• Analytical Science and Technology
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• v.24 no.6
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• pp.510-518
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• 2011

Recent developments and improvements of multiple technological elements including mass spectrometry (MS) instrument, multi-dimensional chromatographic separation, and software tools processing MS data resulted in benefits of large scale proteomics analysis. However, its throughput is limited by the speed and reproducibility of the protein digestion process. In this study, we demonstrated a new method for rapid proteolytic digestion of proteins using acoustic technology. Tryptic digests of BSA prepared at various conditions by super acoustic for optimization time and intensity were analyzed by LC-MS/MS showed higher sequence coverage in compared with traditional 16 hrs digestion method. The method was applied successfully for complex proteins of a breast cancer cells at 30 min of digestion at intensity 2. This new application reduces time-consuming of sample preparation with better efficiency, even with large amount of proteins, and increases high-throughput process in sample preparation state.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1224-1231
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• 2007

Insulin-like growth factor-I (IGF-I) and IGF-II have structure like insulin. In contrast to insulin, however, the bioavaility of IGFs is modulated by the IGF-binding protein (IGFBPs). Each of IGFBPs was different with molecular masses, biological characteristics, and immunological properties.. Human fibroblasts secrete IGFBPs that can modify IGF-I action. In diabetes mellitus, the most study of IGF systems have been investigated in insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, and streptozotocin-in-duced animals in vivo. Recently, a little research regarding the IGFs system has been proposed in por-tion of cell in vitro. In this study, effects of low or high glucose condition on IGFBP-5 in GM10 was investigated. By western blotting analysis, IGFBP-5 level decreased in cells cultured at high glucose, but IGFBP-5 level of mRNA didn't change. IGFBP-5 protease that cleaves IGFBP-5 in conditioned me-dium had was inhibited by EDTA and heparin, like serine protease and metalloprotease. Furthermore, the protease activity was increased in high glucose cultivated condition. In results of gelatin zymog-raphy, molecular weight of proteolytic metalloenzymes was indentified 69-kDa and protease activity was increased in time-dependent manner. Although the mechanism has yet to be determined, IGFBP-5 proteolysis in GM10 cells cultured with high glucose may increase effects of IGFs to decrease the glu-cose level through dissociation of IGFs from IGFBPs. Therefore, we suggest that IGF- I and IGFBPs could be potential models in study of pathophysiology such as diabetes mellitus.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.921-932
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• 2006

Management to improve beef tenderness is always been a historical idea, but during the recent past it has become an issue of prime importance to the meat scientists and the industries as well. Variation in tenderness is the prime explanation for consumer’s dissatisfaction for the concern meat. It has been well documented that both postmortem proteolysis and sarcomere length have significant effect on meat tenderness and its consistency. Electrical stimulation and tenderstretch techniques have been used by a number of countries to underpin carcass quality assurance schemes focused on eating quality. The mechanism(s) by which the postmortem interventions improve tenderness (or prevent toughness) has not been fully elucidated. However, it is evident that electrical stimulation accelerates the development of rigor mortis so that prevention of cold shortening is possible and ageing commences at higher temperatures. On the other hand, tendersretch appears to prevent meat toughness via placing tension of the myofibrils and connective matrix during rigor development. Previous findings indicated that electrical stimulation and tenderstretch improved beef tenderness even for fattened cattle under moderate chilling conditions. Recent studies demonstrate beef tenderness to be one of the most important factors determining satisfaction levels of Korean beef consumers. There are number of studies which reported that electrical stimulation and tenderstretch techniques improved Hanwoo tenderness and color. It is believed that the techniques are mostly useful wherein controls of carcass size, fatness and/or chilling regimes are not easy such as Korean beef industry. However, Korean beef industry is one such area where postmortem intervention techniques have not been adopted so far. Taking into consideration of the Korean beef industry, wherein carcass size and fatness varies the post-slaughter intervention technique could be the most feasible measurement to ensure eating quality. The manuscript attempts to highlight the current knowledge aiming primarily towards the assurance of beef tenderness.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1599-1607
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• 2004

This study examined the effects of pre-slaughter fasting, chasing stress and chiller ageing on objective meat quality, and their relations to the proteome profile of longissimus muscle using 20 male Korean native black pigs. Treatments were composed of two levels of pre-slaughter feed withdrawal, two levels of pre-slaughter stress and four chiller ageing times. A 15 min chasing stress immediately prior to slaughter significantly (p<0.05) decreased detectable levels of $\mu$-calpain activity during rigor development and chiller ageing, but did not have any direct effect on objective meat quality. On the other hand, pigs fed until the morning of slaughter resulted in significantly (p<0.05) higher hunter L* value and cooking loss than those which received an 18 h feed withdrawal prior to slaughter. Cooking loss and hunter L* value were constant during 7 d of chiller ageing, followed by significant increases at 14 d. The fed animals showed a significantly (p<0.05) higher hunter a* value at both 3 and 7 d, while the other group maintained a stable redness for 7 d. WB-shear force was not affected by the pre-slaughter treatments, but had significant (p<0.05) linear reduction from 1 to 7 d. A gelbased proteome analysis was performed on selected animals for low and high hunter L* values at 1 d. Ten and five spots had greater than two-fold spot densities for the low and high hunter L* groups, respectively. The ten spots included chain A, deoxyribounclease I complex with actin, heat shock protein 27 kDa, a protein similar to cardiac $Ca^{2+}$ release channel, and myosin heavy chain, while the five spots included chain A aldehyde dehydrogenase, glycerol-3 phosphate dehydrogenase, and hemoglobin alpha chain. In general, feeding until the morning of slaughter resulted in more desirable meat color, but appeared to reduce palatability due to increased cooking loss. Proteome analysis demonstrated that various proteins were concomitantly involved in the determination of final meat color. The most noticeable observation in the current study was that various isoforms for a particular protein differed in degradation and/or expression rate depending on meat quality.

• Journal of Life Science
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• v.25 no.1
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• pp.1-7
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• 2015

Cyclin D is a member of the cyclin protein family, which plays a critical role as a core member of the mammalian cell cycle machinery. D-type cyclins (D1, D2, and D3) bind to and activate the cyclin-dependent kinases 4 and 6, which can then phosphorylate the retinoblastoma tumor suppressor gene products. This phosphorylation in turn leads to release or derepression of E2F transcription factors that promote progression from the G1 to S phase of the cell cycle. Among the D-type cyclins, cyclin D3 encoded by the CCND3 gene is one of the least well studied. In the present study, we have investigated the biochemistry of the proteolytic mechanism that leads to loss of cyclin D3 protein. Treatment of human prostate carcinoma PC-3-M cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. Additionally, using inhibitors for various proteolytic systems, we show that degradation of cyclin D3 protein involves the $Ca^{2+}$-activated neutral protease calpain. Moreover, the half-life of cyclin D3 protein half-life increased by at least 10-fold in PC-3M cells in response to the calpain inhibitor. We have also demonstrated that the transient expression of the calpain inhibitor calpastatin increased cyclin D3 protein in serum-starved NIH 3T3 cells. These data suggested that the function of cyclin D3 is regulated by $Ca^{2+}$-dependent protease calpain.

• Journal of the Korean Society of Food Culture
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• v.28 no.1
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• pp.78-88
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• 2013

Marinated beef galbi is a traditional Korean dish cooked with soy sauce, pear juice, onion, sesame oil, and sugar. However, there are many differences in beef galbi, including flavor and physicochemical aspects, depending on cooking conditions. Therefore, the physicochemical characteristics of marinated beef galbi prepared through various recipes was evaluated for its effects on pH, texture, aging, proteolysis, heating conditions, cooking time, and flavor compounds (pyrazines, IMPs, or FAAs). There were significant differences in salt concentration (0.8~3.03%), pH (4.89~6.22), and solid soluble contents (1.34-6.31 Brix) between recipes in this study. In the Pearson assay for sensory evaluation, overall preference correlated well with texture (a well-known sensory attribute in meat evaluation). Controlling the pH of meat through soaking in lemon solution, alkali water, phosphate, and baking powder solution, improved water holding capacity as much as 9 to 15% compared with the control. The myofibril index (MFI) of marinated meat stored at $4^{\circ}C$ increased 32% with 24 hours of aging and reached 39% at 48 hours of aging, and its fragmentation was observed through microscopy. SDS-PAGE showed hydrolysis of acid-soluble collagen by the pear juice, possibly related to meat tenderness. On the basis of surface temperature, the cooking time was estimated to be 8 minutes with pan heating at $170^{\circ}C$, 6 minutes at $270{\sim}300^{\circ}C$, and 4 minutes with charcoal at $700{\sim}900^{\circ}C$. Different pyrazine compounds, such as 2-methyl-3-phenylpyrrol(2,3-b) pyrazine (the typical product of the browning reaction) was mainly detected, and IMP (one of the main taste compounds in beef) was in higher amounts with the charcoal treatment, potentially related to its flavor preference among treatments. Our results demonstrate an effective case study and cooking system for beef galbi.

• Development and Reproduction
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• v.3 no.1
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• pp.29-38
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• 1999

The pathogenesis of endometriosis is unknown, but retrograde menstruation is widely accepted as an etiology. Refluxed endometrium from endometriosis patients is more prone to implant and invade peritoneum possibly through the action of extracellular proteolysis. This proteolytic action may involve plasminogen activators and the collagenase system. Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) play a critical role in the breakdown of extracellular matrix components and basement membrane in the processes of implantation and tumor invasion. PAs are inhibited by plasminogen activator inhibitor (PAI) and MMPs activity is inhibited by tissue inhibitor of metalloproteinase (TIMP). To test the hypothesis that lower expression of PAI-1 and TIMP-3 in endometrium from women with endometriosis, we investigated their PAI-1 and TIMP-3 expression by quantitative competitive RT PCR in endometrium from women with and without endometriosis. Endometrial tissues were obtained from 14 patients with severe endometriosis and 14 patients without endometriosis. Total RNA was extracted and reverse transcribed into cDNA, and quantitative competitive PCR (QC PCR) was performed to evaluate PAI-1 and TIMP-3 mRNA expression. Endometrium from patients with endometriosis showed decreased expression of PAI-1 and TIMP-3 mRNA compared to endometrium from control in luteal phase (p<0.05). Our results suggest that endometrium from women with endometriosis expresses lower levels of PAI-1 and TIMP-3 than endometrium from normal women. Endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation than control because of higher PA and MMP enzymatic activity. Thus, increased proteolytic activity may be one of the reasons for the invasive properties of the endometrium resulting in the development of endometriosis.

• Journal of Life Science
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• v.19 no.8
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• pp.1047-1054
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• 2009

Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and SmacjDlABLO from mitochondria to cytosol, andtranslocation of AlF into nucleus were shown. While p53, p21 and $14-3-3{\gamma}$ were upregulated, cyclin Band cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.4
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• pp.398-406
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• 1992

In order to develop a new flavourant using the fish skin gelatin, the proteolytic renditions for the gelatin hydrolysate of the alkali (B-type) and Alcalase (E-type) pretreated flounder (Limanda aspera) skin gelatin were investigated, and some physical properties, molecular weight and amino acid compositions of the hydrolysates were, also, compared with each other. The proteolytic conditions of the gelatins (B-type and E-type) by trypsin were as follows : reaction temperature, 55$^{\circ}C$ : pH, 9.0 : enzyme concentration, 0.1% : re-action time, 4hrs for B-type and 1 hr for E-type. The degrees of hydrolysis of the B-type and E-type gelatin un-der the renditions stated above were 63% and 82%, respectively. The rnajor molecular weights of the hydrolysates were 15,000 dalton for B-type and 12,400 dalton for E-type. Among the amino acids in the hydrolysates, glycine, alanine, proline, hydroxyproline and serine having a sweet taste were responsible for 57% of the total amino acid. But valine, leucine, phenylalanine, tyrosine, methionine, arginine and histidine having a bitter taste were only 18%.

• The Korean Journal of Mycology
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• v.33 no.1
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• pp.11-17
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• 2005

To increase the fibrinolytic activity and production of mycelium, extracts of 7 plant species were supplemented to the growth media of Armillaria mellea, and mycelial growth and enzymatic activity in the mycelium extracts of A. mellea were estimated. The mycelial production of A. mellea was slightly increased by adding ASH-R, UDVN or RGR extract, whereas KG extract significantly affected the growth. Supplement of ASH-S, UDVN and RGR extracts increased proteolytic activity from 36.8 to 46.1% Fibrinolytic activity was increased to $50{\sim}65%$ by supplement with RVS, ASH-S and RGR extracts, respectively. Enzyme extracts of the fungus grown with RGR extract supplement degraded all chains of fibrinogen within 2 hours, whereas control was required 3 hours. Degradation of fibrin fragments by the enzyme extracts was also observed through microscopy.