• Korean Journal of Microbiology
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• v.52 no.3
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• pp.260-268
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• 2016

The aim of this work was to investigate the cellular responses and proteomic analysis of Bacillus cereus MH-2 exposed to EGCG. Strain MH-2 was isolated from commercial Ssamjang and has the hemolytic activity. Survival of the MH-2 strain with time in the presence of different concentrations of EGCG under sublethal conditions was monitored. The amount of alginate from MH-2 strain decreased depending on the increasing concentrations of EGCG and increased depending on the exposure time at any particular EGCG concentration. Analysis of SDS-PAGE and Western blot using anti-DnaK and anti-GroEL revealed that two stress shock proteins, 70 kDa DnaK and 60 kDa GroEL were found to decrease in proportion to the EGCG concentration in exponentially growing cultures. Scanning electron microscopic analysis demonstrated the presence of protrusions and fused rod forms on the cells treated with EGCG. 2-DE of soluble protein fractions from MH-2 cultures showed 20 protein spots changed by EGCG exposure. These proteins involved in enterotoxins (hemolysin BL lytic component L1 and hemolysin BL-binding protein), chaperons (DnaK and GroEL), cell defense (peptidase M4 family proteins), and various biosynthesis and energy metabolism were identified by peptide mass fingerprinting using MALDI-TOF. These results provide clues for understanding the mechanism of EGCG-induced stress and cytotoxicity on B. cereus MH-2.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.262-268
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• 2005

Intoxication of murine macrophages (RAW 264.7) with the anthrax lethal toxin (LeTx 100 ng/ml) results in profound alterations in the host cell gene expression. The role of LeTx in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional polyacrylamide gel electrophoresis to analyze the protein profile of murine macrophages treated with the LeTx, and have coupled this to protein identification using MALDI-TOF mass spectrometry. Interpretation of the peptide mass fingerprint data has relied primarily on the ProFound database. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase (Mek1) and glucose-6-phosphate dehydrogenase were increased in the LeTx treated macrophages. Mek1 acts as a negative element in the signal transduction pathway, and G6PD plays the role for the protection of the cells from the hyper-production of active oxygen. Our results suggest that this proteomic approach is a useful tool to study protein expression in intoxicated macrophages and will contribute to the identification of a putative substrate for LeTx.

• Journal of Applied Biological Chemistry
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• v.63 no.4
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• pp.387-392
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• 2020

Pseudomonas tolaasii is a pathogen causing brown blotch disease in cultivated mushrooms. In previous study, various strains of P. tolaasii were isolated from the mushrooms with disease symptoms and they were further divided into Ptα, Ptβ, and Ptγ subtypes according to the 16S rRNA gene analysis. To investigate the secretion of peptide toxins, tolaasin and its analog peptides, culture extracts of Pt group strains were analyzed by gel permeation chromatography. Those of Ptα subtype strains contained two chromatographic peaks, band A and B. Meanwhile, those of Ptβ and Ptγ subtype strains contained mainly band A component and a little of band B. Molecular weights of toxic peptides of culture extracts were measured by MALDI-TOF mass spectrometry. In Ptα subtype strains, the peptide compositions of band A and B were same including tolaasin I (1,987 Da), tolaasin II (1,943 Da), and its two analog peptides, 1,973 Da and 2,005 Da. The strains of Ptβ and Ptγ subtype secreted many components of MW 1,100-1,200 Da, but they did not synthesize any tolaasin-like peptides. These results suggest that the only Ptα subtype strains secrete tolaasin and its analog peptide toxins and the strains of Ptβ and Ptγ subtypes have different pathogenic characters causing brown blotch disease.

• Applied Chemistry for Engineering
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• v.21 no.6
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• pp.638-642
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• 2010

After phthalonitrile derivatives were synthesized by the introduction of phenoxy, 2-naphthoxy or 4-trityl phenoxy group on ${\alpha}$- and ${\beta}$-position, oxovanadyl phthalocyanine (VOPc) derivatives containing electron-rich substituent group at different position were synthesized successfully in this investigation. The chemical structure of samples was determined by the means of $^1H$-NMR, MALDI-TOF mass spectroscopy, and FT-IR spectrometer. Also, optical and chemical properties were determined by the means of UV-Vis spectrometer, X-ray diffractometry, and thermo gravimetry. It was found that the maximum absorbing wavelength of VOPc derivatives ranged from 684 to 726 nm. Also, their solubility and Q-band were enhanced and shifted by the introduction of substitute group, respectively.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.217-221
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• 2010

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1~2 mm, 2~6 mm and 6~10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2~6 mm follicle and 5 spots from follicle fluid of 6~10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2~6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6~10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

• BMB Reports
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• v.38 no.5
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• pp.545-549
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• 2005

Acidithiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize $Fe^{2+}$ or sulphide as energy source. Growth curves for Acidithiobacillus ferrooxidans under phosphate starvation and normal condition have been tested, showing lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Acidithiobacillus ferrooxidans cultivated with normal cultivating condition and from 20 to 60 hrs for Acidithiobacillus ferrooxidans cultivated phosphate starvation. Differences of protein patterns of Acidithiobacillus ferrooxidans growing in case of normal or phosphate starvation were separately investigated after cultivation at $30^{\circ}C$ by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization (MALDI)-Mass spectrometry. There were total 6 protein spots identified, which were Recombination protein recA, RNA helicase, AP2 domain-containing transcription factor, NADH dehydrogenase I chain D, Hyothetical protein PF1669, and Transaldolase STY3758. From the 6 identified protein spots, 3 proteins were found to be decreased in expression at the cultivating condition of phosphate starvation, while another three upregulated.

• Food Science of Animal Resources
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• v.32 no.6
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• pp.763-768
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• 2012

This study has been performed to measure the prevalence and microbial flora on chicken slaughtering as well as the processing process from the months of October to November. Whole-chicken rinsing technique was used in order to analyze the incidence of microorganisms on chicken carcass at the stage before chilling (after evisceration), after chilling and after cutting. The swab technique was used on processing the processed samples, such as working plates and cutting knives. Brine and cooling water from four cooling tubs were taken from each processing processes and were used as samples. Furthermore, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool was used to identify the isolated microorganisms. Of the tested samples and processes, brine ($4.50{\pm}0.64$ Log CFU/mL) and chicken carcass before chilling ($4.15{\pm}0.46$ Log CFU/mL) showed the highest population of microorganisms; the predominant microbial flora of them were Moellerella wisconsensis (54.84%), a member of the Enterobacteriaceae family, and Escherichia coli (60.36%), respectively. However, the predominant microbial flora of cut carcass was changed to Staphylococcus aureus (27.32%), which is a kind of pathogenic microorganism that can cause a food-borne illness. Therefore, the slaughtering and processing procedure of chicken are needed to be controlled more hygienically.

• BMB Reports
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• v.37 no.3
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• pp.298-303
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• 2004

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.254-263
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• 2014

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative ${\alpha}$-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in high-level expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at $40^{\circ}C$. The ${\alpha}$-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its ${\alpha}$-amylase can be useful in lowering EP molecular weight and in starch processing.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.59 no.3
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• pp.359-363
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• 2014

Soybean seed is a good source of plant protein in human consumables such as baby formula and protein concentrate. The seeds contain an abundance of storage proteins, namely ${\beta}$-conglycin and glycinin that account for ~ 70-80% of the total seed protein content. Proteome profiling has been proved to be an efficient way that can help us to investigate the seed storage proteins. In the present study, the seeds were removed from the pods and the cotylendonary tissues were separated from the testa for proteome analysis in order to investigate the seed storage proteins. A systematic proteome profiling was conducted through one-dimensional gel electrophoresis followed by MALDI-TOF-TOF mass spectrometry in the seeds (cotyledonary tissue) of soybean genotypes. Two dimensional gels stained with CBB, a total of 10 proteins were identified and analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. A total of ten proteins such as glycinin Gy4 precursor, glycinin G3 precursor, glycinin G1 precursor, glycinin chain A2B1a precursor, glycinin chain A2B1a precursor were identified in our investigation. However, the glycinin subunit may be considered to play important roles in soybean breeding and biochemical characterization. In addition, the improved technique will be useful to dissect the genetic control of glycinin expression in soybean.