• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.207-211
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• 2004

The purpose of the present study was to evaluate the expression of cardiac marker protein in rabbit cardiac tissue that was exposed to ischemic preconditioning (IPC), or ischemiareperfusion injury (IR) using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). We compared 2DE gels of control (uninjured) cardiac tissue with those of IPC and IR cardiac tissue. Expression of one protein was detected in IR heart tissue, however the protein was not detected in the samples of control and IPC tissue. To further characterize the detected protein molecule, the protein in the 2D gel was isolated and subjected to trypsin digestion, followed by MALDI-MS. The protein was identified as myoglobin, which was confirmed also by Western blot analysis. These results are consistent with previous studies of cardiac markers in ischemic hearts, indicating myoglobin as a suitable marker of myocardial injury. In addition, the present use of multiple techniques indicates that proteomic analysis is an appropriate means to identify cardiac markers in studies of IPC and IR.

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.97-108
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• 2000

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.

• Food Science of Animal Resources
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• v.26 no.2
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• pp.263-268
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• 2006

Probiotics including Lactobacillus acidophilus, refer to a group of nonpathogenic organisms that protect the human host against gastrointestinal(GI) infections by pathogenic bacteria such as Shiga toxin-producing E. coli(STEC). In the study, the inhibitory effects of STEC ATCC 43894 adhesion by L. acidophilus A4 was investigated on the HT-29 epithelial cells. Specific proteins regulated by cell Iysates of L. acidophilus A4 on STEC ATCC 43894 were also characterized by proteomic analysis. Both cell mass and Iysate of L. acidophilus A4 have exhibited the profound inhibitory activity on the HT-29 cells(about 1.5 log scale reduction). Two-dimensional gel electrophoresis(2-DE) revealed seven proteins that were up-regulated by cell Iysates of L. acidophilus A4 and three proteins that were down-regulated. In addition, three protein spots were only detected in the presence of cell Iysates. These results suggest that inhibitory effects of STEC adhesion by L. acidophilus may be due to the regulation of specific protein of STEC.

• Applied Chemistry for Engineering
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• v.10 no.1
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• pp.160-165
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• 1999

Agarose gels can be used as reference standards for the measurement of fluid properties in porous media because the relaxation properties of the gel reference standard and those of the fluid in porous media can be closely matched. The use of reference standard to determine porosity and saturation is discussed and the requirements for gel NMR properties given. The relaxtion times of agarose gels measured at 2.0 Tesla are illustrated as a function of agarose and paramagnetic impurity ($CuSO_4$) concentrations. This work shows an empirical result between agarose gel composition and gel relaxtion times. The average value for the porosity distribution is 17.7%, which compares well with the value calculated with the gravimetric analysis. Finally, two phase immiscible displacement using agarose gels as a reference standard was performed. The saturation profiles appear to be consistent with what one might calculate for a one-dimensional displacement in a uniform porous media.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.39-44
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• 1999

Protein profiles of beef loin were constructed by comparing two-dimensional gel electrophoresis patterns of the proteins from different growth stages of Hanwoo, Korean cattle. Proteins from the lean muscle of 0, 6, 12 and 24 months old Hanwoo were separated by isoelectric focusing (IEF) on 16 cm tube gels, and further processed second dimensionally by 12% SDS-polyacrylamide gel electrophoresis (PAGE) using $18{\times}20$ cm gel. Proteins with pI values ranging 3.0 to 9.0 and molecular weight of 15 to 100 kDa could be clearly detected on gel by silver staining. Interestingly, many of the proteins significantly increased and decreased during growth happened to be low molecular ones. To isolate the increased proteins, the soluble proteins were obtained from the tissue by 1% Triton X-100 extraction, then, fractionated by 30% and 50% ammonium sulfate. The isolation condition of each particular protein was determined. According to the conditions, two of the increased proteins were isolated, and transferred to PVDF membrane and microsequenced.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.90-90
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• 2003

체세포 핵이식에 의한 복제기술은 매우 낮은 성공률 나타내고 있어 실용화에 지장을 초래하고 있다. 이것은 후생적인 유전현상인 reprogramming이 불완전하게 이루어지기 때문인 것으로 추측되어지고 있다(Reik et al., Theriogenology 2003, 59: 21-32; Han et al, Theriogenology 2003, 59: 33-44). 체세포 핵이식 후에 태아사망의 원인이 태반의 비정상적인 기능과 관계가 있는 것으로 추정되는데 복제시 태아사망의 원인을 찾기 위해 본 연구를 시행하였다. 한우에서 체세포 복제 후 임신 말기에 태아가 사망한 태반조직 3개와 IVF 수정란 이식 후 동일한 시기에 제왕절개술을 실시한 태반조직 2개를 실험에 이용하였다. 태반 protein을 Two-Dimensional electrophoresis와 Mass spectrometer를 이용하여 분석 비교하였다. IPG-system을 이용하여 pH 4~7, pH 6~9에서 1차 전기영동을 한 후, 8~l6%의 SDS-PAGE gel에 2차 전기영동을 실시하였고 G-250 Coomassie로 염색하였다. gel 이미지는 Malanie III program을 이용하여 분석하였다. 전체 gel에서 약 1800개의 구분 가능한 protein spot이 나타났다. pH 4~7 범위에서 양적으로 차이나는 것 15개 중 복제한우 태반에서 증가되는 protein spot 5개와 감소하는 protein spot 10개를 골라 protein identification을 실시하였다. MALDI-TOF-MS를 이용하여 동정한 결과 phosphatidylinositol transfer protein-$\alpha$와 interleukin-18 등의 protein이 복제태반에서 발현이 증가되었고, 복제한우에서 발현이 감소되는 것으로는 vimentin, Rho-GDI-$\beta$, TRAST $\beta$-chain, ovarian sterol carrier protein 2, triosephosphate isomerase, tropemyosin beta chain, Aldose reductase 등으로 나타났다. 이러한 protein들은 inositol 지질 신호전달과 면역시스템, 세포분열, 산소 운반, steroidogenic 세포에서의 콜레스테롤 이동, 촉매 작용, 대사 작용 등에 중요한 역할을 하는 것으로 알려져 체세포 복제에 의한 태아사망 원인은 태반에서 이러한 protein들의 비정상적인 발현에 기인된 것으로 추정된다.

• Journal of Microbiology
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• v.44 no.4
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• pp.369-374
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• 2006

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis $D-8^T$. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain $D-8^T$ are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and $Imagemaster^{TM}$ 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, S proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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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.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.166-174
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• 1993

Streptomyces coeUc%r (Muller) cells were treated with $100 \mu$M hydrogen peroxide for I hour and proteins synthesized during hydrogen peroxide stress were labeled with L-[$^{35}S$]-methionine. Total cellular proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. In exponential growth phase, synthesis of about 100 proteins was increased by hydrogen peroxide treatment. These proteins were named as Pin (£eroxide-inducib]e) proteins and classified into 4 subgroups according to their induction time after hydrogen peroxide treatment. About 60 of them were found to be induced within 20 minutes and maintained throughout I hour of treatment. In stationary growth phase. synthesis of 62 proteins was increased by hydrogen peroxide and 21 of them were the same Pins found in exponential growth phase. Proteins from the mutants which are resistant to hydrogen peroxide were obtained in exponential growth phase and compared with those from the wild type on two-dimensional gel. The three mutants, N7, N9. and N24, were found to have higher constitutive leve]s of ]5, 17, and 15 Pin proteins respectively, than the wild type. 9 of these Pin proteins (D74.7a, E76.0c, E23.3. F50.7, F47.2a. F25.5, G39.6b, G24.0, H39.6a) increased in two of the three mutants and 3 proteins (F39.7, H6I.7. 120.8) increased in all of the three mutants. These proteins might play important roles in the response of S. coelic%r to hydrogen peroxide.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.1
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• pp.68-72
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• 2007

Flounder is one of the major aquacultured fish and of an economically important item in Korean fisheries. Recently, there are trends of research worldwide that aim to analyze and characterize a whole genome or a whole proteome of interesting species. The data are utilized for the understanding and development of preventive and curative technologies for the serious diseases. However, there are very limited information of proteome for marine organisms, we optimized first the conditions for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with serum form a marine fish, flounder (Paralichthys olivaceus). A pre-treatment of serum and an optimization of protein concentration analyzed were surveyed for enhancing the separation for proteins. A statistical analysis was performed on the overall 1,820 protein spots to overcome the variability among individual fishes.