight, while saline injected animals didn't.In conclusion, our study demonstrated the phenotypical and biochemical similarities of the knockout mouse to a mucolipidosis type II patient and showed the therapeutic potential of the lysosome enriched fraction. We admit that a larger scale animal study will be needed; however, the disease model and the therapeutic potential of the lysosome enriched fraction will highlight the hope for a novel treatment approach to mucopolipidosis type II, for which no therapeutic modality is available.

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Postchilling Accumulation of Superoxide in Cells and Chilling Injury in Rice Plant (Superoxide의 세포내 축적과 벼냉해의 발현)

  • Kim, Jong-Pyung;Hyun, Il;Jung, Jin
    • Applied Biological Chemistry
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    • v.30 no.4
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    • pp.364-370
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    • 1987
  • The $O_2^-$ level of the extract from young rice leaves, which was cold treated for 2 days and then placed at room temperature for a period of time significantly higher than that from tissues untreated. $O_2^-$ level in leaves was practically unchanged during cold treatment for 48 hours. But it started to increase to arrive at maximum in 8 hours, once the plants were placed under room temperature. The abnormal production of $O_2^-$ in mitochondria during postchilling process was interpreted as a biochemical consequence of accumulation of glycolysis product(s) in cytosol and/or NADH in mitochondrial matrix due to disruption of catabolic balance at low temperature. Mitochondria isolated from the chilling injured tissue was found to have lost considerably their respiratory activity. This fact may imply the involvement of intramitochondrial accumulation of $O_2^-$ in the inactivation of electron transport chain system. The observation that mitochondria in the presence of the $O_2^--producing$ enzymatic system (Xanthine/Xanthine oxidase) lost their respiratory activity supports this inference. It was also found in this work that Superoxide dismutase (SOD) is a substrate inducible enzyme, and that SOD is a possible protective agent in plant cell against chilling injury.

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Antagonistic Mode of Action of Fenoxaprop-P-ethyl Phytotoxicity with Bentazon (Fenoxaprop-P-ethyl의 제초활성에 대한 Bentazon의 길항작용기구)

  • Ma, S.Y.;Kim, S.W.;Chun, J.C.
    • Korean Journal of Weed Science
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    • v.18 no.2
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    • pp.161-170
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    • 1998
  • Antagonistic mode of action of fenoxaprop-P-ethyl [ethyl(R)2-4-{(6-chloro-2-benzoxazolyloxy) phenoxy}propionate] with bentazon was investigated with respect to absorption, translocation, metabolism, and change in target site response of fenoxaprop-P-ethyl using four-leaf stage of rice(Oryza sativa L.) and barnyardgrass [Echinochloa eras-galli (L.) P. Beauv.]. Shoots of rice and barnyardgrass was more sensitive to fenoxaprop-P-ethyl than the roots. More than 90% of fenoxaprop-P-ethyl was absorbed within 6 hours after treatment and 30% of the absorbed was acropetally and basipetally translocated at 24 hours after treatment. Fenoxaprop-P-ethyl was rapidly transformed to its acid form, fenoxaprop(2-[4-(6-chloro-2-benzoxazolyloxy)phenoxy]propionic acid), which was subsequently metabolized to polar conjugates. However, changes in absorption, translocation, and metabolism of fenoxaprop-P-ethyl by bentazon treatment were not found in both species. Background activity of acetyl-CoA carboxylase(ACCase) in rice and barnyardgrass was 26.5 and 23.2nmol/min/mg, respectively. Concentration required to inhibit fifty percent enzyme activity$(I_{50})$ in vitro was 6.5~7.4${\mu}M$ of fenoxaprop-P-ethyl and more than 500${\mu}M$ of bentazon. There were no significant differences in $I_{50}$ value between two treatments of fenoxaprop-P-ethyl alone and its bentazon mixture. However, bentazon reduced ACCase activity in vivo and inhibited electron transport in chloroplast thylakoid. Based on the results obtained, it is concluded that the antagonistic effect of bentazon occurs due not to direct effect on target site of fenoxaprop-P-ethyl, but to indirect involvement in reducing herbicidal activity of fenoxaprop-P-ethyl through physiological disturbances caused by bentazone at whole chloroplast level.

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Inhibitory Effects of Novel Hexapeptide on Melanogenesis by Regulating MITF in B16F10 Melanoma Cells (B16F10 멜라닌 세포에서 신규 헥사펩타이드의 MITF 조절을 통한 멜라닌 생성 저해 효과)

  • Lee, Eung Ji;Kim, Jandi;Jeong, Min Kyeong;Lee, Young Min;Chung, Yong Ji;Kim, Eun Mi
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.46 no.1
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    • pp.11-22
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    • 2020
  • In this study, we investigated anti-pigmentation effect of a hexapeptide. The peptide significantly reduced melanin contents and inhibited tyrosinase activity in a dose-dependent manner, in which tyrosinase is a key enzyme in melanogenesis. The peptide also significantly reduced the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and their upstream transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, the peptide suppressed the phosphorylation level of cAMP-response element binding protein (CREB), a transcription factor of MITF, and increased the phosphorylation level of extracellular signal-regulated kinase (ERK), a kinase mediates MITF phosphorylation and proteasomal degradation. The peptide significantly inhibited the expression of Rab27A, Melanophilin, and MyosinVa, the components of motor complex involved in intracellular movement of melanosome. These results suggest that Hexapeptide could be used as an effective whitening agent that has inhibitory effect on melanin production and melanosome transport by regulating expression and degradation of MITF in melanocytes.

Electrochemical Immobilization of Osmium Complex onto the Carbon Nano-Tube Electrodes and its Application for Glucose sensor (전기화학적인 방법을 이용한 탄소나노튜브 전극상의 오스뮴 착물의 고정화 및 혈당센서에 관한 응용)

  • Choi, Young-Bong;Jeon, Won-Yong;Kim, Hyug-Han
    • Journal of the Korean Electrochemical Society
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    • v.13 no.1
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    • pp.50-56
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    • 2010
  • The multi-wall carbon nano-tube composite mixed with carbon paste electrode presented more sensitive and selective amperometric signals in the oxidation of glucose than general screen-printed carbon electrodes(SPCEs). Redox mediators to transport electrodes from enzyme to electrodes are very important part in the biosensor. A novel osmium redox complex was synthesized by the coordinating pyridine group containing primary amines which were electrochemically immobilized onto the MWCNT-SPCEs surface. Electrochemical studies of osmium complexes were investigated by cyclic voltammetry, chronoamperometry. The surface coverage of osmium complexes on the modified carbon nano-tube electrodes were significantly increased at 100 time (${\tau}_0=2.0\;{\times}\;10^{-9}\;mole/cm^2$) compared to that of the unmodified carbon electrodes. It's practical application of the glucose biosensor demonstrated that it shows good linear response to the glucose concentration in the range of 0-10 mM.

Enzymatic Properties of Cytochrome Oxidase from Bovine Heart and Rat Tissues

  • Lee, Jae-Yang;Lee, Sang-Jik
    • BMB Reports
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    • v.28 no.3
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    • pp.254-260
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    • 1995
  • Cytochrome oxidase was purified from bovine-heart mitochondria and its enzymatic properties were examined. The purified cytochrome oxidase was identified by its absorption spectrum and chromatogram through gel filtration. The specific activity, purification degree and yield of purified cytochrome oxidase were 18 nmol/mg/ml/min, 24.83 fold and 0.93%, respectively. The activity of the enzyme assayed by a ferrocytochrome $c-O_2$ system was optimized at $25^{\circ}C$ and pH 6.5. Examining the effect of nonionic detergents established that cytochrome oxidase was deactivated by Triton X-100. The oxidase was activated by Tween 80 and deactivated by Tween 20. The Michaelis constant and maximum velocity of the oxidase for ferrocytochrome c were 0.032~0.044 mM and 0.019~0.021 mM/min, respectively. After adaption to basal diet for a week, experimental diets containing 6 mg Cu/kg, or zero mg Cu/kg, or 12 mg Cu/kg were fed to a control group, a copper-free group and a copper-rich group of Sprague-Dawley rats, respectively, for 4 weeks. The specific activities assayed for the ferrocytochrome $c-O_2$ system of isolated cytochrome oxidase from the rat liver of control, copper-free, and copper-rich group were 1.00, 1.19, and 0.878 nmol/mg/ml/min, respectively. Their degrees of purification were 11.38, 10.82 and 8.78 fold, respectively. The specific activities for liver and heart mitochondrial cytochrome oxidase of copper-free/copper-rich groups assayed using the ferrocytochrome $c-O_2$ system were 81.4% and 96.4%/64.1% and 61.1%, respectively, compared with those of the control.

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Genomic Analysis of the Moderately Haloalkaliphilic Bacterium Oceanobacillus kimchii Strain X50T with Improved High-Quality Draft Genome Sequences

  • Hyun, Dong-Wook;Whon, Tae Woong;Kim, Joon-Yong;Kim, Pil Soo;Shin, Na-Ri;Kim, Min-Soo;Bae, Jin-Woo
    • Journal of Microbiology and Biotechnology
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    • v.25 no.12
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    • pp.1971-1976
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    • 2015
  • Oceanobacillus kimchii is a member of the genus Oceanobacillus within the family Bacillaceae. Species of the Oceanobacillus possess moderate haloalkaliphilic features and originate from various alkali or salty environments. The haloalkaliphilic characteristics of Oceanobacillus advocate they may have possible uses in biotechnological and industrial applications, such as alkaline enzyme production and biodegradation. This study presents the draft genome sequence of O. kimchii X50T and its annotation. Furthermore, comparative genomic analysis of O. kimchii X50T was performed with two previously reported Oceanobacillus genome sequences. The 3,822,411 base-pair genome contains 3,792 protein-coding genes and 80 RNA genes with an average G+C content of 35.18 mol%. The strain carried 67 and 13 predicted genes annotated with transport system and osmoregulation, respectively, which support the tolerance phenotype of the strain in high-alkali and high-salt environments.

Effect of Rhodiola Sachalinensis Administration and Endurance Exercise on Insulin Sensitivity and Expression of Proteins Related with Glucose Transport in Skeletal Muscle of Obese Bucker Rat (홍경천 섭취와 운동수행이 비만 쥐의 인슐린 민감도와 골격근내 당수송 관련 단백질 발현에 미치는 영향)

  • Oh Jae-Keun;Shin Young-Oh;Jung Hee-Jung;Lee Jung-Eun
    • Journal of Nutrition and Health
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    • v.39 no.4
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    • pp.323-330
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    • 2006
  • Peripheral insulin resistance in obese/type II diabetes animals results from an impairment of insulin-stimulated glucose uptake into skeletal muscle. Insulin stimulate the translocation of GLUT4 from intracellular location to the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) is implicated in mediation of fusion of GLUT4-containing vesicle with the plasma membrane. Present study investigated regulatory effects of Rhodiola sachalinensis administration and exercise training on the expression of GLUT4 protein and SNAREs protein in skeletal muscles of obese Zucker rats. Experimental animals were randomly assigned into one of five groups ; lean control(LN), obese control(OB), exercise-treated(EXE), Rhodiola sachalinensis-treated(Rho), combine of Rho & EXE (Rho-EXE). All animals of exercise training (EXE, Rho-EXE) performed treadmill running for 8 weeks, and animals of Rho groups (Rho, Rho-EXE) were dosed daily by gastric gavage during the same period. After experiment, blood were taken for analyses of glucose, insulin, and lipids levels. Mitochondrial oxidative enzyme (citrate synthase, CS ; $\beta$-hydroxyacyl-CoA dehydrogenase, $\beta$-HAD) activity were analysed. Skeletal muscles were dissected out for analyses of proteins (GLUT4, VAMP2, syntaxin4, SNAP23). Results are as follows. Exercise and/or Rhodiola sachalinensis administration significantly reduced body weight and improved blood lipids (TG, FFA), and increased insulin sensitivity. Endurance exercise significantly increased the activity of mitochondrial enzymes and the expression of GLUT4 protein, however, administration of Rhodiola sachalinensis did not affect them. The effect of exercise and/or Rhodiola sachalinensis administration on the expression of SNARE proteins was unclear. Our study suggested that improvement insulin sensitivity by exercise and/or Rhodiola sachalinensis administration in obese Zucker rats is independent of expression of SNARE proteins.