• Title/Summary/Keyword: enzymatic assay

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Optimization of γ-Aminobutyric Acid Production Using Lactobacillus brevis spp. in Darae Sap (Lactobacillus brevis 균주를 이용한 다래 수액에서의 감마아미노뷰티르산 (γ-Aminobutyric Acid) 생산 최적화)

  • Jeong, Myeong-Kyo;Jeong, Ji-Hee;Kim, Kwang-Yup
    • Korean Journal of Food Science and Technology
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    • v.48 no.3
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    • pp.214-222
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    • 2016
  • This study was performed to increase the production of ${\gamma}$-aminobutyric acid (GABA) by lactic acid bacteria (Lactobacillus brevis CFM11) and manufacture an optimum medium using the sap from Darae (Actinidia arguta). The concentration of GABA in the fermented sap was determined using GABase enzymatic assay. The isolated L. brevis CFM11 produced $605.67{\mu}g/mL$ GABA after incubation for 24 hours at $37^{\circ}C$ in broth. The sap was fermented by L. brevis CFM11 under optimum conditions of $32^{\circ}C$ for 48 hours with 40% rice bran extract, 1.0% sucrose, 3.0% soytone, 0.2% magnesium sulfate, and 0.2% MSG. The fermented sap produced a concentration of $1366.13{\mu}g/mL$ GABA. These results demonstrate that fermenting Darae sap using L. brevis CFM11 can produce a fermented sap beverage with increased GABA content.

In vitro Comparision of Biological Activities of Solvent Fraction Extracts from Orostachys japonicus (와송(Orostachys japonicus) 용매별 분획 추출물의 항산화, 항균 및 암세포 독성 비교)

  • Kim, Seung Mi;Park, Jeong Hun;Boo, Hee Ock;Song, Sang Gi;Park, Hyeon Yong
    • Korean Journal of Plant Resources
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    • v.30 no.2
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    • pp.133-143
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    • 2017
  • This study was conducted to evaluate the contents of total polyphenol and flavonoid, and the effect of antioxidant, antimicrobial activities and cytotoxicity in vitro by different solvent fractions from Orostachys japonicus. The ethylacetate fraction extract for O. japonicus contained $634.48{\mu}g/g$ polyphenol and $205.20{\mu}g/g$ flavonoid. The ABTS radical scavenging ability of ethylacetate fraction extract at 1 mg/ml was higher than 95% which is comparable to ascorbic acid of 97%. The APX enzymatic activity and CAT activity were $1125.89{\mu}mol$ ascorbate oxidized/min/mg protein and 119.87 H2O2 decomposed/min/mg protein, respectively. In disc agar plate diffusion assay, the extract gave rise to a larger inhibition circle with Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus and Malassezia furfur strains compared with antibiotics kanamycin suggestive of high antibiotic activity. The cytotoxicity of extracts of O. japonicus was significant differences between solvent fractions. That is, the cytotoxic effect against human cancer cell was higher in ethylacetate fraction extract than other fraction extracts. These results suggest that fraction extract of O. japonicus might be very effective and economical in developing natural antioxidant and antimicrobial.

Post-irradiation Changes in Antioxidant Activity of Spindle Tree Leaves from Different Areas (서식지에 따른 사철나무 잎의 이온화 방사선 조사 후 항산화 활성 변화 차이)

  • Kim, Jin-Kyu;Cha, Min-Kyoung;Wilhelmova, Nad'a;Mukherjee, Anita
    • Korean Journal of Environmental Biology
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    • v.29 no.4
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    • pp.280-285
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    • 2011
  • As the plants grow in a fixed place, they can be a good indicator which reflects the level of environmental pollution. It is necessary for them to develop a strategy to cope with stress under unfavorable environmental conditions. In this study, spindle trees ($Euonymus$ $japonica$) were collected from a clean area (Kijang) as well as a heavily polluted area (Onsan) to check applicability of irradiation combined with plant bioassay to environmental monitoring. The leaves were irradiated with 0, 50 and 100 Gy of gamma rays, and then evaluated for antioxidative capacity with 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide dismutase (SOD) analysis. The result shows that there was no significant changes in SOD and EDA (Electron Donationg Ability) in the samples collected from a polluted area. In the meanwhile, SOD increased in the samples from a clean area until 6 to 10 hours after irradiation, then it decreased gradually until 24 hours after irradiation. In conclusion, the plants in the polluted area have developed higher resistance to oxidative stress induced by ionizing radiation than those in the relatively clean area. Irradiation combined with plant bioassay on enzymatic activities and free radical scavenging capacity has proven to be a possible tool for biomonitoring the environmental pollution.

Effect of Growth Hormone Releasing Hormone on the Proliferation of Cultured Cells Derived from Rat Anterior Pituitary Gland (배양중인 흰쥐 뇌하수체 전엽 세포의 증식에 미치는 Growth Hormone Releasing Hormone (GHRH)의 영향)

  • Lee, Sung-Ho
    • Development and Reproduction
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    • v.4 no.2
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    • pp.237-242
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    • 2000
  • Growth hormone releasing hormone (GHRH), the major hypothalamic stimulus of GH secretion from the anterior pituitary gland, has been found to be present in several extrahypothalamic sites including placenta testis, ovary and anterior pituitary gland. The present study was performed to elucidate the role of pituitary GHRH on proliferation of cells derived from rat anterior pituitary gland. The GHRH content of pituitary tissue, cultured pituitary cells, and the conditioned media was evaluated by radioimmunoassay (RIA). Primary cultures of pituitary cells derived from adult rats were prepared by enzymatic dispersion. Significant amounts of GHRH-like molecules were detected in both pituitary tissue and cell cultures by GHRH RIA. Competition curves with increasing amounts of tissue extracts and conditioned media were parallel with those of standard peptide, indicating that the pituitary GHRH-like material is similar to authentic GHRH. To analyze specific cell types responsible for producing GHRH in anteroior pituitary, cell fractionation technique combined with GHRH RIA was performed. In cell fractionation experiment, the highest level of GHRH content was found in gonadotrope enriched-fraction and followed by somatotrope-, lactotrope- and thyrotrope-fraction. Treatment of pituitary cells with GHRH resulted in a dose-dependent increase in [$^3$H] thymidine incorporation. The mitogenic effect of GHRH could be mediated by typical oncogenic activation since the GHRH induced transient increase in c-fos mRNA levels with peak response at 30 minutes. The present study demonstrated that i) the pituitary GHRH expressed in the rat anterior pituitary gland can be secreted, ii) among the various cell types, gonadotropes and somatotorpes are the major GHRH source, and iii) the GHRH treatment increased the [$^3$H] thymidine incorporation and c-fos transcriptional activity in the pituitary cell culture. These findings suggested that GHRH could participated in the paracrine and/or autocrine regulation of cell proliferation, as well as promoting growth hormone secretion.

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α-Glucosidase, Tyrosinase, and Elastase Inhibitory Effects of Enzymatic Extracts from Ecklonia cava and its Alcohol Metabolizing Activity (감태(E. cava Kjellman) 효소분해산물의 항당뇨 및 알코올 분해능과 미용효과)

  • Kim, Hye-Youn;Cho, Eun-Kyung;Kang, Su-Hee;Bae, Jeong-Mi;Choi, Young-Ju
    • Journal of Life Science
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    • v.22 no.6
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    • pp.751-759
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    • 2012
  • Microbulbifer sp. was used to acquire the degrading products from Ecklonia cava (DPEC) and the products were investigated to determine the physiological activities. Firstly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity and superoxide dismutase (SOD) assay were about 84.1% and 89.6% at 2.5 mg/ml, respectively. In addition, nitrite scavenging ability was shown to be 56.3% at 0.5 mg/ml on pH 1.2. ${\alpha}$-Glucosidase inhibitory activity was increased in a dose-dependent manner and was about 58.7% at 2.5 mg/ml. To determine the influence of DPEC on alcohol metabolism, the generating activity of reduced-nicotinamide adenine dinucleotide (NADH) by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were measured. Facilitating rates of ADH and ALDH activities by DPEC were 123.3% and 215.2% at 2.5 mg/ml, respectively. For analyses of anti-wrinkling and whitening effects, its elastase and tyrosinase inhibitory activities were measured and were about 73.1% and 42.2% at 2.5 mg/ml, respectively. These results indicated that DPEC has valuable biological attributes owing to its antioxidant, nitrite scavenging, and alcohol metabolizing activities and ${\alpha}$-glucosidase, elastase, and tyrosinase inhibitory activities.

Effect of Anti-Phospholipid Antibodies and Phospholipids on In Vitro Fertilization and In Vitro Development of Mouse Oocytes (항인지질 자가항체 및 각종 인지질의 처리가 Mouse 난자의 체외수정 및 초기 배발생에 미치는 영향)

  • Ko, J. J.;Chung, H. M.;Shim, S. W.;Kim, N. K.;Lim, J. M.;Lee, H. K.;Park, C.;Kim, S. Y.;Cha, K. Y.
    • Journal of Embryo Transfer
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    • v.13 no.2
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    • pp.89-96
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    • 1998
  • Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

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The Anti-calcification Effect of Dithiobispropionimidate, Carbodiimide and Ultraviolet Irradiation Cross-linking Compared to Glutaraldehyde in Rabbit Implantation Models

  • Park, Samina;Kim, Soo Hwan;Lim, Hong-Gook;Lim, Cheong;Kim, Yong Jin
    • Journal of Chest Surgery
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    • v.46 no.1
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    • pp.1-13
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    • 2013
  • Background: Glutaraldehyde (GA) is a widely used cross-linking agent for improving mechanical properties and resistance to enzymatic degradation of collagenous tissue, but it has several drawbacks such as calcification and cytotoxicity. The aim of this study was to find the alternative effective cross-linking methods to GA. Materials and Methods: Bovine pericardium was processed with GA with ethanol+octanol and glycine detoxification, and polyethylene glycol (PG) space filler, dimethyl 3,3'-dithiobispropionimidate (DTBP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) treatment, and the physical fixation of ultraviolet irradiation were done. The biologic material properties of variously treated pericardial tissues were assessed by biochemical, mechanical and histological tests. Treated pericardial tissues were also implanted subcutaneously or intramuscularly into the rabbit for 10 weeks to assess the xenoreactive antibody response of immunoglobulin G and M, their anti-calcification effect. Results: The biochemical and mechanical properties of EDC fixed pericardial tissues were comparable to the GA fixed tissue. The cytotoxicity was lowest in space filler treated GA fixed group. In rabbit subcutaneous or intramuscular implantation models, decellularization, space filler, EDC treatment group showed significantly lower calcium content than GA only and DTBP treatment group (p<0.05, analysis of variance). The titer of anti $Gal{\alpha}1-3Gal{\beta}1$-4GlcNAc-R antibodies did not change in the postimplantation serial enzyme-linked immunosorbent assay. Hematoxylin and eosin and von Kossa staining showed that decellularization, space filler, EDC, and ultraviolet treatment had less inflammatory cell infiltration and calcium deposits. Conclusion: The decellularization process, PG filler, and EDC treatments are good alternative cross-linking methods compared to GA only fixation and primary amine of DTBP treatment for cardiovascular xenograft preservation in terms of the collagen cross-linking stability and in vivo anti-calcification effects.

Transition Temperature of Mitochondrial Respiratory Activity as an Index for Chilling Susceptivity of Varieties of Rice Plant (미토콘드리아 호흡활성 전이온도의 측정에 의한 벼품종별 상대적 내냉성판정)

  • Jung, Jin;Kim, Hee-Jung
    • Applied Biological Chemistry
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    • v.30 no.4
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    • pp.357-363
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    • 1987
  • The transition temperature of respiratory activity of mitochondria isolated from panicles of rice plant was measured with 41 varieties bred in Korea, which had been agronomically classified into five groups based on their relative cold-tolerance; S (strong cold-tolerance), S-M (moderate to strong cold-tolerance), M (moderate cold-tolerance), M-W (moderate to weak cold-tolerance) and W (weak cold-tolerance). The temperature was found to be $11.5{\sim}13.5^{\circ}C$ for group S, $13.5{\sim}16^{\circ}C$ for group S-M, $16{\sim}17^{\circ}C$ for group M, $17{\sim}18^{\circ}C$ for group M-W, and ${\geq}18^{\circ}C$ for group W respectively with a few exceptions of minor deviation, demonstrating a close correlation between the transition temperature and the chilling susceptivity of a variety. This observation supports the suggestion that the transition temperature of respiratory activity of panicles be adopted as a reliable index for grading a rice variety as to its overall cold tolerance. Also studied is on the methodology for measurement of the transition temperature in the aspect of analytical biochemistry. In addition to the usual polarographic method, a spectrophotometric method which is based on the enzymatic assay of membrane-bound cytochrome C oxidase was examined. This enzyme was found previousely and confirmed again in the present work to be a key factor governing the respiratory trantion at least in plant mitochondria. The optical method was turned out to be very successful, although it was a little more complicate and time-consuming than the electrochemical method.

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Identification and Characterization of Protein Encoded by orf382 as $\small{L}$-Threonine Dehydrogenase

  • Ma, Fei;Wang, Tianwen;Ma, Xingyuan;Wang, Ping
    • Journal of Microbiology and Biotechnology
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    • v.24 no.6
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    • pp.748-755
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    • 2014
  • In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

The Relationship between Serum Lipid Levels and Psychologic Characteristics (혈청 지질 농도와 심리적 특성의 관련성)

  • Lee, Jun-Suk;Lee, Jang-Han;Yang, Byung-Hwan;Ji, Yong-Jin
    • Korean Journal of Psychosomatic Medicine
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    • v.9 no.2
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    • pp.194-202
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    • 2001
  • Objectives : Many studies have shown an association between serum lipid concentrations and psychologic characteristics. However, conflicting results have also been reported. This study was designed to find the relationship between serum lipid levels(cholesterol and triglycerides) and psychologic characteristics(impulsiveness, aggressiveness, depressiveness) in adolescents. Methods : Serum cholesterol concentration, serum triglycerides concentration, complete blood count, electrolytes, liver function test, and blood sugar level were measured by overnight fasting blood sampling and urinalysis was also conducted in 407 high school students. Impulsiveness level was measured by Barratt Impulsiveness Scale(BIS). Aggressiveness level was measured by Buss-Durkee Hostility Inventory(BDHI). Depressiveness level was measured by Beck Depression Inventory(BDI). Serum cholesterol was measured by standard enzymatic assay and serum triglycerides was measured by Boehringer Mannheim method. Results : 1) Low cholesterol group(<15 percentile) were found to have significantly higher BIS score than high cholesterol group(>85 percentile) in female. Female low cholesterol group were also found to have significantly higher motor impulsivity score, a subscale of BIS, than high cholesterol group. 2) High triglycerides group(>85 percentile) were found to have significantly higher BDI score than low triglycerides group(<15 percentile) in male and female separately. Especially, male high triglycerides group were also found to have significantly higher BIS and motor impulsivity score than low triglycerides group. Conclusion : These results support the previous hypothesis that serum lipid levels(cholesterol and triglycerides) affect human psychologic characteristics.

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