• Reproductive and Developmental Biology
• /
• 제33권4호
• /
• pp.237-242
• /
• 2009

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The uterine proteins were separated using 2-DE, Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty-one spots were identified as differentially expressed proteins, of which 10 were up-regulated proteins such as alpha-fetoprotein, chloride intracellular channel 1, transgelin, heat-shock protein beta-1, and carbonic anhydrase II, while 11 were down-regulated proteins such as X-box binding protein, glutathione S-transferase omega 1, olfactory receptor Olfr204, and metalloproteinase-disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.

• Reproductive and Developmental Biology
• /
• 제33권4호
• /
• pp.243-248
• /
• 2009

To examine the differential expression of proteins during the cycling (70~80% confluences) and G0/G1 (full confluences) phases in porcine fetal fibroblast cells, we used a global proteomics approach by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. Cycling cell were harvested at approximately 70% to 80% confluent state while cells in G0/G1 phase were recovered after maintenance of a confluent state for 48 hr. Cellular proteins with isoelectric points ranging between 3.0~10.0, were analyzed by 2-DE with 2 replicates of each sample. A total of approximately 700 spots were detected by 2.D gels stained with Coomassie brilliant blue. On comparing the cell samples obtained from the cycling and G0/G1 phases, a total of 13 spots were identified as differentially expressed proteins, of which 8 spots were up-regulated in the cycling cell and 5 were up-regulated in the G0/G1 phase. Differentially expressed proteins included K3 keratin, similar to serine protease 23 precursor, protein disulfide-isomerase A3, microsomal protease ER-60, alpha-actinin-2, and heat-shock protein 90 beta. The identified proteins were grouped on the basis of their basic functions such as molecular binding, catabolic, cell growth, and transcription regulatory proteins. Our results show expression profiles of key proteins in porcine fetal fibroblast cells during different cell cycle status.

• Journal of Microbiology
• /
• 제45권5호
• /
• pp.402-408
• /
• 2007

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around $50^{\circ}C$. Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of $Ca^{2+},\;Zn^{2+},\;Mg^{2+}$, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.

• 한국축산식품학회지
• /
• 제33권2호
• /
• pp.281-286
• /
• 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.

• 대한전기학회:학술대회논문집
• /
• 대한전기학회 2006년도 제37회 하계학술대회 논문집 C
• /
• pp.1629-1630
• /
• 2006

This paper describes a micro total analysis system ($\mu$ TAS) for detecting and digesting the target protein which includes a bead based temperature controllable microchip and computer based controllers for temperature and valve actuation. We firstly combined the temperature control function with a bead based microchip and realized the on-chip sequential reactions using two kinds of beads. The PEG-grafted bead, on which RNA aptamer was immobilized, was used for capturing and releasing the target protein. The target protein can be chosen by the type of RNA aptamer. In this paper, we used the RNA aptamer of HCV replicase. The trypsin coated bead was used for digesting the released protein prior to the matrix assisted laser desorption ionization time of flight mass spectrometer (MALDI TOF MS). Heat is applied for release of the captured protein binding on the bead, thermal denaturation and trypsin digestion. PDMS microchannel and PDMS micro pneumatic valves were also combined for the small volume liquid handling. The entire procedures for the detection and the digestion of the target protein were successfully carried out on a microchip without any other chemical treatment or off-chip handling using $20\;{\mu}l$ protein mixture within 20 min. We could acquire six matched peaks (7% sequence coverage) of HCV replicase.

• 한국환경농학회지
• /
• 제30권4호
• /
• pp.395-401
• /
• 2011

BACKGROUND: Potassium (K) is one of the macronutrients which are essential for plant growth and development. Its deficiency in paddy soils is becoming one of the limiting factors for increasing rice yield in Asia. METHODS AND RESULTS: To investigate physiological symptoms under K-starvation (NP) compared with complete media (NPK) condition, we measured shoot/root length, weight, nutrients, and patterns of protein expression. The shoot growth was significantly reduced, but root growth was not affected by K-starvation. However, biomasses were decreased in both shoot and root. Uptake of K was reduced up to 85%, while total concentrations of P, Ca, Mg, Na were increased in root and shoot. To better understand the starved K mechanism of rice, comparative proteome analysis for proteins isolated from rice leaves was conducted using 2-DGE. Five spots of differentially expressed proteins were analyzed by MALDI-TOF MS. Analysis of these K-starvation response proteins suggested that they were involved in metabolism and defense. CONCLUSION(s): Physiological and 2-DGE based proteomics approach used in our study results in observation of morphology or nutrients change and identification of K-starvation responsive proteins in rice root. These results have important roles in maintaining nutrient homeostasis and would also be useful for further characterization of protein function in plant K nutrition.

• 한국균학회지
• /
• 제38권1호
• /
• pp.57-61
• /
• 2010

본 연구에서는 버섯으로부터 새로운 항비만 치료(예방) 효과를 갖는 물질을 추출, 정제하여 그 특성을 규명하고, 이를 기능성식품 개발의 소재로 용용하고자 lipase 저해 활성이 높은 상황버섯(Phellinus linteus)의 lipase 저해 물질을 각종 chromatography로 정제 하였다. 먼저 상황버섯(Phellinus linteus) 자실체 추출물에 대한 용매 계통분획을 실시하여 저해 활성이 가장 높은 에틸아세테이트 추출물을 얻었고 이를 3차례의 sillicas gel column chromatography와 분취용 HPLC를 실시하여 $IC_{50}$값이 175 ng의 lipase 저해활성을 가진 연한 연두색 분말의 정제된 lipase 저해 물질을 얻었다. 정제한 lipase 저해물질을 일련의 NMR spectrometry를 실시한 결과 분자량이 523.06 Da으로 추정되었고 225.1 nm에서 최대 흡수 파장을 가진 물질이었다.

• 방사선산업학회지
• /
• 제6권2호
• /
• pp.129-138
• /
• 2012

Proteomic analysis was performed in order to identify proteomic changes by salt stress between the Japonica cv. Donganbyeo (WT) and two salt-tolerant (ST) mutant lines by using the SDS-PAGE and 2-DE. Two salt tolerant rice mutant lines, ST-87 and ST-301, were selected by in vitro mutagenesis with gamma-ray. Three-week-old seedlings were treated with 171 mM NaCl for 7 days. In the SDS-PAGE, three proteins with molecular weights of 27, 46 and 58 kDa were highly increased under salt treatment. Total proteins from shoots of both WT and ST-lines were separated by two-dimensional gel electrophoresis. In 2-DE, 201, 226, 217 and 213 protein spots were detected in the untreated-or treated-WT and untreated- or treated-ST-87, respectively. Of theses, 17 and 10 protein spots were up- and down-regulated under salt stress in the WT, respectively. While, 16 and 8 protein spots were up- and down-regulated under salt stress in the ST-87, respectively, compared with the untreated plants. High intensity or de novo synthesized proteins were analyzed by MALDI-TOF/MS analysis.

• 한국육종학회지
• /
• 제41권3호
• /
• pp.205-212
• /
• 2009

Wheat (Triticum aestivum L.) grain texture is an important determinant of milling properties and end product use. Two linked genes, puroindoline a (PINA) and puroindoline b (PINB), control most of the genetic variation in wheat grain texture. Wheat seed proteins were examined to identify PINA and PINB gene using two pre-harvest sprouting wheat cultivars; Jinpum (resistant) and Keumgang (susceptible).Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses and complete gene sequence were found by UniProt database. Puroindoline a and puroindoline b that is responsible for grain texture related with baking performance and roughness. Two spots were found Pin b (16.7 kDa) and Pin a (16.3 kDa) in Jinpum compare to seven spots were identified Pin a (16.1 kDa, 16.3 kDa) and Pin b (16.7 kDa, 9.5 kDa and 14.4 kDa) in Keumgang. Some selected spots were identified puroindoline like grain softness protein (16.9 kDa, 17 kDa and 18.1 kDa) in Keumgang. Moreover, to gain a better inferring the identification of puroindoline related proteins using proteomics, we accomplished a complete gene sequence of PINA and PINB gene in pre-harvesting sprouting wheat seeds between resistant (Jinpum) and susceptible (Keumgang).

• Journal of Microbiology and Biotechnology
• /
• 제33권6호
• /
• pp.753-759
• /
• 2023

The genus Paenibacillus contains a variety of biologically active compounds that have potential applications in a range of fields, including medicine, agriculture, and livestock, playing an important role in the health and economy of society. Our study focused on the bacterium SS4^T (KCTC 43402^T = GDMCC 1.3498^T), which was characterized using a polyphasic taxonomic approach. This strain was analyzed using antiSMASH, BAGEL4, and PRISM to predict the secondary metabolites. Lassopeptide clusters were found using all three analysis methods, with the possibility of secretion. Additionally, PRISM found three biosynthetic gene clusters (BGC) and predicted the structure of the product. Genome analysis indicated that glucoamylase is present in SS4^T. 16S rRNA sequence analysis showed that strain SS4^T most closely resembled Paenibacillus marchantiophytorum DSM 29850^T (98.22%), Paenibacillus nebraskensis JJ-59^T (98.19%), and Paenibacillus aceris KCTC 13870^T (98.08%). Analysis of the 16S rRNA gene sequences and Type Strain Genome Server (TYGS) analysis revealed that SS4^T belongs to the genus Paenibacillus based on the results of the phylogenetic analysis. As a result of the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) results, SS4^T was determined to belong to the genus Paenibacillus. Comparing P. marchantiophytorum DSM 29850^T with average nucleotide identity (ANI 78.97%) and digital DNA-DNA hybridization (dDDH 23%) revealed values that were all less than the threshold for bacterial species differentiation. The results of this study suggest that strain SS4^T can be classified as a Paenibacillus andongensis species and is a novel member of the genus Paenibacillus.