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Site-specific and deletional mutagenesis for two regions of Verotoxin-2 A gene encoding enzymatically active domain (Verotoxin-2 A 유전자의 효소활성 부위에 대한 위치특이적 변이 및 결손변이유발)

  • Kim, Yong-hwan;Kim, Sang-hyun;Cha, In-ho;Kim, Kyoung-shook;Lee, Young-choon
    • Korean Journal of Veterinary Research
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    • v.37 no.3
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    • pp.541-546
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    • 1997
  • There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

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The amino acid contents of wild mushrooms in Korea (국내 야생수집 버섯의 아미노산 함량 분석)

  • An, Gi-Hong;Cho, Jae-Han;Han, Jae-Gu
    • Journal of Mushroom
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    • v.18 no.1
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    • pp.107-114
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    • 2020
  • Fifteen species of wild mushrooms, which are classified as edible, medicinal, poisonous, or unknown types, were analyzed for their free amino acid contents. The average total amino acid content was 1,120.0 mg/kg in the edible type, 1,037.8 mg/kg in the medicinal type, and 1,010.8 mg/kg in the poisonous or unknown type, respectively. The edible type wild mushrooms showed the highest average content of total essential amino acids; conversely, the poisonous or unknown type showed the lowest content. Leucopaxillus giganteus (OK811) showed the highest total amino acid (1,212.4 mg/kg) and essential amino acid (490.2 mg/kg) contents among edible type, while Pycnoporus sanguineus (OK1071) showed the highest total amino acid (1,233.7 mg/kg) and essential amino acid (412.8 mg/kg) contents among the medicinal type. Among the poisonous or unknown type, Hypholoma fasciculare (OK826) showed the highest total amino acid (1,223.4 mg/kg) and essential amino acid (442.8 mg/kg) contents. The amino acid contents of wild mushrooms are fundamental data for the screening of useful genetic resources with nutritionally superior properties.

Chemical Components of Hololeion maximowiczii Root and Change by Drying Methods (께묵뿌리의 화학성분과 건조방법에 따른 변화)

  • Choi, Dong-Yeon;Kim, Young-Hee;Park, Hoon;Yang, Cha-Bum
    • Korean Journal of Food Science and Technology
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    • v.25 no.5
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    • pp.421-425
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    • 1993
  • Hololeion maximowiczii root was analyzed for proximate composition, free sugar, fatty acids of crude lipid, amino acids and minerals after freeze dried (FD), hot air dried (HAD) and hot air dried after blanched (HADB), respectively. Proximate composition was little different among drying methods. Free sugar was abundant in order of sucrose $(28{\sim}29mg/g\;dw)$, fructose $(0.8{\sim}3mg/g\;dw)$, glucose and xylose (trace). Fructose was decreased in HADB. In fatty acid composition of crude lipids, linoleic acid was highest $(55{\sim}59%)$ and then palmitic and linolenic acid in abundance. These three fatty acids made $88{\sim}90%$ of total lipid, Low molecular fatty acids showed decreasing tendancy in HAD and HADB. Amino acid composition of hydrolyzed sample showed abundancy in order of arginine $(34{\sim}42mg/g)$, lysine, glutamic acid, glycine, aspartic acid, valine and histidine, while that of free amino acids was arginine, histidine, lysine, aspartic acid, serine, glutamic acid, isoleucine. Glycine and glutamic acid in hydrolyzed sample showed slight decreasing tendency in HADB. Mineral content was abundance in order of Ca $(1927{\sim}2379\;ppm),\;K\;(1159{\sim}1259ppm)$ and $Mg(760{\sim}777\;ppm)$. No difference was found in drying methods.

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The Movement of Boron Compound by Infusion Method and Combination of Injection and Bandage-Wrapping

  • DAMAYANTI, Ratih;SRIBUDIANI, Evi;SOMADONA, Sonia;Djarwanto, Djarwanto;TARMADI, Didi;AMIN, Yusup;YUSUF, Sulaeman;SATITI, Esti Rini;ARSYAD, Wa Ode Muliastuty;SULAEMAN, Rudianda;Syafrinal, Syafrinal;PRAMASARI, Dwi Ajias
    • Journal of the Korean Wood Science and Technology
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    • v.48 no.4
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    • pp.513-526
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    • 2020
  • The existing preservation methods are difficult to be applied to a large dimension log which is needed for making traditional wooden ship 'Jalur' in Riau Province. Novel techniques to provide the use of readily available species to replace traditional species alternative were investigated. These included infusion and a combination of injection and bandage-wrapping methods for preserving living trees of Balam (Macaranga conifera (Rchb.f. & Zoll.) Müll.Arg.) and Bintangor (Calophyllum soulattri Burm.f.). Water-based boron compounds were applied as wood preservatives. In total, 18 discs from the bottom, middle, and top of four trees and two controls were used. Trees undergoing treatment were also used to see how wood anatomical structure might affect the boron penetration. The overall aim was to identify the best method for use in Jalur manufacturing. The results showed that in infused Balam tree where the hose position for the preservative intake was deep (10-15 cm from the bark), no boron compound was observed in the outer sapwood. Combination of injection and bandage-wrapping method gave higher percentage of boron penetration at bottom and middle of Balam tree. However, infused Bintangor showed 100% boron penetration. The larger vessel diameter, the absence of tyloses, and the simple perforation plates in Bintangor wood were likely to have contributed to the higher penetration of boron. The combination of bandage-wrapping and infusion, or alternatively by infusing the living trees close to the bark, and at as low as position in the stem gives better protection when treatments are applied to living trees.

A Missense Mutation in Exon 5 of the Bovine Growth Hormone Gene (소 성장호르몬 유전자의 Exon 5번에서의 새로운 다형성 연구)

  • Yoon, D. H.;Kim, T. H.;Lee, K. H.;Park, E. W.;Lee, H. K.;Cheong, I. C.;Hong, K. C.
    • Journal of Animal Science and Technology
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    • v.45 no.1
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    • pp.13-22
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    • 2003
  • Growth Hormone (GH) gene is a member of gene family through the evolutionary process from a small common ancestral gene by a series of gene duplications. The role of the GH in growth and performance controls has been extensively studied in human, mice and livestock. Many researchers have considered GH as a strong candidate gene for evaluation of genetic polymorphisms that could be associated with economic traits in cattle. We report here a novel missense mutation within the exon 5 of the bovine Growth Hormone (bGH) gene. We could amplified 522 bp fragments from eight unrelated Hanwoo cattle by PCR, then, subsequently cloned and sequenced. An Msp I RFLP corresponding to a C to T transition was observed at position 2258 nt. From this result, we could predict a missense mutation (Arg to Trp) at codon 166 in a highly conserved region among many mammals. Codominant Mendelian segregation of the two alleles, Msp I (+) and Msp I (-), was observed in two full-sib F2 families (n = 32, African taurine Bos taurus ${\times}$ African zebu Bos indicus) and eight half-sib Hanwoo families. For the availability of genetic marker, we have performed PCR-RFLP with a large number of individual animals from 15 different cattle breeds (European and Asian taurines, and African indicines). Consideration of breed frequencies of Msp I (-) allele in relation to breed type and their geographic origins, shows higher frequencies in humped breeds or Asian cattle breeds than in humpless or European breeds. This result indicates that the missense mutation can be contributed the functional significance such as the signal transduction through the receptor binding, also may be used as a marker for selection of the economic traits in Hanwoo.

Evaluation of energy and amino acids of brown rice and its effects on laying performance and egg quality of layers

  • An, Byoung-Ki;An, Su Hyun;Jeong, Han-Seul;Kim, Kwan-Eung;Kim, Eun Jip;Lee, Sang-Rak;Kong, Changsu
    • Journal of Animal Science and Technology
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    • v.62 no.3
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    • pp.374-384
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    • 2020
  • Two experiments were conducted to determine apparent metabolizable energy (AME), nitrogen-corrected AME (AMEn), and ileal digestible amino acid (AA) content of brown rice (BR) and to investigate the effect of dietary supplementation of BR on laying performance and egg quality of laying hens. In Exp. 1, 72 Hy-line Brown layers (49-week-old) were allocated to two treatments using a completely randomized block design, and each treatment included six cages per treatment and six hens per cage. A semi-purified diet was formulated to include BR as the sole source of AA and energy and an N-free diet was used to determine basal endogenous loss of AA. The hens were fed a commercial layer diet for adaptation to the experimental environment and diet for 7 days from d 0, and then fed experimental diets for 5 days from d 7. Excreta were collected from d 10 to 11 and ileal digesta were collected on d 12. On a dry matter (DM) basis, the AME and AMEn of BR was determined at 3,773 and 3,729 kcal/kg, respectively. The apparent ileal digestibility (AID) of BR ranged from 32.7% for Thr to 73.7% for Arg. The range of the standardized ileal digestibility (SID) value was between 79.4% for Met and 96.6% for Lys. In Exp. 2, 252 Hy-line Brown layers (44-week-old) were divided into four groups, comprising seven replicates of nine birds each and assigned to four experimental diets containing 0 (Control), 5%, 10%, or 15% BR for 5 weeks. The BR-containing diets were formulated to be equal in the content of AMEn and digestible AA to those of the diet without BR. No significant differences were observed in laying performances. Egg quality and blood profiles were not linearly or quadratically affected by dietary treatments. These results suggest that up to 15% BR can be included into layer feed without any adverse effects on laying performance and egg quality, if its energy and digestible AA values are well evaluated.

Effect of Preventing the Maillard Reaction between Casein and Glucose with Corn Starch and Sucrose (옥수수 전분 및 자당에 의한 Casein 과 포도당 간의 갈변반응 저지효과)

  • Lee, Jong-Tae;Woo, Kang-Lyung
    • Korean Journal of Food Science and Technology
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    • v.20 no.4
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    • pp.526-535
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    • 1988
  • To estimate the effect of preventing the Maillard reaction between casein and glucose coated by freeze drying with corn starch slurry or mixture slurry of corn starch and sucrose, the reactants were treated into five groups of nonbrowning material(control), uncoated browning material, browning material coated casein alone with starch slurry, browning material coated both of reactants with starch slurry and browning material coated both of reactants with mixtrue slurry. All samples had a moisture content of about 15%. Browning reaction was carried out by storage for 30 days at $37^{\circ}C$, 67% RH. In vitro available lysine contents were decreased by the browning regardless of coating the reactants and were higher about 20.5% in the browning materials coated with mixture slurry than in the uncoated browning materials. Fructosyl-lysine contents were increased about tenfold by the browning regardless of coating and were decreased about 15.8% in the browning materials coated with mixture slurry as compare with the uncoated browning materials. The materials showing the greatest resistance to the browning reaction in the coated materials were those in which both of reactants were coated with the mixture slurry of corn starch and sucrose.

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Brewing of Acid-hydrolyzed Soy Sauce with Defatted Soybeans and Wheat Flour Koji (탈지대두와 밀가루 코오지를 이용한 산분해간장의 양조)

  • Sun, Sung-Kyun;Han, Eun-Mi;Lee, Taik-Soo;Lee, Myung-Whan
    • Applied Biological Chemistry
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    • v.30 no.2
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    • pp.147-152
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    • 1987
  • Soy sauce which was prepared with a mixture of defatted soybeans ant wheat flour koji added into acid-hydrolyzed soy sauce was evaluated for the chemical changes in levulinic acid, ethanol and amino acids composition during fermentation, Results showed that the highest reducing sugar content of $12.13{\sim}15.76%$ was found after $20{\sim}40\;days$ fermentation. The contents of total nitrogen, amino nitrogen, and ethanol increased with increase in fermentation time. A high levulinic acid content was fount at the initial fermentation period which was gradually decreased with further fermentation. The content of lactic acid also increased during fermentation. Amino acids such as Glu, Leu, Ala, Phe, Asp, Thr, Ser, Gly, Met, Tyr, Lys, His, Arg and Pro were detected in all tested groups. Higher values were found particularly for Glu, Leu, Ala and Phe and lower values for His, Tyr and Met. The most of amino acids increased in their contents after 120 days of fermentation.

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Chemical Compositions of the Green and Ripened Pumpkin(Cucurbita moschata Duch.) (미숙호박과 완숙호박의 화학성분)

  • Cho, Gyu-Seong
    • Korean Journal of Food Science and Technology
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    • v.29 no.4
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    • pp.657-662
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    • 1997
  • An attempt was made to investigate the chemical compositions of green pumpkin, edible flesh and seed of ripened pumpkin (Cucurbita moschata Duch.). The proximate compositions were moisture 9.34%, 11.98%, protein 12.70%, 13.38%, lipid 11.31%, 0.85%, carbohydrate 64.32%, 62.18%, fiber 6.31%, 4.54% and ash 6.05%, 7.76% in green and ripened pumpkin, respectively. The compositions of free sugar were glucose, fructose, sucrose, lactose and maltose in green and ripened pumpkin, respectively. During flesh growth, glucose, maltose and lactose was increased but sucrose and fructose was decreased in pumpkin. Pattern of 15 amino acid compositions in green and ripened pumpkin was shown to be of similarity. Major amino acids were glutamic acid, aspartic acid and alanine in green pumpkin and edible flesh of ripened pumpkin. And major amino acid in seed of ripened pumpkin were glutamic acid, arginine, aspartic acid and leucine. The predominant fatty acids were palmitic acid, linolenic acid, linoleic acid and oleic acid in green and ripened pumpkin, respectively. And those in seed of ripened pumpkin were linoleic acid, palmitic acid and oleic acid. The richest mineral contained in the green and ripened pumpkin was shown to be K and followed by Ca, Mg, Na and Fe in order.

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Mutations in the gyrB, parC, and parE Genes of Quinolone-Resistant Isolates and Mutants of Edwardsiella tarda

  • Kim, Myoung-Sug;Jun, Lyu-Jin;Shin, Soon-Bum;Park, Myoung-Ae;Jung, Sung-Hee;Kim, Kwang-Il;Moon, Kyung-Ho;Jeong, Hyun-Do
    • Journal of Microbiology and Biotechnology
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    • v.20 no.12
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    • pp.1735-1743
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    • 2010
  • The full-length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity), and Salmonella Typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinolone-resistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83$\rightarrow$Arg). However, an alteration in the QRDR of ParC (Ser84$\rightarrow$Ile) following an amino acid substitution in GyrA (Asp87$\rightarrow$Gly) was detected in E. tarda mutants selected in vitro at $8{\mu}g/ml$ ciprofloxacin (CIP). A mutant with a GyrB (Ser464$\rightarrow$Leu) and GyrA (Asp87$\rightarrow$Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.