• Journal of Life Science
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• v.14 no.4
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• pp.657-663
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• 2004

Tobacco transformants harboring cinnamic acid 4-hydroxylase gene (C4H) fused with susquiterpene cyclase promoter was developed in order to regulate biosynthesis of phenolic compounds by the expression of the introduced gene. Twenty transformants for each specific promoter were used to analyze the incorporation of the chimeric genes by PCR and Southern blot analysis. PCR products of NPTII(neomycin phosphotransferase) gene (553bp) were detected in the transgenic tobacco plants. The incorporation of the chimeric gene was confirmed in the Southern blot analysis. C4H activity in the transgenic plants was elevated by UV-irradiation and its level was higher compared to that of control plants.

• Preventive Nutrition and Food Science
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• v.2 no.4
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• pp.333-337
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• 1997

Trans-cinnamic acid-4-hydroxylase(tC4H) is the first cytochrome P450-dependent monooxygenase of the phenylpropanoid pathway. The roots of avocado seedlings were wounded and examined to determine whether the tC4H would be activated in response to wounding and/or whether tC4H activity be modulated by the application of exogenous p-coumarate. At the specified length of times, the wounded and treated roots were either frozen in liquid nitrogen or used immediately to extract microsomal proteins. The microsomal proteins were subjected to immunoblot analysis using polyclonal antibodies against CYP73 of tC4H gene. In this study, tC4H was induced in wounded roots sealed in bags within 6 hours, and in low level({TEX}$10^{-8}${/TEX}M) of p-coumarate solution within 24 hours, whereas the olution without p-coumarate and high levels of p-coumarate solution repressed tC4H induction in wounded roots. These results indicate that tC4H is induced by wounding in the root of avocado, and is inhibited by the application of exogenous p-coumarate.

• Archives of Pharmacal Research
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• v.19 no.3
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• pp.219-222
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• 1996

Ephedra olistachya cultures have been known to accumulate $rho-coumaroylamino$ acids by elicitor treatment. Based on their chemical structures, the biosynthetic pathway of$rho-coumaroylamino$acids was postulated and phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (4-CH) and p-coumaroyl CoA: D-Ala p-coumaroyltransferase ($rho-CT$) were supposed to be involved in the pathway. The time course inductions of these enzymes were investigated after treatment of yeast extract, yeast-derived mannan glycopeptide and D-Ala. They were detectable at only 4 hours and reached to their maximum level at 9 hours after onset of elicitor treatment. The activities of PAL and 4-CH were almost disappeared within 24 hours, however, that of $rho-CT$was remained up to 48 hours irrespective of the kind of elicitors. $rho-CT$ showed substrate specificity to D-Ala at crude enzyme extract level.

• Plant Biotechnology Reports
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• v.5 no.1
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• pp.79-88
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• 2011

Vitis vinifera cv. Koshu is a traditional grape cultivar that has been grown for centuries in Japan. The Koshu grape has pink-colored skin and Koshu wines have slight astringency. We demonstrated for the first time the characterization of hydroxycinnamic acids, flavan-3-ols, and flavonoids in Koshu grape using high-performance liquid chromatography and liquid chromatography-mass spectrometry. The gross weight of phenolic compounds excluding anthocyanins and proanthocyanidins in Koshu grape at harvest was higher than those in Sauvignon Blanc, Chardonnay, and Merlot grapes. In addition, hydroxycinnamic acid and monomeric flavonol contents in Koshu grape were also higher than those in the other grape cultivars. Transcription analysis of cinnamic acid 4-hydroxylase, p-coumarate 3-hydroxylase, caffeate methyltransferase, and flavonol synthase genes indicated high accumulation of hydroxycinnamic acids and flavonols in Koshu grape skin compared with the other cultivars. These findings obtained by chemical and molecular approaches partially explained the phenolic characteristics and the peculiar astringency of Koshu grape.

• Korean Journal of Weed Science
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• v.17 no.1
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• pp.59-65
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• 1997

Bentazon 6-hydroxylase (B6H) and cinnamic acid 4-hydroxylase (CA4H) activities were determined in rice (Oryza sativa L.) microsomes to study methods of enhancing cytochrome P-450 mediated aryl hydroxylation of bentazon by hydoxylase inducing compounds. Pretreating rice seeds with 1,8-naphthalic anhydride at 0.5-2% and fenclorim at 5 and 10 ${\mu}M$ increased B6H and CA4H activities. Treatments of rice seedling with ethanol 2.5% enhanced B6H and CA4H activities, and with phenobarbital at 12 mM enhanced B6H activity, and CA4H activity was enhanced at 2 mM. B6H activity was synergistically enhanced by combined treatments of ethanol 2.5 or 5% and phenobarbital 8 or 12mM and also that of 1,8-naphthalic anhydride 0.5 or 1% and phenobarbital 8 or 12 mM, but CA4H activity was decreased by combined treatment. Five-day-old rice seedlings showed higher B6H and CA4H activities which decreased with seedling age.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.501-507
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• 1997

Chloroplasts isolated from Stevia rebaudiana Bertoni leaves contained an enzyme activity which catalyzed hydroxylation of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA) to steviol (ent-13-hydroxy kaur-16-en-19-oic acid), the diterpenoid carboxylic alcohol which is the aglycone of sweet stevioside-related glycosides. $[^(14)C]-methylated$ ent-KA was used to localize ent-KA hydroxylase. $[^(14)C]-methyl-KA$ was most actively was transformed into methyl-steviol in chloroplast. The enzymatic activity was found in stroma fraction but not in thylakoid membrane in Stevia rebaudiana Bertoni. However, ent-KA 13-hydroxylase activity was not detected in stroma fraction of either Spinacia oleracea and Solidago altissima. The reaction products using $[^(14)C]-methyl-KA$ were purified and identified on TLC autoradiogram. The hydroxylation of ent-KA from stromal protein to form steviol required NADPH and oxygen. FAD and riboflavin stimulated the enzyme activity 1.5-and 1.7-fold, respectively. It also turned out that the activity of this enzyme using methyl-KA as a substrate was 16.7% that of ent-KA. The purified ent-KA 13-hydroxylase did not act on t-cinnamic acid, 4-hydroxyphenyl acetic acid, choline and resorcinol, known as monooxygenase and hydroxylase substrates.

• 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.

• Korean Journal of Plant Tissue Culture
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• v.24 no.4
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• pp.233-238
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• 1997

Salicylic acid (SA) is involved in the establishment of systemic acquired resistance (SAR) in many plant including tobacco. Considering the important role of SA in disease resistance, biosynthetic and metabolic pathways of SA in tobacco have been studied extensively: The initial step for biosynthetic pathway of SA is conversion of phenylalanine to trans-cinnamic acid, followed by decarboxylation of trans-cinnamic acid to benzoic acid and ie subsequent ring hydroxylation at the C-2 position to form SA. In TMV inoculated tobacco, most of the newly synthesized SA is glucosylated or methylated. Methyl salicylate has been identified as a biologically active, volatile signal. In contrast, the two glucosylated forms accumulate in the vicinity of lesions and consist of SA glucoside, a major metabolite, and SA glucose ester, a relatively minor from. Two enzymes involved in SA biosynthesis and metabolism have been purified and characterized : benzoic acid 2-hydroxylase which catalyzes conversion of benzoic acid to SA; UDP-Glucose: SA 1-O-D glucosyltransferase which converts SA to SA glucose ester. Further studies of the biosynthetic and metabolic pathways of SA will help to elucidate the SAR signal transduction pathway and provide potential tools for the manipulation of disease resistance.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.46 no.1
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• pp.22-28
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• 2001

The induction of enzymes and the accumulation of their end products associated with self-defense mechanism in rice were investigated. When rice leaves were irradiated with UV light, activities of diterpene cyclase, phenylalanine ammonia-lyase (PAL), and cinnamic acid 4-hydroxylase (CA4H) were induced and rice phytoalexin, momilactone A was accumulated. The content of p-coumaric acid in rice leaves was closely correlated with self-defense or allelopathic potential against barnyardgrass. UV-challenged rice leaves gave rise to the inhibition of barnyardgrass growth.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.20 no.2
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• pp.37-43
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• 2020

Phenylketonuria is the most prevalent disorder caused by an inborn error in aminoacid metabolism. It results from mutations in the phenylalanine hydroxylase (PAH) gene. If untreated or late treated, results in profound and irreversible mental disability. Newborn screening test identify patients with phenylketouria. The early initiation of a phenylalanine restricted diet very soon prevents most of the neuropsychiatric complications. However, the diet therapy is difficult to maintain and compliance is poor, especially in adolescents and adulthood. Since 2015, American Medical College of Medical Genetics and Genomics (ACMG) recommended more strong restrictive diet therapy for target blood level of phenylalanine (<360 umol/L). For over four decades the only treatment was a very restrictive low phenylalanine diet. This changed in 2007 with the approval of cofactor therapy (Tetrahydrobiopterin, BH4) which is effective in up to 30% of patients. Data from controlled clinical trials with sapropterin dihydrochloride indicate a similar occurrence of all-cause adverse events with this treatment and placebo. Large neutral aminoacids (LNAA) competes with phenylalanine for transport across the blood-brain-barrier and have a beneficial effect on executive functioning. A new therapy has just been approved that can be effective in most patients with PAH deficiency regardless of their degree of enzyme deficiency or the severity of their phenotype. Phenylalanine ammonia lyase (PAL-PEG) was approved in the USA by FDA in May of 2018 for adult patients with uncontrolled blood phenylalanine concentrations on current treatment. Nucleic acid therapy (therapeutic mRNA or gene therapy) is likely to provide longer term solutions with few side effects.