• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.298-303
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• 2005

This experiment was carried to investigate the optimum time and times of abscisic acid (ABA) treatment for the coloration in 'Kyoho' grapes. The L-phenylalanine ammonia-lyase (PAL) enzyme activities were showed highly in both ABA treatments at veraison and 10 days after veraison. However, PAL enzyme was slightly higher in ABA treatment at 10 days after veraison than in that at veraison. Anthocyanin content showed a tendency that were increased during fruit development after veraison in all treatments, and was the highest in ABA treatment at 10 days after veraison. Fructose and glucose as soluble sugars were detected by HPLC and showed little differences in all treatments. In times of ABA treatment, PAL activity showed a tendency that decreased after increased in all treatments. PAL activity in 2 times treatment of ABA was higher than other treatments. Also, anthocyanin content was highest in 2 times treatment of ABA, as 5 folds of control and folds of 1 time treatment of ABA. Both fructose and glucose contents in all ABA treatments was slightly higher than control.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.205-212
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• 2011

Phenylalanine ammonia-lyase (PAL) from the maize pathogen Ustilago maydis was analysed immunologically to obtain insights into the structural relationships between plant PAL and fungal PAL and between PAL and histidine ammonia-lyase (HAL). Cross-reactivity was found among all the PAL proteins from different species tested, using antibodies raised against both plant and fungal PALs. Both anti-Alfalfa and anti-popular PAL antibodies strongly recognized plant PALs but only weakly recognized fungal PALs. Antibodies raised against U. maydis PAL only weakly recognized the Rhodotorula glutinis yeast PAL. The anti-U. maydis PAL antibodies showed low affinity for the plant PALs but they bound strongly to Pseudomonas bacterial HAL. Significant cross-reactivity between the two plant PAL antibodies and the bacterial HAL was also observed. Both the anti-Ustilago PAL and the anti-poplar PAL antibodies displayed similar enzyme inhibition patterns, including moderate inhibition of bacterial HAL activity. However, the bacterial HAL antibody inhibited only Ustilago PAL. The PAL and HAL antibodies tested showed no inhibition against yeast PAL. This is first report on the immunological relationships between PAL and HAL.

• Journal of Life Science
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• v.12 no.3
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• pp.274-280
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• 2002

The metabolites related to phenylpropanoid pathway play an important role in the self-defense of plants and induced by environmental stress like wounding, pathogen attack, UV-irradiation and so on. The mRNA level of rite phenylalanine ammonia lyase (PAL) was increased at 12 h to 48 h, however it was gradually decreased 48 h to 60 h after UV irradiation. The PAL enzyme activities in rice were peaked at the time of 24 h after UV irradiation, on the other hand, it was not affected by wounding. The PAL enzyme activities in pepper were raised high at 24 h and 10 h by UV irradiation and wounding respectively. The cinnamic acid 4-hydroxylase (C4H) activities were increased by wounding treatment and were detected from 12 h to end time point of experiment, while UV-irradiation didn't affect the C4H activity in rice and pepper. These results were assumed that the action of isoflavonid has an alternative effect on the defenses which include wounding and UV irradiation and on the diverse roles in rice and hot pepper.

• Current Research on Agriculture and Life Sciences
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• v.20
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• pp.65-70
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• 2002

This study was conducted to investigate allelopathic potential of various crosses of rice cultivars using their inhibitory effect on barnyardgrass growth under field conditions, and to determine PAL activity and phenolic compounds involved in phenylpropanoid pathway from selected crosses of rice cultivars. Under field conditions, Kouketsumochi, Woo co chin yu possessed higher allelopathic potential inhibiting over 90% of barnyardgrass growth. Crosses of Kouketsumochi/Woo co chin yu, Dongjinbeyo/Kouketsumochi, Dongjinbeyo/Woo co chin yu showed over 80% inhibitory effects on barnyardgrass growth. The highest PAL activity, $63.46{\mu}kats/kg$ proteins was detected in Kouketsumochi which is the most important enzyme in phenylpropanoid pathway and also higher PAL activity in crosses with Kouketsumochi. Content of cinnamate was $2.64{\mu}g/g$ f.w. in Kouketsumochi which was 2 to 5 times higher than other rice cultivars tested, indicating that higher PAL activity resulted in more cinnamate. The similar trends in cinnamate content and PAL activity were observed in crosses of rice cultivars with Kouketsumochi.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.293-297
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• 2005

This experiment was carried to investigate the optimum concentration of abscisic acid (ABA) treatment for enhancing fruit coloration of 'Kyoho' grapes. Cluster and berry weights showed a tendency that increased in proportion to concentration of ABA treatment, but were not significant in all treatments. Also, soluble solids and titratable acidity were not significant during fruit development in all treatments. L-phenylalanine ammonia-lyase (PAL) activity showed a tendency that decreased after rapidly increased in all treatments, and was the highest in 1000 mg/l ABA treatment at final harvest. Anthocyanin and total phenolics contents were high in proportion to ABA treatment concentration, and anthocyanin content in 1000 mg/l ABA treatment was 2.5 folds of that in control. Fructose and glucose as soluble sugars were detected, but sucrose was not detected. Both fructose and glucose contents increased during fruit development, but showed little difference in all treatments.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.57-57
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• 1995

1) 효소원의 조제 : 류마티스 관절염환자의 관절액 및 rat platelet PLA$_2$는 장등의 방법으로 조재하여 사용하였으며, 기질은 1-pal- (1-$^{14}$ C) linoleoyl PE로 하여 Dole 변법으로 효소활성을 측정하였다. 2) Histamine 유리반응 ; Rat복강으로부터 정제한 비만세포를 항원-항체 복합체로 자극하거나, $A_{23187}$등의 처리로 유리되는 histamine 을 methylation 시킨 후 유기용매법으로 추출한 후 scintillation counter로 측정하였다. 결과 : \circled1 천연물로부터 분리한 5종의 biflavonoid (amentoflavone 및 그 유도체)의 PLA$_2$ 저해 활성을 검토한 결과 거의 유사한 IC50 (약 3$\mu\textrm{m}$)을 나타내었다. \circled2 이들 중 amentoflavone은 염증성 PLA$_2$(Group II형 PLA$_2$)에 비교적 특이성을 나타내었다. 또한 제해양식은 비경쟁적 이면서 비가역적이였다. \circled3 비만세포에서 histamine 유리 억제반응은 자극제의 종류에 관계없이 억제작용을 나타내었으며, 기존에 임상적으로 사용되고 있는 Tranilast나 DSCG(disodium chromoglycate)에 비하여 10배 이상 강한 histamine 유리 억제작용을 나타내었다.다.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Current Research on Agriculture and Life Sciences
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• v.8
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• pp.19-27
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• 1990

This study was conducted to investigate the effect of sulfur containing pesticides, captan, edifenphos, EPN, acephate, asulam, bentazone on arylsulfatase activity in soil incubated at $30^{\circ}C$ for 42 days with or without urea addition. The results obtained were as follows: When pesticides, captan, edifenphos, EPN, acephate, asulam, bentazone were treated in urea added and unadded soil, the activity of arylsulfatase was the highest at 7 days of incubation. The arylsulfatase activity in urea added soil was kept higher as compared with that of the urea unadded soil. When pesticides, captan, edifenphos, acephate, asulam, bentazone, were treated in urea added and unadded soil, the activity of arylsulfatase was inhibited at the entire experimental period. By the treatment of EPN in urea added and unadded soil, the arylsulfatase activity was decreased at the early stage of treatment, but increased after 28 days of incubation.

• Journal of dental hygiene science
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• v.12 no.4
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• pp.303-309
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• 2012

This study was designed to provide basic data for developing a standardized caries potentiality index to help to choose snack foods with a low score on the index by investigating adolescents' snack intakes and measuring them in total saccharinity, pH, and acidogenic potential and glucosidase activity of Streptococcus mutans (S. mutans). Total 28 snack foods were selected and measured for total saccharinity (Pocket refractometer PAL-1, ATAGO) and pH with a pH meter (Mentor, Seoul, Korea). Artificial saliva was added to each sample. The experiment group was administered with S. mutans (ATCC 3692), cultured in a constant temperature incubator at $37^{\circ}C$, and measured for pH changes over five times including after 10 minutes, 30 minutes, 1 hour, and 24 hours. Each sample of 0.3 ml was added to tubes containing sucrose, galactose, or glucose to measure the glucosidase activity of S. mutans. They were then observed for glucosidase activity with colorimetry after 24 hours of culture in a constant temperature incubator at $37^{\circ}C$. The mean pH by the acidogenic potential of S. mutans was pH 5.33. The experiment group dropped in pH more than the control group due to the increasing acidogenic potential of S. mutans by glucosidase activity, recording pH 5.27 after 10 minutes, pH 5.21 after 30 minutes, pH 5.15 after 1 hour, and pH 4.80 after 24 hours. The observation results of glucosidase activity of S. mutans with colorimetry show that most of the samples were positive in orange and yellow with glucose, sucrose, and glactose recording activity of 78.58%, 75%, and 71.42%, respectively.