• Journal of Embryo Transfer
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• v.26 no.4
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• pp.223-227
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• 2011

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (${\alpha}$-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.7
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• pp.950-955
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• 2012

This study was conducted to determine how the isolation method of the porcine preantral follicles influenced the following follicular growth in vitro. Mechanical and enzymatical isolations were used for retrieving the follicles from prepubertal porcine ovaries, and in vitro-growth of the follicles and the expression of folliculogenesis-related genes were subsequently monitored. The enzymatic retrieval with collagenase treatment returned more follicles than the mechanical retrieval, while the percentage of morphologically normal follicles was higher with mechanical retrieval than with enzymatic retrieval. After 4 days of culture, mechanically retrieved, preantral follicles yielded more follicles with normal morphology than enzymatically retrieved follicles, which resulted in improved follicular growth. The mRNA expression of FSHR, LHR Cx43, DNMT1 and FGFR2 genes was significantly higher after culture of the follicles retrieved mechanically. These results suggest that mechanical isolation is a better method of isolating porcine preantral follicles that will develop into competent oocytes in in vitro culture.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.1
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• pp.10-16
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• 2023

Intermuscular fat is essential for enhancing the flavor and texture of cultured meat. Mesenchymal stem cells derived from intermuscular adipose tissues are a source of intermuscular fat. Therefore, as a step towards developing a platform to derive intermuscular fat from mesenchymal stem cells (MSCs) for insertion between myofibrils in cultured beef, an advanced protocol of intermuscular adipose tissue dissociation effective to the isolation of MSCs from intermuscular adipose tissues was developed in cattle. To accomplish this, physical steps were added to the enzymatic dissociation of intermuscular adipose tissues, and the MSCs were established from primary cells dissociated with physical step-free and step-added enzymatic dissociation protocols. The application of a physical step (intensive shaking up) at 5 minutes intervals during enzymatic dissociation resulted in the greatest number of primary cells derived from intermuscular adipose tissues, showed effective formation of colony forming units-fibroblasts (CFU-Fs) from the retrieved primary cells, and generated MSCs with no increase in doubling time. Thus, this protocol will contribute to the stable supply of good quality adipose-derived mesenchymal stem cells (ADMSCs) as a fat source for the production of marbled cultured beef.

• Mycobiology
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• v.33 no.4
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• pp.188-193
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• 2005

Twenty nine samples of pigeon droppings (n = 12) and soil contaminated with avian excreta (n = 19), collected from different sites in Busan, were examined for isolation and characterization of Cryptococcus neoformans. Of these samples, 5 strains of C. neoformans were recovered from pigeon droppings (5/12 : 41.7%). All isolates were belonged to C. neoformans var. grubii (serotype A). The extracellular enzyme activities of the strains by using the API-ZYM system showed two different enzymatic patterns. The genetic variability among C. neoformans isolates was analyzed by random amplified polymorphic DNA (RAPD) using three 10-mer primers. Two different RAPD patterns, which clearly distinguished the isolates, were identified. Analysis of RAPD patterns provided a good characterization of environmental strains of C. neoformans serotype A as a heterogeneous group and were in good agreement with enzymatic profiles.

• Animal cells and systems
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• v.15 no.1
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• pp.19-27
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• 2011

Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.35 no.1
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• pp.34-40
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• 2003

This study was carried out to select the useful bacteria which secret extracellula enzymes for enzymatic deinking agent of old newspaper. CMCase, FPase and xylanase activities of the bacteria liquid culture were measured at optimal growth conditions. Clear zone test was checked on the solid culture. The results of this study were as follow: Eight strains of 28 bacteria isolated from a paper mill soil ground were shown strong CMCase and xylanase activity with the clear zone test. The optimal pH and temperature for culture growth were 6~8 and 26~$34^{\circ}C$, respectively and optimal culture period were less than 60 hours. Based on CMCase, FPase and xylanase activity, strain No. 18, 21, 22 and 28 which were relatively higher than the other strains, were selected for further enzymatic deinking research.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.93-96
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• 2010

The isolation of preantral follicles from the ovaries of bovine was performed under mechanical and enzymatic methods. A significant increase in the total number of follicles retrieved was detected when tissue chopper was used. Micro-dissection could supply good quality, larger sized follicles (400 to $700{\mu}m$) but with the lowest yield ($9.0{\pm}1.0$). The isolated preantral and early antral follicles were cultured for 14 days. Follicles isolated by the mechanical method had a greater growth during a culture period than follicles collected enzymatically. Morphologically normal bovine oocytes from early antral follicles after 14 days culture were 59.6% after culture and after 24 h of maturation culture, 12.9% of in vitro-grown oocytes reached the second metaphase. In conclusion, this study showed that mechanical method can be used effectively to isolate intact preantral follicles from bovine ovaries.

• Korean Journal of Agricultural Science
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• v.39 no.4
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• pp.561-568
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• 2012

The aim of this study was to introduce a simple method for isolation of ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin from cow's milk, and peptides produced by enzymatic hydrolysis of ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin with alcalase. Whey protein were precipitated from whey by ammonium sulfate and, ${\alpha}$-lactalbumin and ${\beta}$-lactoglobulin were isolated using Hi Prep 26/60 Sephacryl S-100 column gel filtration chromatography. Bovine serum albumin and ${\beta}$-lactoglobulin were isolated by Mono-Q 5/50 GL column anion exchange chromatography of the 50% Ammonium Sulfate-supernatant. Isolated whey proteins were hydrolyzed by proteolytic alcalase. Tricine SDS-PAGE and reverse-phase HPLC analyses revealed that almost hydrolyzed all the ${\alpha}$-lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin with alcalase. Molecular weight of various peptides derived from alcalase hydrolysate were small molecular weight than 3.5 kDa.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.36 no.3
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• pp.9-14
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• 2004

The useful fungi which secret extracellular enzymes was selected for deinking agent of old newsprint. Five fungal strains were isolated from a paper mill soil ground. The CMCase, FPase and xylanase activities of fungi on the liquid culture were investigated at optimal growth conditions. The results of this study were as follow: The optimal pH and temperature for culture growth were 4～8 and 27～$35^{\circ}C$, respectively. For screening of extracellular enzymes at optimal culture conditions the optimal culture period were less than 6-7 days. Fusarium pallidoroseum and Aspergiilus niger which shows relatively higher CMCase, FPase and xylanase activities than the other species were selected for further enzymatic deinking research.

• Korean Journal of Pharmacognosy
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• v.19 no.1
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• pp.28-33
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• 1988

To identify biologically active enzymatic components of the higher fungi in Korea, the dried carpophores of Cryptoderma citrinum was smashed with cool distilled water, extracted, salted out and the precipitate was purified by dialysing with visking tube and dissolved with pH 7.8 ammonia aqua. The fraction of the filtrate was lyophilized to obtain as light brown powder and then cellulase activity was determined. Cellulolytic potency of Cryptoderma citrinum was 750 unit/g. The optimum condition for the enzymatic reaction was pH 4.5 and $40^{\circ}$. The enzyme activity was activated in the presence of $Ca^{2+}$, $Fe^{2+}$ and $Co^{2+}$.