• Reproductive and Developmental Biology
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• 제37권3호
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• pp.103-108
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• 2013

An uterus is female reproductive tract organ that affected estrus cycle. During a various changes occur at uterus in estrus cycle, one of them is body fluids secretion be called uterine fluid. Therefore, the objective of this study was to investigate the changes of protein patterns using two-dimensional gel electrophoresis in uterus fluids during the follicular and luteal phases in estrus cycle of pigs. In changes of protein spots were confirmed during the follicular and luteal phases. The 136 spots were expressed in follicular phase, the 57 spots of them showed reproducibility. On the other hand, the 140 spots were expressed in luteal phase, the 73 spots of them showed reproducibility. Also, spots expressed in follicular phase were number 69 and 94 spots and spots expressed in luteal phase only were number 156, 157, 184~187, 190 and 191 spots. The spots which of higher expression levels in the luteal phase than in follicular phase were number 76 and 79 spots. In conclusion, the spots expressed in follicular and luteal phases were confirmed with difference levels and these differences are function of RNA resolving, protein synthesis and cytoskeletal architecture.

• Journal of mucopolysaccharidosis and rare diseases
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• 제3권1호
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• pp.20-27
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• 2017

Purpose: Mucopolysaccharidosis IV (MPS IV) is a disease characterized by deficient activity of N-acetylgalactosamine-6-sulfatase (GALNS) causing excessive lysosomal storage of keratan sulfate (KS). The identification of the relevant disaccharide units of KS after keratanase II digestion followed by liquid chromatography/tandem mass spectrometry detection (LC-MS/MS) is validated and applicable for the preliminary diagnosis of MPS IV. Methods: A total of 67 urine samples were collected and analyzed from 11 MPS IV patients comprising 10 MPS IVA and one MPS IVB patients, and 56 normal controls. Urinary glycosaminoglycan was first precipitated by the Alcian blue method followed by a digestion of keratanase II. The protonated species of the digested disaccharide products were detected by using multiple reaction monitoring experiment. Results: One particular disaccharide of KS was selected. The transition mass-to-charge (m/z) of the parent ion and its daughter ion after collision was $462.0{\rightarrow}97.0$, whereas the chondrosine used as an internal standard in this assay was m/z $353.9{\rightarrow}73.0$. The results corresponded well with the two-dimensional electrophoresis method. The quantities of urinary KS were significantly raised in confirmed MPS IV patients when comparing with those of normal controls ($170.2{\pm}81.1$ vs. $4.06{\pm}1.92{\mu}g/mL$). Conclusion: The LC-MS/MS method for MPS IVA determination is specific, sensitive, validated, and applicable for urinary KS quantification. This method can be used not only as a first-line biochemistry examination of MPS IVA, but also as an outcome survey after enzyme replacement therapy.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.248-254
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• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• Applied Biological Chemistry
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• 제42권1호
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• pp.39-44
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• 1999

각각 다른 성장기의 한우 등심에서 추출한 단백질을 이차원 전기영동법으로 분리하여 젤 상의 단백질 전개 양상을 비교하였다. 성장 0, 6, 12, 24 개월령의 한우 등심 단백질들을 길이 16 cm 튜브젤에서 등전점에 따라 분리하고, 이차원적으로 $18{\times}20$ cm, 12% SDS-polyacrylamide gel 전기영동 하여 단백질을 분리하였다. 등전점 3.0에서 9.0 그리고 분자량 15,000에서 100,000 Da 사이의 단백질들이 분리되어 Silver 염색법으로 명확히 구분할 수 있었다. 흥미롭게, 성장과정에서 단백질 발현이 증가했거나 감소한 단백질들은 저분자 단백질들 이었다. 성장 과정 중 증가된 단백질들을 분리하기 위해 수용성 단백질들을 조직으로부터 1% Triton X-100 으로 추출하였다. 그리고 이를 30%와 50% 황산암모니아로 분획하였다. 이와 같이하여 각 단백질들의 분리조건을 결정하였다. 이들 조건을 이용하여 발현이 증가된 단백질들을 분리하고 PVDF membrane에 옮겨서 아미노산 서열을 결정하여 단백질을 규명하였다.

• 미생물학회지
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• 제31권2호
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• pp.166-174
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• 1993

Streptomyces coelicolor (Muller) 의 세포에 $100 \mu$M 의 과산화수소를 1시간 동안 처리하여 과산화수소 스트레스중에 생성되는 단백질을 L-[$^{35}S$] methionine 을 이용하여 순간 표지하였다. 단백질을 추출하여 2차원 겔 전기영동으로 분석한 결과 지수 성장기의 세포가 가지는 총세포 단백질 중 약 100개의 단백질의 합성이 과산화수소에 의해 증가되는 것을 관찰하였다. 이들을 Pin(peroxide inducible) 단백질이라고 명명하고 과상화수소 처리 후 발현이 증가되는 시간에 따라 세 그룹으로 나누었다. 약 60 개의 Pin 단백질은 과산화수소 처리후 20분 이내에 발현이 증가하여 1시간동안 지속적으로 다량 합성되었다. 정체 성장기의 세포에서는 62개의 단백질의 합성이 과산화수소에 의해 증가되었으며, 이 중 21 개의 단백질은 지수성장기의 Pin 단백질과 일치하였다. 과산화수소에 대한 저항성이 증가한 세가지 돌연변이체의 단백질을 지수 성장기에서 추출하여 2 차원 겔 전기영동으로 분석한 결과, 각각의 경우 15, 17, 15개의 Pin 단백질을 야생형보다 항상적으로 다량합성하는 것을 관찰하였다. 이 pin 단백질 중 9개 (D74.7 a, E76.0c, E23.3, F50.7, F47.2a, F25.5, G39.6b, G24.0, H39.6a) 는 두가지 돌연변이체 모두에게 증가되었다. 따라서 이 단백질들은 S. coelicolor 가 과산화수소 스트레스에 대응하는데 있어 중요한 역할을 담당한다고 추정된다.

• 한국식물병리학회지
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• 제11권4호
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• pp.318-323
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• 1995

The effect of AF-toxin I produced by the strawberry pathotype of Alternaria alternata on the protein synthesis of susceptible strawberry protoplasts was examined by using the radiolabeled amino acids. The incorporation of the radiolabeled amino acids into newly synthesized proteins in the strawberry protoplasts was stimulated by the toxin treatment at relatively low concentrations (2.2$\times$10-11 to 2.2$\times$10-9 M), but not at higher concentrations (2.2$\times$10-8 to 2.2$\times$10-6 M). An one-dimensional SDS-polyacrylamide gel electrophoresis revealed no detectable differences in the proteins synthesized in both the toxintreated and untreated protoplasts. The susceptible strawberry protoplasts were treated with AF-toxin I and stained with Fluostain I to detect the extracellular polysaccharides. The toxin treatment induced the accumulation of extracellular polysccharides in a dose-dependent manner. These results indicate a transient activation of cellular metabolism in the susceptible cells by the toxin exposure.

• Mycobiology
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• 제43권3호
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• pp.272-279
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• 2015

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without ${\alpha}$-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an ${\alpha}$-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at $70^{\circ}C$ for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no ${\alpha}$-crystalline domain in their sequences.

• 한국군사과학기술학회지
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• 제7권4호
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• pp.87-90
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• 2004

In order to elucidate the mechanism of infection on human macrophages, we peformed the 2-dimensional electrophoresis and the western blot analysis using the infected human macrophages with the spores of live and inactivated Sterne. We confirmed P21-activated kinase 2 protein which related to cell death(apoptosis) human macrophages at the early stage events. The inhibition of the P21-activated protein kinase 2 protein will be reduced apoptosis on infected human macrophages with Sterne spores.

• 한국작물학회지
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• 제60권1호
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• pp.29-32
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• 2015

Two-dimensional electrophoresis (2-DE) was executed to separate the seed storage proteins from the buckwheat. The proteins extracted from the whole seed proteins were better separated and observed in the use of lysis buffer. Using this method, the highly reproducible isoelectric focusing (IEF) can be obtained from polyacrylamide gels, and IEF from the polyacrylamide gel at all the possible pH range (5.0-8.0) was more easily separated than IPG (immobilized pH gradient) gels. The polyacrylamide gels in the first dimension in 2-DE was used to separate and identify a number of whole seed proteins in the proteome analysis. In this new apparatus using 2-DE, 27cm in length of plate coated with polyacrylamide gel was used and the experiment was further investigated under the various conditions.