• 한국수산과학회지
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• 제38권5호
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• pp.286-290
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• 2005

Oxytocin (OT)-related peptide, isotocin was purified from the brain extract of conger eel (Conger myriaster) using reverse-phase, ion-exchange and size exclusion high performance liquid chromatography (HPLC). The sequence of the peptide, with a molecular weight of 967.30 Da, was determined as Cys-Tyr-Ile-Ser-Asn­Cys-Pro-Ile-Gly-$NH_2$, where the Cys between 1st and 6th residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis and MALDI-TOF mass spectrometry. The sequence was confirmed by identical elution with the purified and synthetic peptide using the HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of OT -superfamily member, isotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-7}M$ on the intestinal smooth muscle of goldfish (Carassius auratus).

• 한국수산과학회지
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• 제44권5호
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• pp.478-483
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• 2011

An antimicrobial material was purified from the acidified whole body extract of the jellyfish Nemopilema nomurai by using C18 reversed phase and cation-exchange high performance liquid chromatography (HPLC). Whole body extract and the purified compound (JAP-1) showed potent antimicrobial activities against a wide range of microorganisms including Escherichia coli D31, Bacillus subtilis, Streptococcus iniae and Candida albicans, without significant hemolytic activity. Treatment of JAP-1 with trypsin completely abolished all antibacterial activity against Bacillus subtilis, suggesting that JAP-1 is likely to be a proteinaceous antibiotic. The molecular weight of JAP-1 was determined to be 680.10 Da by MALDI-TOF mass spectroscopy.

• Asian-Australasian Journal of Animal Sciences
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• 제28권6호
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• pp.788-795
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• 2015

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2206-2213
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• 2016

Recently, several studies have revealed that commercial microbial identification systems do not accurately identify the uncommon causative species of candidiasis, including Candida famata, Meyerozyma guilliermondii, and C. auris. We investigated the accuracy of species-level identification in a collection of clinical isolates previously identified as C. famata (N = 38), C. lusitaniae (N = 1 2), and M. guilliermondii (N = 5) by the Vitek 2 system. All 55 isolates were re-analyzed by the Phoenix system (Becton Dickinson Diagnostics), two matrix-assisted laser desorption ionization-time of flight mass spectrometry analyzers (a Vitek MS and a Bruker Biotyper), and by sequencing of internal transcribed spacer (ITS) regions or 26S rRNA gene D1/D2 domains. Among 38 isolates previously identified as C. famata by the Vitek 2 system, the majority (27/38 isolates, 71.1%) were identified as C. tropicalis (20 isolates) or C. albicans (7 isolates) by ITS sequencing, and none was identified as C. famata. Among 20 isolates that were identified as C. tropicalis, 17 (85%) were isolated from urine. The two isolates that were identified as C. auris by ITS sequencing originated from ear discharge. The Phoenix system did not accurately identify C. lusitaniae, C. krusei, or C. auris. The correct identification rate for 55 isolates was 92.7% (51/55 isolates) for the Vitek MS and 94.6% (52/55 isolates) for the Bruker Biotyper, as compared with results from ITS sequencing. These results suggest that C. famata is very rare in Korea, and that the possibility of misidentification should be noted when an uncommon Candida species is identified.

• Plant Biotechnology Reports
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• 제3권2호
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• pp.167-174
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• 2009

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.237-242
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• 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
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• 제33권4호
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• pp.243-248
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• 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.

• Bulletin of the Korean Chemical Society
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• 제34권11호
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• pp.3243-3248
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• 2013

The tissue inhibitor of metalloproteinase-2 (TIMP-2) inhibits matrix metalloproteinases activity and modulates cellular proliferation and apoptosis. The human serum albumin-TIMP-2 with folic acid conjugate (termed HT2-folate) was synthesized to promote uptake through folate receptors (FRs), and a corresponding radio-labeled compound was prepared for tumor diagnosis by positron emission tomography (PET). $^{68}Ga$-NOTA-HT2-folate was synthesized from $^{68}Ga$ and the NOTA chelator with HT2-folate. The fusion protein was identified using MALDI-TOF mass spectrometry. The radioligand was prepared with a high radiochemical yield. Cell-surface association of $^{68}Ga$-NOTA-HT2-folate significantly increased over time in FR-positive tumor cells. In animal PET and biodistribution studies, tumor uptake was very high as early as 1 h after radioligand injection. Folate conjugation enhanced the selective receptor-targeting efficacy of HT2 in FRexpressing tumors, and its radioligand will be useful as an in vitro tool and for in vivo tumor diagnosis by PET imaging.

• International Journal of Oral Biology
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• 제34권1호
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• pp.29-36
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• 2009

Cyclosporin A (CsA) has been used clinically as an immunosuppressive drug to prevent organ transplant rejection and in basic research as a mitochondrial permeability blocker. It has been reported that CsA has a protective role in severed neurons and a neurotrophic effect in neuronal cells. However, the molecular mechanisms underlying the stimulation of neuronal cell proliferation by CsA have not yet been elucidated. In our current study, we investigated CsA responsive proteins in PC12 cells using a systematic proteomic approach. The viability of these cells following CsA treatment increased in a dose- and time-dependent manner. Proteins in the CsA-treated PC12 cells were profiled by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadupole time-of-flight mass spectrometries (EIQ-TOFMS). This differential expression analysis showed significant changes for 10 proteins (6 up-regulated and 4 down-regulated) upon CsA treatment that were related to cell proliferation, metabolism and the stress response. These proteomics data further our understanding of the proliferation mechanisms of PC12 cells exposed to CsA and demonstrate that our methodology has potential to further elucidate the mechanisms and pathways involved.

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