• Title/Summary/Keyword: electrophoretic method

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Downregulation of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in human keratinocytes by melanogenic inhibitors

  • Ahn, Kwang-Seok;Lee, Jinseon;Kim, Yeong-Shik
    • Proceedings of the SCSK Conference
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    • 2003.09a
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    • pp.780-803
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    • 2003
  • Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB ($\textrm{NF}_{-{\kappa}}\textrm{B}$) activators [e.g. tumor necrosis factor-$\alpha$, interleukin-1, lipopolysaccharides, and ultraviolet light] leads to phosphorylation and degradation of the inhibitory protein, $\textrm{I}_{{\kappa}}\textrm{B}$. Liberated $\textrm{NF}_{-{\kappa}}\textrm{B}$ is translocated into the nucleus where it can change or alter expression of target genes, resulting in the secretion of extracellular signaling molecules including melanotrophic factors affecting melanocyte. In order to demonstrate the possible role of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of $\textrm{NF}_{-{\kappa}}\textrm{B}$ induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with $\textrm{pNF}_{-{\kappa}}\textrm{B}$-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the $\textrm{NF}_{-{\kappa}}\textrm{B}$ activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then ultraviolet B (UVB) was irradiated. $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the Mis tested, kojic acid ($IC_{50}$/ = 60 $\mu$M) was found to be the most potent inhibitor of UVB-upregulating $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in transfectant HaCaT cells, which is followed by niacinamide ($IC_{50}$/= 540 $\mu$M). Pretreatment of the transfectant HaCaT cells with the Mis, especially kojic acid and niacinamide, effectively lowered $\textrm{NF}_{-{\kappa}}\textrm{B}$ binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion level of IL-6, one of melanotrophic factors, triggered by UV-radiation of the HaCaT cells. These observations suggest that Mis working at the in vivo level might act partially through the modulation of the synthesis of melanotrophic factors in keratinocyte.

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Comparisons of Physiological Characteristics in Coriolus versicolor Intraspecific Strains (한국산 구름버섯의 균주간(菌株間) 생리적(生理的) 특성(特性) 비교(比較))

  • Park, Young-Do;Whang, Wan-Kyunn;Huh, Jae-Doo;Kim, Seong-Hwan;Park, Won-Mok
    • The Korean Journal of Mycology
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    • v.17 no.1
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    • pp.7-13
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    • 1989
  • This study was executed to investigate characterization of physiological genetic in Coriolus versicolor, basidiomycetes. The optimal media for mycelia culture were PDA, CVT-I and MES as solid media, 927 and CVT-III were good as liquid media, respectively. The optimal condition for mycelial culture was pH 5.6 and $25-30^{\circ}C$. Electrophoretic isozymes and protein patterns from mycelia identified very similar in 16001 and KD88001 strain, but the other species were very diffrent in band patterns. Especially, pattern of esterase seemed to be valuable tool as taxonomic techniques for indentifying species of Coriolus versicolor. Fruiting body was cultivated with artificial logs cultivation method; 16001 and KD88001 were very similar to fruiting body shapes and colours but 16001 and CVT-80 were different in their shapes, colours and making primordia. Therefore, 16001 and KD88001 were assumed to the same strain, but 16001, 16002 and CVT-80 had the different genetic background.

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Proteomic analysis of human serum from patients with temporal lobe epilepsy (측두엽 간질환자의 혈청에서 프로테오믹스기법을 활용한 질병관련 단백질 동정)

  • Lee, Chang Woo;Yu, Seung Taek;Choi, Ha Young;Koh, Bun Jeong;Kwak, Yong Guen
    • Clinical and Experimental Pediatrics
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    • v.52 no.5
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    • pp.567-575
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    • 2009
  • Purpose : Epilepsy affects more than 0.5% of the world's population. It has a large genetic component and is caused by electrical hyperexcitability in the central nervous system. Despite its prevalence, the disease lacks definitive diagnostic serological biomarkers. To identify potential biomarkers for epilepsy by a convenient method, we analyzed the expression of serum proteins, reflecting alterations in the patient's proteomes. Methods : We compared two-dimensional electrophoretic band patterns of human sera from eight patients with temporal lobe epilepsy (TLE) with those of eight control subjects. The differentially expressed bands were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. esults : Twelve proteins were differentially expressed in the TLE group, of which 6 were identified. Expression of haptoglobin Hp2, PRO2675, immunoglobulin heavy chain constant region gamma 2, an unnamed protein, and three unidentified proteins were upregulated in serum from the patients with TLE, whereas those of major histocompatibility complex (MHC) class I antigen, plasma retinol-binding protein precursor, and three unidentified proteins were downregulated in these patients. After resection of the epileptogenic zone, the expressions of MHC class I antigen, immunoglobulin heavy chain constant region gamma 2, two of the downregulated unidentified proteins, and one of the upregulated unidentified proteins returned to the normal range. Conclusion : The 12 serum proteins in this study are potentially useful biomarkers for the diagnosis and monitoring of TLE.

Studies on Soybean Protein [Part ll]-Isolation and Subunit Composition of Multiple 7S Globulins- (대두(大豆) 단백질(蛋白質)에 관(關)한 연구(硏究) 제2보[第二報]-7S Globulin중의 복합단백질(複合蛋白質)의 분리(分離) 및 그 구성(構成) Subunit에 대하여)

  • Lee, C.Y.;Kim, I.S.;Kim, S.U.
    • Applied Biological Chemistry
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    • v.20 no.1
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    • pp.26-32
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    • 1977
  • The multiple 7S globulins composed of two fractions (A and B) in the electrophoresis with Davis' method were isolated at different stages of the soybean seed development. Electrophoresis of their subunits liberated in PAWU solvent [phenol-acetic acid-water (2 : 1 : 1) solution plus 5M urea] yielded 4 major bands. Observation of both the electrophoretic bands of the multiple 7S fractions(7S-A and 7S-B) and those of their subunits was suggestive of a similarity of the subunit pattern between two 7S fractions. The two fractions in multiple 7S globulins were isolated with DEAE-Sephadex A-50 column$(2.0{\sim}100cm)$ chromatography. They were separated into 2 fractions in a linear gradient concentration of 0.28 to 0.40M NaCl with phosphate buffer (pH 7.8) containing 10mM ${\beta}-mercaptoethanol$(ME). The isolated protein was dissociated into subunits with two different solvent systems; in PAWU solvent and in Tris-HCl buffer(pH 8.0) containing 1% sodium dodecyl sulfate (SDS) and 40mM ME. The dissociated subunits were subjected to electrophoresis in PAWU-treated 7.5% acrylamide gel and in 1% SDS-treated 5.6% acrylamide gel. In PAWU gel electrophoresis, total 7S globulin was separated into 5 major bands, two of which were occupied in common by two 7S fractions(7S-A and 7S-B). In SDS gel electrophoresis, total 7S globulin was separated into 7 major bands, three of which were overlapped with the subunit of the two 7S fractions. The above results alluded us to the presence of a common and/or similar subunit between the multiple 7S globulins.

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Characterization and Comparative Evaluation of Milk Protein Variants from Pakistani Dairy Breeds

  • Yasmin, Iqra;Iqbal, Rabia;Liaqat, Atif;Khan, Wahab Ali;Nadeem, Muhamad;Iqbal, Aamir;Chughtai, Muhammad Farhan Jahangir;Rehman, Syed Junaid Ur;Tehseen, Saima;Mehmood, Tariq;Ahsan, Samreen;Tanweer, Saira;Naz, Saima;Khaliq, Adnan
    • Food Science of Animal Resources
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    • v.40 no.5
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    • pp.689-698
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    • 2020
  • The aim of study was to scrutinize the physicochemical and protein profile of milk obtained from local Pakistani breeds of milch animals such as Nilli-Ravi buffalo, Sahiwal cow, Kajli sheep, Beetal goat and Brela camel. Physicochemical analysis unveiled maximum number of total solids and protein found in sheep and minimum in camel. Buffalo milk contains the highest level of fat (7.45%) while camel milk contains minimum (1.94%). Ash was found maximum in buffalo (0.81%) and sheep (0.80%) while minimum in cow's milk (0.71%). Casein and whey proteins were separated by subjecting milk to isoelectric pH and then analyzed through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed heterogeneity among these species. Different fractions including αS1, αS2, κ-casein, β-casein and β-lactoglobulen (β-Lg) were identified and quantitatively compared in all milk samples. Additionally, this electrophoretic method after examining the number and strength of different protein bands (αS1, αS2, β-CN, α-LAC, BSA, and β-Lg, etc.), was helpful to understand the properties of milk for different processing purposes and could be successfully applied in dairy industry. Results revealed that camel milk was best suitable for producing allergen free milk protein products. Furthermore, based on the variability of milk proteins, it is suggested to clarify the phylogenetic relationships between different cattle breeds and to gather the necessary data to preserve the genetic fund and biodiversity of the local breeds. Thus, the study of milk protein from different breed and species has a wide range of scope in producing diverse protein based dairy products like cheese.

Fractionation and Electrophoretic Patterns of Rice Proteins (쌀단백질의 분획 및 전기영동)

  • Kim, Su-Il;Jo, Do-Hyun
    • Applied Biological Chemistry
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    • v.26 no.1
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    • pp.65-72
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    • 1983
  • The composition of four rice protein groups is greatly affected by the extraction conditions. The extraction amounts of albumins and glubulines primarily depended on the temperature rather than the method of extraction. The total amount of glutelins, the major components of rice storage proteins, could be extracted by a successive extraction processes, extraction with 0.5% SDS-0.1M borate buffer(pH 8.3) followed by extraction with 0.5% SDS-0.6% ${\beta}-mercaptoethanol-0.1M$ borate buffer(pH 8.3). The extracted amounts of glutelin with these solvents were 54.1 and 45% respectively. The further purification of SDS soluble glutelins was achieved by Sephadex G-150 gel column chromatography. The molecular weight of the components in four protein groups has been estimated by SDS-polyacrylamide gel electrophoresis with or without ${\beta}-mercaptoethanol.$ The comparison of albumins and globulins by starch gel electrophoresis at pH 3.1 permitted us to identify seven rice varieties. However, at pH 8.95, the specific bands for Japonica type rice varieties were observed.

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Effects of Packaging Method on Quality of Chilled Plaice Muscle (포장방법이 냉장어육의 품질에 미치는 영향)

  • 신완철;송재철;최석영;김미숙
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.32 no.8
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    • pp.1292-1296
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    • 2003
  • The purpose of this study is to investigate the changes of physico-chemical properties of chilled plaice muscle, stored at 4$^{\circ}C$ for 0 ∼ 21 days, with different packaging methods (vacuum packaged with PVDC and aerobic packaged with HDPE). pH value in aerobic packaged plaice muscle (APPM) decreased from 6.3 to 6.09 at first 2 day storage, and then increased gradually during storage time. Although pH pattern of vacuum packaged plaice muscle (VPPM) was similar to that of APPM, change of pH value during storage time was slower and lower than APPM. VBN value in aerobic packaged one increased during storage time. Especially it increased significantly after 7 days of storage. While VBN value in VPPM increased only a little to 14 days. TBA value showed significant difference between APPM and VPPM. WHC of APPM was higher than that of VPPM after 7 days of storage. In electrophoretic pattern of myofibril of APPM stored for 14 days hydrolysis of heavy chain and tropomyosin was observed. However, in VPPM, some hydrolysis occurred only in heavy chain. SDS-PAGE analysis showed that hydrolysis of VPPM occurs later than that of APPM.

Effects of High Pressure Treatment on the Microbiological and Chemical Properties of Milk (초고압 처리가 우유의 미생물학적 및 이화학적 특성에 미치는 영향)

  • Lee, Jieun;Choi, Eun-Ji;Park, Sun Young;Jeon, Ga Young;Jang, Ja-Young;Oh, Young Jun;Lim, Seul Ki;Kim, Tae-Woon;Lee, Jong-Hee;Park, Hae Woong;Kim, Hyun Ju;Jeon, Jung Tae;Choi, Hak-Jong
    • Microbiology and Biotechnology Letters
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    • v.42 no.3
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    • pp.267-274
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    • 2014
  • High pressure processing (HPP) is a non-thermal method used to prevent bacterial growth in the food industry. Currently, pasteurization is the most common method in use for most milk processing, but this has the disadvantage that it leads to changes in the milk's nutritional and chemical properties. Therefore, the effects of HPP treatment on the microbiological and chemical properties of milk were investigated in this study. With the treatment of HPP at 600 MPa and $15^{\circ}C$ for 3 min, the quantity of microorganisms and lactic acid bacteria were reduced to the level of 2-3 log CFU/ml, and coliforms were not detected during a storage period of 15 d at $4^{\circ}C$. An analysis of milk proteins, such as ${\alpha}$-casein, ${\beta}$-casein, ${\kappa}$-casein, ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin by on-chip electorophoresis revealed that the electrophoretic pattern of the proteins from HPP-treated milk was different from that of conventionally treated commercial milk. While the quantities of vitamins and minerals in HPP-treated milk were seen to be comparable to amounts found in raw milk, the enzyme activity of lipase, protease and alkaline phosphatase after HPP treatment was reduced. These results suggest that HPP treatment is a viable method for the control of undesirable microorganisms in milk, allowing for minimal nutritional and chemical changes in the milk during the process.

Effect of Proteinase Activity on the Cheddar Cheese Quality (단백분해 효소 활성(蛋白分解 酵素 活性)이 Cheddar Cheese의 품질(品質)에 미치는 영향(影響))

  • Kim, Min-Bae
    • Journal of Dairy Science and Biotechnology
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    • v.14 no.2
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    • pp.157-164
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    • 1996
  • This study aimed increase the quality during ripening of Cheddar cheese made with proteinase-negative mutant of Streptococcus lactis KCTC 1913 selected by curing. The degradation of protein during cheese ripening were investigated by electrophoresis and chromatography. The results were summarized as follow ; 1. The number of lactic acid bacteria decreased with the ripening stage, and that of the control cheese decreased faster than that of the cheese made with mutant. 2. Polyacrylamide gel electrophoretic analysis of cheese caseins revealed no difference between the cheese made with mutant and the control cheese, but differences along with the ripening stage were evident. 3. On Sephadex G-25 column chromatography, the extracts of bitter components from the green cheese and 3 month ripended cheese were fractionated into 3 fractions. With the progress of ripening, bitter peptides were degraded to rather small peptides or free amino acids. 4. Sensory evaluation of the 3 month ripended Cheddar cheese found no significant differences in color but the cheese made with mutant evidenced higher palatability in flavor and better texture than the control cheese. 5. The yields of the cheddar cheese made with mutant was 0.14% higher than that of the control cheese.

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