• Asian-Australasian Journal of Animal Sciences
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• v.18 no.7
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• pp.922-926
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• 2005

The present study was aimed to investigate proteome affected by Panax ginseng extracts in chicken muscles. The whole muscle proteins from chicken fed boiled extracts of 0% (control), 1%, 3%, and 5% Panax ginseng in water were separated by two-dimensional electrophoresis (2-DE) gels using immobilized non-linear gradient (pH 3-10) strips. More than 300 protein spots were detected on silver staining gels. Among them, four protein spots were distinctively up-regulated by Panax ginseng treatments and further investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The obtained MS data were searched against SwissProt database using the Mascot search engine. The up-regulated proteins were finally identified as $\alpha$-tropomyosin (2 spots), triosephosphate isomerase, and one unknown protein. Based on the known functions of the identified proteins, they are highly related to muscle development and enhanced immunity in chickens. These proteins can give valuable information of biochemical roles for Panax ginseng in chicken meats.

• Food Science of Animal Resources
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• v.33 no.2
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• pp.281-286
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• 2013

Salmonella Gallinarum (SG) is known as an important pathogen that causes fowl typhoid in chickens. To investigate SG outer-membrane proteins (OMPs) as a vaccine candidate, we used proteomic mapping and database analysis techniques with extracted OMPs. Also, extracted OMPs were evaluated in several aspects to their safety, immune response in their host and protective effects. Our research has established a proteomic map and database of immunogenic SG-OMPs used as inactive vaccine against salmonellosis in chickens. A total of 22 spots were detected by 2-dimensional gel electrophoresis and immunogenic protein analysis. Eight spots were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprinting (PMF) and categorized into four different types of proteins. Among these proteins, OmpA is considered to be an immunogenic protein and involved in the hosts' immune system. To estimate the minimum safety dose in chickens, 35 brown layers were immunized with various concentrations of OMPs, respectively. Consequently, all chickens immunized with more than a $50{\mu}g$ dose were protected against challenges. Moreover, intramuscular administration of OMPs to chickens was more effective compared to subcutaneous administration. These results suggest that the adjuvanted SG-OMP vaccine not only induces both the humoral and cellular immune response in the host but also highly protects the hosts' exposed to virulent SG with $50{\mu}g$ OMPs extracted by our method.

• Proceedings of the Korean Society of Life Science Conference
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• 2002.12a
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• pp.20-47
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• 2002

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed In human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected on silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained by colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsln, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser dosorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, maps of lower resolution, i.e. overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.2005-2017
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• 2007

Diabetic nephropathy remains a major cause of morbidity and mortality in the diabetic population and is the leading cause of end-stage renal failure. Despite current therapeutics including intensified glycemic control and blood pressure lowering agents, renal disease continues to progress relentlessly in diabetic patients, albeit at a lower rate. Since synthetic drugs for diabetes are known to have side effects, fungal mushrooms as a natural product come into preventing the development of diabetes. Our previous report showed the hypoglycemic effect of extracellular fungal polysaccharides (EPS) in streptozotocin (STZ)-induced diabetic rats. In this study, we analyzed the differential expression patterns of rat kidney proteins from normal, STZ-induced diabetic, and EPS-treated diabetic rats, to discover diabetes-associated proteins in rat kidney. The results of proteomic analysis revealed that up to 500 protein spots were visualized, of which 291 spots were differentially expressed in the three experimental groups. Eventually, 51 spots were statistically significant and were identified by peptide mass fingerprinting. Among the differentially expressed renal proteins, 10 were increased and 16 were decreased significantly in diabetic rat kidney. The levels of different proteins, altered after diabetes induction, were returned to approximately those of the healthy rats by EPS treatment. A histopathological examination showed that EPS administration restored the impaired kidney to almost normal architecture. The study of protein expression in the normal and diabetic kidney tissues enabled us to find several diabetic nephropathy-specific proteins, such as phospholipids scramblase 3 and tropomyosin 3, which have not been mentioned yet in connection with diabetes.

• Journal of The Korean Society of Grassland and Forage Science
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• v.34 no.4
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• pp.262-268
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• 2014

In order to reveal the aluminum (Al) stress tolerance mechanisms in alfalfa plant at low pH soil, a proteomic approach has been conducted. Alfalfa plants were exposed to Al stress for 5 days. The plant growth and total chlorophyll content are greatly affected by Al stress. The malondialdehyde (MDA) and $H_2O_2$ contents were increased in a low amount but free proline and soluble sugar contents, and the DPPH-radical scavenging activity were highly increased. These results indicate that antioxidant activity (DPPH activity) and osmoprotectants (proline and sugar) may involve in ROS ($H_2O_2$) homeostasis under Al stress. In proteomic analysis, over 500 protein spots were detected by 2-dimentional gel electrophoresis analysis. Total 17 Al stress-induced proteins were identified, of which 8 protein spots were up-regulated and 9 were down-regulated. The differential expression patterns of protein spots were selected and analyzed by the peptide mass fingerprinting (PMF) using MALDI-TOF MS analysis. Three protein spots corresponding to Rubisco were significantly down-regulated whereas peroxiredoxin and glutamine synthetase were up-regulated in response to Al stress. The different regulation patterns of identified proteins were involved in energy metabolism and antioxidant / ROS detoxification during Al stress in alfalfa. Taken together, these results provide new insight to understand the molecular mechanisms of alfalfa plant in terms of Al stress tolerance.

• Korean Journal of Veterinary Research
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• v.50 no.4
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• pp.285-295
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• 2010

Protein expression patterns in Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strains with diverse phenotypes, such as phage type, antibiotic resistance pattern and plasmid profiles were examined. For detailed analysis of proteins expressed by different S. Typhimurium strains, protein fractions were divided into detergent-rich phase (DP) and aqueous phase (AP) using triton X-114 detergent. The two phases were subjected to two-dimensional gel electrophoresis (2-DE), followed by protein identification using peptide mass fingerprinting (PMF). In the results, PMF showed that DP fractions consisted mainly of outer membrane proteins, whereas the AP fractions included cytosolic proteins. Comparison of 2-DE profiles of DP did not show any distinct protein spots which could be correlated with phage type, antibiotic resistance pattern or plasmid profile. However, comparisons of 2-DE profiles of the AP revealed differences in the protein spots, which could be correlated with the plasmid profile and phage types. Among these protein spots, flagellin was specific for strains containing a 90 kb plasmid. Compared to DT193 phage type, three protein spots in the range of pI 5.0-5.5 and MW 8-15 kDa of AP 2-DE profiles were absent in the DT104 phage types. Additionally, a protein spot with PI in the range of 4.5-5.0 and molecular weight (MW) between 51-69 kDa was specific for phage type DT104, while a protein spot with pI in the range of 4.0-4.8 and MW between 18-20 kDa was specific for DT193 phage type. These protein spots may be useful for discriminating phage types of S. Typhimurium.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.32-32
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• 2003

소 난자의 체외성숙 과정에서 세포질 내 단백질의 생산과 축적의 변화는 핵 및 세포질 성숙과 밀접한 관계가 있다는 보고가 있지만, 난자의 성숙과 관련된 특정 단백질의 종류에 대한 보고는 없었다. 따라서 본 연구는 한우 난자의 체외성숙과 관련된 단백질의 생산 및 축적의 변화와 그 종류에 대해서 검토하였다. 체외성숙 시간(4.5, 9, 13.5, 18 및 24시간)에 따른 배양액 내의 단백질 합성의 변화는 2D gel electrophoresis를 이용하였고, 단백질 spot에 대해서는 peptide mass fingerprinting(PMF) 방법을 이용하였다. 또한 단백질 측정 시간에 신선 체외성숙 배양액으로 교환 후 난포란의 핵성숙과 배발달율을 검토하였다. 그 결과 한우 난포란의 체외성숙 시간에 따라 배양액에서 단백질의 양 및 질적인 변화를 확인하였다. 그리고 총 296개 단백질 spot들을 확인하였고, 그 중 30개 spot에서 유의적인 변화가 인정되었다. 또한 유의적인 변화를 보인 spot에 대한 PMF 분석을 통하여 Apolipoprotein A-1 precursor, Alpha enolase, Aldose reductase, 43kDa collectin precursor, Heat shock 27kDa protein, Plasminogen activator inhibitor-1 precursor, Thrombospondin 1 및 Transitional endoplasmic reticulum ATPase가 동정되었다 그리고 총 단백질 합성 경향은 0∼4.5 시간에는 감소하였고, 13.5∼18시간에 증가 한 후 다시 감소하는 경향을 나타내었으며, 단백질의 종류도 시간대별로 현저한 변화가 있었다. 한편 단백질을 측정하는 시기에 신선 체외성숙 배양액으로 교환한 후 난포란의 핵성숙 및 배발달율을 검토한 결과 18시간 체외성숙군에서 9시간째의 교환이 유의적으로 높은 핵성숙을 나타내었으나, 배발달율에서는 유의성이 인정되지 않았다. 그러나 24시간 체외성숙군에서 18시간째의 배양액 교환은 8세포기 및 배반포 발달율이 유의적(P<0.05)으로 높았다. 연구 결과로부터 소 난자의 체외성숙 시간에 따른 단백질 합성 경향의 차이를 확인하였고, 유의적인 변화를 나타낸 8가지의 단백질을 분리할 수 있었으며, 향후 이들 작용기전에 대한 연구가 필요하다고 사료된다.

• KSBB Journal
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• v.31 no.4
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• pp.228-236
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• 2016

The aim of this study was to characterize the hemolytic Aeromonas sp. MH-8 exposed to green tea catechin, epigallocatechin gallate (EGCG). Initially, the hemolytic Aeromonas sp. MH-8 was enriched and isolated from stale fish. Bactericidal effects of MH-8 exposed to EGCG ranging from 1 mg/mL to 4 mg/mL were monitored, and complete bactericidal effects were achieved within 3 h at 3 mg/mL and higher concentrations. SDS-PAGE with silver staining revealed that the amount of lipopolysaccharides increased or decreased in the strain MH-8 treated to different concentrations and exposing periods of EGCG in exponentially growing cultures. The stress shock proteins (70-kDa DnaK and 60-kDa GroEL), which might contribute to enhancing the cellular resistance to the cytotoxic effect of EGCG, were induced at different concentrations of EGCG exposed to cell culture of MH-8. Scanning electron microscopic analysis demonstrated the presence of irregular rod shapes with umbilicated surfaces for cells treated with EGCG. 2-DE of soluble protein fractions from MH-8 cultures showed 18 protein spots changed by EGCG exposure. These proteins involved in chaperons (e.g., DnaK, GroEL and trigger factor), enterotoxins (e.g., aerolysin and phospholipase C precursor), LPS synthesis (e.g., LPS biosynthesis protein and outer membrane protein A precursor), and various biosynthesis and energy metabolism were identified by peptide mass fingerprinting using MALDI-TOF. In consequence, EGCG was found to have substantial antibacterial effects against food-poisoning causing bacterium, hemolytic Aeromonas sp. MH-8. Also the results provide clues for understanding the mechanism of EGCG-induced stress and cytotoxicity on Aeromonas sp. MH-8.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.516-525
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• 2012

LAB were isolated from makgeolli locally produced around Jinju, Gyeongnam, S. Korea during spring of 2011. Randomly selected 11 isolates from MRS agar plates were identified first by API CHL 50 kits and then 16S rRNA gene sequencing. All 11 isolates were identified as Lactobacillus plantarum. Among them, ST4 grew in MRS broth with ethanol up to 10%, showing the highest alcohol resistance. L. plantarum ST4 was moderately resistant against acid and bile salts. When cellular proteins of L. plantarum ST4 under ethanol stress were analyzed by two-dimensional gel electrophoresis (2DE), the intensities of 6 spots increased, whereas 22 spots decreased at least 2-fold. Those 28 spots were identified by peptide mass fingerprinting (PMF). FusA2 (elongation factor G) increased 18.8-fold (6% ethanol) compared with control. Other proteins were AtpD (ATP synthase subunit beta), DnaK, GroEL, Tuf (elongation factor Tu), and Npr2 (NADH peroxidase), respectively. Among the 22 proteins decreased in intensities, lactate dehydrogenases (LdhD and LdhL1) were included.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.532-541
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• 2010

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.