• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.33-33
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• 2001

본 연구는 한우 난포발달에 있어서 난포액 및 inhibin의 생리적 역할을 검토하기 위해 수행하였다. Anti-inhibin serum(AIS) 생산을 위해 사용된 항원은 porcine inhibin-$\alpha$-subunit 19~32의 peptide를 사용하여 adjuvant 용액을 1:3의 비율로 혼합하여 앙고라종 토끼 5두(체중 2.5kg)에게 주 2회 간격으로 8회 실시후 ELISA Leader로 항혈청의 역가를 확인하였다. 난포액(bFF; bovine follicular fluid)은 도축장에서 도축되는 한우 난소로부터 직경 1.0cm 이하의 난포로부터 회수하여 스테로이드를 제거하기 위해 10% chacoal solution(50 mg/$m\ell$, Norrit-A, Fisher Sci., USA)을 처리하여 45분간 배양후 원심분리후 상층액을 회수하여 실험에 공시하였다. 과립막세포의 체외배양을 위해 D-MEM용액(10% FCS와 antibiotics를 첨가)을 배양액으로 하여 1 $\times$ $10^{6}$ cells/$m\ell$로 조절하였다. 호르몬은 RIA 및 ELISA법으로 분석하였다. 그 결과 항원-항체반응은 항원처리후 24일째부터 항체가를 확인할 수 있었으며 52일째에서 높은 항원-항체반응을 보였다. 한편, 난포크기별 난포액의 progesterone 및 Estradiol-l7$\beta$을 농도를 분석한 결과, Progesterone 난포크기가 직경 1.0 cm부터 유의적으로 증가하기 시작하여 직경 2.0cm의 난포액에서는 높은 progesterone이 존재하고 있는 것으로 나타났다. 이에 반해, 난포크기별 난포액중 estradiol 17$\beta$농도는 직경 1.0cm구에서 가장 높게 나타났다. 난포직경이 1cm일 경우에 난포액내 etradiol-17$\beta$가 가장 많이 존재하고 있음이 나타났다. 과립막세포의 체외배양에서 배양 24시간에서는 과립막세포의 progesterone분비는 약 40ng/1$\times$$10^{6}$ cell/well/$m\ell$ 전후로 나타났으며 bFF 5%, AI 5% 및 bFF＋AI 첨가구에 따라 유의적인 차이는 없었다 반면에 48시간배양구에서는 24시간에 비해 유의적으로 높게 분비되었으며, bFF 5%처리구와 bFF5%＋AI5%처리구에서는 progesterone을 대조구보다 유의적으로 억제되었으나 AI 5%단독처리구에서는 대조구와 큰 차이가 없었다. 한편, 각 세포질을 SDS-PAGE로 분리하여 nitro cellulose membrane에 transfer하여 Western blotting법에 의해 검토한 결과, 직경 1.0 cm의 성숙난포의 granulosa cell에 특이하게 Inhibin이 존재하고 있음이 확인되었다. 이상의 결과로, 한우에 있어서 성숙난포에 존재하는 Inhibin은 난포발달 및 난포세포의 스테로이드호르몬합성에 중요하게 관여하고 있는 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.757-764
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• 2018

A cellulolytic fungus, YDJ14, was isolated from compost and identified as an Aspergillus sp. strain. Three extracellular ${\beta}$-glucosidases, BGL-A1, BGL-A2, and BGL-A3, were separated using ultrafiltration, ammonium sulfate fractionation, and High-Q chromatography. The molecular masses of the three enzymes were estimated to be 100, 45, and 40 kDa, respectively, by SDS-PAGE. The optimum pH and temperature of BGL-A3 were 5.0 and $50^{\circ}C$, respectively, whereas the optimum pH and temperature of BGL-A1 and BGL-A2 were identical (4.0 and $60^{\circ}C$, respectively). The half-life of BGL-A3 at $70^{\circ}C$ (2.8 min) was shorter than that of BGL-A1 and BGL-A2 (12.1 and 8.8 min, respectively). All three enzymes preferred p-nitrophenyl-${\beta}$-$\text\tiny{D}$-glucopyranoside (pNPG) and hardly hydrolyzed cellobiose, suggesting that these enzymes were aryl ${\beta}$-glucosidases. The $K_m$ of BGL-A3 (1.26 mM) for pNPG was much higher than that of BGL-A1 and BGL-A2 (0.25 and 0.27 mM, respectively). These results suggested that BGL-A1 and BGL-A2 were similar in their enzymatic properties, whereas BGL-A3 differed from the two enzymes. When tilianin (a flavone glycoside of acacetin) was reacted with the three enzymes, the inhibitory activity for monoamine oxidase, a target in the treatment of neurological disorders, was similar to that shown by acacetin. We conclude that these enzymes may be useful in the hydrolysis of flavone glycosides to improve their inhibitory activities.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.301-310
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• 1997

Diabetes mellitus is associated with a wide range of pathophysiological in the kidney. This study was designed to examine the effects of high glucose concentration on IGF-I binding and glucose transporters in renal proximal tubule cells. The results were as follows : The binding of $^{125}I-IGF-I$ reached the peak at the 30 minutes and gradually decreased by the time dependent manner. The binding of $^{125}I-IGF-I$ was inhibited by the unlabelled IGF-I($10^{-14}{\sim}10^{-8}M$) in a concentration dependent manner. The relative affinity of IGF-I receptor for IGF-I, IGF-II and insulin exhibited typical type 1 binding(IGF-I > insulin > IGF-II). However IGF-II did not compete for the cultured cell membrane $^{125}I-IGF-I$ binding site at $10^{-14}{\sim}10^{-8}M$. Under optimal conditions, IGF-I binding to the membranes from 5mM and 20mM glucose treated cells was analyzed. It was found that 20mM glucose treated cells exhibited higher binding activity for IGF-I. In order to further substantiate this increase in IGF-I binding sites, we performed affinity-labelling studies. The cross-linked cell membrane subjected to SDS-PAGE; labelled material was detected by autoradiography. 20mM glucose treated cells exhibited higher levels. The initial rate of $methyl-{\alpha}-D-glucopyranoside({\alpha}-MG)$ uptake was significantly lower($74.41{\pm}6.71%$) in monolayers treated with 20mM glucose than those of 5mM glucose. However, 3-O-methyl-D-glucose(3-O-MG) uptake was not affected by glucose concentration in culture media. IGF-I significantly increased ${\alpha}-MG$ uptake in both 5mM and 20mM glucose treated cells. However, 3-O-MG uptake was not affected by IGF-I in both conditions. In conclusion, 20mM glucose increased binding sites of $^{125}I-IGF-I$, inhibited Na/glucose cotransporter activity. But 20mM glucose did not change facilitated glucose transporter.

• Journal of agricultural medicine and community health
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• v.16 no.1
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• pp.79-89
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• 1991

A systematic study was conducted to identify and isolate a serologically pertinent antigen with high specific activity and low cross reactivity from Cysticercus parenchymal antigen. Differential centrifugation of the homogenate yield three particulate and one soluble fractions ; the $480{\times}G$ pellets($CyL_2$), the $7650{\times}G$ pellet($CyL_3$), the $100000{\times}G$ pellet($CyL_4$), and $100000{\times}G$ supernatant($CyL_6$). We compared antigenicity of these antigens to that or cystic fluid antigens($CyF_1$), saline extract of cystic wall($CyL_1$), and n-butanol treated $GyL_4$ antigen ($CyL_6$) based on SDS-PAGE and immunoblot techniques. The data obtained were as follows : 1) The ratio of O.D. value of ELISA against cysticercosis positive pool sera to that of negative pool sera was highest when using $CyF_1$ as antigen. However the ratio was relatively low in case of $CyL_{3.4}$ and $CyL_5$. 2) We have noted in previous paper that most strong antigenic activities are present in 63Kd band with low cross reactivities. An effective serologic reagent must contain components that are recognized by most infected sera. 63Kd band met this criteria and could be considered as a reliable band for the diagnosis of cysticercosis. As far as 63Kd band concern, $CyL_5$ showed most strong activities without disturbance of cross reaction by EITB in spite of low applicability to microplate ELISA. 3) $CyL_5$ could detect the serum antibody of cysticercosis even in very low titers, around cut-off values of microplate ELISA, by immunoblot. It also could detect the cross reactivities of Echinococcus species, which showed high absorbance value in micro plate ELISA and some sparganosis cases. Further purification of this antigen will be able to represents a antigen that can be used in the diagnosis of cysticercosis.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.292-300
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• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.818-825
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• 2013

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

• Korean Journal of Food Science and Technology
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• v.44 no.5
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• pp.600-606
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• 2012

A Bacillus sp. that produces fibrinolytic enzyme was isolated from Cheonggukjang, a traditional Korean soybean-fermented food. According to 16S rRNA gene base sequencing, the bacillus was identified as a variety of Bacillus subtilis, and named Bacillus subtilis IDCC 9204. Fibrinolytic enzyme NK-IL9204 was stable up to $60^{\circ}C$ and within pH range of 5-10. Purified NK-IL9204 was detected through fibrin zymography. The molecular weight and isoelectric point of the enzyme were estimated to be 27.7 kDa and 6.7 by SDS-PAGE and 2D electrophoresis, respectively. Its amino acid sequence was similar to that of nattokinase (identities 99.5%) and different from that of nattokinase BPN (identities 86.4%). The plasma fibrinolytic activity of NK-IL9204 was measured by euglobulin clot lysis times (ECLT). The NK-IL9204 was orally administered to SD rats for 3 weeks (1,000 FU/rat/day). The ECLT was significantly shortened by supplementation of NK-IL9204.

• Korean Journal of Veterinary Research
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• v.48 no.4
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• pp.431-440
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• 2008

Brucellosis is one of the most important zoonosis in worldwide. As one of the control measures, attempts have been made to develop new diagnostic methods using filed isolates as a national policy in many countries. Currently, bovine brucellosis in Korea have been received attention in both public health and economical aspects due to sudden increase of outbreak. Based on the situation, we compared standard strain (B. abortus 1119-3) with field isolates to reveal the differences among them. Biological and biochemical charateristics, antibiotic resistance profiles, outer membrane proteins (OMPs) and lipopolysaccharide analysis of the strains were included in this study. For the diagnostic purpose, an attempt was made to find out a novel antigen from the Korean isolates by serological analysis. There were differences about 55 kDa, 36-38 kDa and 20 kDa in analysis of OMPs by SDS-PAGE and Western blot with positive sera ($\geq$ 1:400 in SAT titer). Also, a serological diagnostic method, ELISA was conducted using OMPs of the strains as novel antigen. Relationships between O.D. and SAT titer were analyzed using field sera showing different SAT titer. High correlation coefficient was observed between SAT titer and ELISA. Results from this study suggested that a new diagnsotic method should be developed using their own field isolates in each country.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1670-1674
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• 2007

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as monomers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity ($K_D=1.02{\times}10^{-7}\;M$). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and playa critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.