• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.

• Bulletin of the Korean Chemical Society
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• v.16 no.1
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• pp.42-47
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• 1995

Interpolymer complex formation between basic polypeptide poly(L-proline) Form Ⅱ (PLP(Ⅱ)) and acidic polypeptides poly(L-glutamic acid) (PLGA) and poly(L-aspartic acid)(PLAA) has been studied in water-methanol (1:2 v/v) mixed-solvent by viscometry, potentiometry, light scattering and circular dichroism (CD) measurements. It has been found that polymer complexes between PLP(Ⅱ) and PLGA (or PLAA) are formed via hydrogen bonding with a stoichiometric ratio of PLP(Ⅱ)/PLGA (or PLAA)=1:2 (in unit mole ratio) and that PLP(Ⅱ) forms polymer complex more favorably with PLGA than with PLAA. In addition, the minimum (for pH 5.0) and the maximum (for pH 3.2) in reduced viscosity of dilute PLP(Ⅱ)-PLGA mixed solutions are observed at 0.67 unit mole fraction of PLGA (i.e., [PLP(Ⅱ)]/[PLGA]=1/2). These findings could be explained in terms of molecular structure (or conformation) of the complementary polymers associated with the complex formation.

• Nutritional Sciences
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• v.8 no.3
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• pp.169-174
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• 2005

This study investigated the effect of allium vegetable intake on the storage and utilization of energy substrates before, during, and after exercise in tissues of rats. Ninety mts were fed either a control diet or a diet with added allium sativum (AS), allium cepa (AC), allium fistulosum (AF), or allium tuberosum (AT) for 4 weeks and then subdivided into 3 groups: before-exercise (BE) during-exercise (DE) after-exercise (AE). The DE group exercised on treadmill for 1 hour just before being sacrificed at the end of the 4th week of the dietary treatment Rats in the AE group were allowed to recuperate for 2 horns after being exercised like the DE group. Pyriooxal 5'-phospwe (PLP) levels were compared in plasma, liver and skeletal muscle of rats. There was no difference between AS animals and control animals in plasma PLP levels regardless of exercise. The plasma PLP levels of AC animals were higher than those of control animals before exercise but this PLP was decreased with exercise and lower than that of control animals after exercise. The plasma PLP levels of AF animals were higher than those of control animals during exercise but there was no difference before and after exercise. The plasma PLP levels of AT animals were higher than those of control animals regardless of exercise. Compared to those of control mts, the PLP levels of liver and muscle were significantly lower in AS, AC, AF and AT mts before exercise. The levels of liver PLP were significantly decreased in control mts while not changed in AS, AC, AF and AT mts during exercise. The levels of liver PLP tended to decrease in AS, AC and AF mts after exercise. The levels of muscle PLP were significantly decreased in control rats, while not changed in AS, AC and AF mts during exercise. The levels of muscle PLP were decreased in control mts but not changed in AS, AC and AF mts after exercise. Thus, it is suggested that the changes of PLP concentrations in plasma and tissues induced by exercise are affected by allium vegetable diet and demonstrated that allium vegetable intake induced an alteration in the redistribution of PLP among tissues.

• Nutritional Sciences
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• v.3 no.1
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• pp.31-35
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• 2000

The purpose of this study was to compare the changes in PLP concentrations induced by regular, moderate, and abrupt, high-intensity exercise in the plasma and tissues of vitamin B6 deficient and normal rats. Forty-eight rats were fed either a vitamin B6 deficient (-B6) diet or a normal (+B6) diet for 5 weeks and were subdivided into 4 groups:non-exercise(NE) group: regular, moderate-intensity exercise (RME) group; abrupt, high-intensity exercise (AIE) group; abrupt, high-intensity exercise and recuperation(IRE) group. The RME group was exercised on treadmill ($10^{\circ}$, 0.5-0.8km/h) for 2 hours just before sacrifice at the end of 5th week on the diet and the IER group was recuperated for three days on the diet after being exercised like the AIE group. Pyridoxal 5 -phosphate(PLP) levels were compared in the plasma, liver and skeletal muscle of the rats. Plasma PLP concentration tended to decrease in -B6 rats and tended to increase in +B6 rats with AIE. Plasma PLP concentration in both +B6 rats with AIE and no change in both -B6 and +B6 rats with RME. Muscle PLP concentration decreased in +B6 rats, showed no change in -B6 rats with AIE. Muscle PLP concentrations in both +B6 and -B6 rats did not change with RME. Plasma PLP, liver PLP and muscle PLP concentration of IER returned to those of NE in both +B6 and -B6 rats. These results suggest that changes in PLP concentration in plasma, liver and muscle occur with exercise and are affected by exercise intensity and vitamin B6 status. These changes may be due to interorgan redistribution of PLP.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.18 no.1
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• pp.99-132
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• 1992

Polypeptide has been used broadly as an active ingredient in cosmetics We thought it is very important to investigate the adsorption behavior of polypeptide in order to pre-estimate the effect of these polypeptides. For the study of polypeptide adsorption, we have investigated complex formation of basic homopolypeptides, poly(L-proline) Form I [PLP(I)], Form II [PLP(II)] and poly(4-hydroxy-L-proline) (PHLP) with acidic homopolypeptides, poly(L-glutamic acid) (PLGA), poly(D-glutamic acid) (PDGA) and poly(L-aspartic acid) (PLAA) through hydrogen bonding in a hydroalcoholic medium with viscometer, 1ight scatter, pH meter and circular dicroism (CD). The polypeptides used in this study have helical structure in some conditions. The result exhibited that al 1 the complexes were formed as the composition of basic/acidic homopolypeptide : L:2 irrespective of the complex systems used. A more favorable complex is formed in the PLP(II)-PLGA system than PHLP-PLGA because PLP(II) has a more flexible helical conformation, whereas PHLP has a more rigid helical conformation. The right-handed helix PLGA formed the complex favorably and quickly with the left-handed helix PLP(II), whereas the left-handed helix PDGA formed the complex favorably with the right-handed helix PLP(I). The effect of side chain of the acidic homopolypeptides on the complexation was also studied. The result showed that more favorable condition for the complexation was PLGA-PLP(II) system which has longer side chain at acidic homopolypeptide than PLAA - PLP(II). All the above facts were well supported by CD measurement for the complex systems. By the CD spectra for the complexes we could deduce the conformational change of each homopolypeptide in the complexes On the basis of the above results, we performed the adsorption test of PLP(I, II) and PHLP on the hair having a left-handed helix. The adsorption amount of each polypeptide was analyzed by HPLC. The result showed that PLP(I) was adsorbed more than PLP(II), PLP(II) was adsorbed more than PHLP on the hair. On adsorbing polypeptides having a helical structure on the hair through hydrogen bonding, it could be concluded that the helical polypeptides having the opposite directional structure to the hair are adsorbed more than those having the same directional structure with the hair and also the polypeptides having a flexible conformation are adsorbed more than those having a rigid conformation.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.3
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• pp.350-355
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• 2003

This study was carried out to clarify the effect of addition of perilla leaf powder (PLP) and carcass grade on the quality and palatability of pork sausage. The chemical composition, pH, calorie, water holding capacity, surface color, textural properties and amino acid composition of the samples were determined, and sensory characteristics were evaluated. Moisture and crude ash were not different among sausage of four type. Crude fat of grade B sausage was higher than that of grade E sausage, and grade B sausage added PLP was higher than grade B sausage without PLP. Crude Protein of grade E sausage was higher than that of grade B sausage. The pH of grade E sausage added PLP was highest, and calorie of grade B sausage without PLP was highest among sausage of four type. Water holding capacity of grade B added and free PLP was significantly higher than grade I sausage. The residual nitrite of sausage added PLP was significantly lower than sausage without PLP. In case of Hunter's $L^{*}$, grade B sausage and free PLP sausage were significantly higher than grade E sausage and sausage added PLP, respectively. In case of Hunter' $a^{*}$, grade E sausage and free PLP sausage were significantly higher than grade B sausage and sausage added PLP. Hunter's $b^{*}$ of sausage added PLP was higher than that of free PLP sausage. Textural properties, hardness, springiness, cohesiveness and chewiness were significantly different among sausage of four type, but gumminess of grade E added PLP was lowest among sausage of four type. Total amino acid was not significantly different among sausage of four type. Sensory color and texture of grade B sausage were superior to grade I sausage, palatability of free PLP sausage was superior.sage was superior.

• Korean Journal of Acupuncture
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• v.39 no.2
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• pp.43-53
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• 2022

Objectives : Paeonia lactiflora Pallas (PLP) have been reported to have pharmacological effects such as anti-inflammatory and analgesic. However, it is not yet known whether PLP extract has anti-inflammatory effect on HaCaT cells, human keratinocyte. Methods : To confirm the anti-inflammatory effect of PLP on keratinocyte, TNF-𝛼/IFN-𝛾-stimulated HaCaT cells were used. HaCaT cells were pre-treated with PLP for 1h before stimulation with TNF-𝛼/IFN-𝛾. Then HaCaT cells were stimulated with TNF-𝛼/IFN-𝛾 for 24 h, the cells and media were harvested to measure the inflammatory cytokines levels. Granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1𝛽), and TNF-𝛼 were analyzed by enzyme-linked immunosorbent assay (ELISA), and the mRNA expression of thymus and activation-regulated chemokines (TARC), IL-6, and IL-8 were measured by reverse transcription-polymerase chain reaction (RT-PCR). We also investigated the inhibitory mechanism of the mitogen-activated protein kinase (MAPKs) including ERK, JNK, and p38 and nuclear factor-kappaB (NF-𝜅B) by PLP using western blot. Results : PLP did not show cytotoxicity in HaCaT cells. In TNF-𝛼/IFN-𝛾-stimulated HaCaT cells, PLP significantly inhibited the expression of GM-CSF, MCP-1 IL-1𝛽, TNF-𝛼, TARC and IL-6. PLP inhibited the phosphorylation of ERK and translocation of NF-𝜅B into the nucleus. Conclusions : These results indicate that PLP could ameliorate the TNF-𝛼/IFN-𝛾-stimulated inflammatory response through inhibition of MAPK and NF-kB signal pathway. This suggests that PLP could be used beneficial agent to improve skin inflammation.

• Journal of Nutrition and Health
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• v.28 no.5
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• pp.426-434
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• 1995

The purpose of this study were to investigate the effect of fasting and vitamin B6 repletion on tissue concentration of pyridoxal 5-phosphate and urinary excreteion of 4-pyridoxic acid in vitamin B6 deficient rats. Sixty six rats(6 per group) were fed either a vitamin B6 deficient diet (-B6) or a control diet (+B6) for 6 weeks and then rats were repleted with +B6 diet for 2 weeks. Rats were fasted for 1 and 3 days and for 3 days after repletion. Pyridoxal 5-phosphate (PLP) concentration in plasma, liver, skeletal muscle, and heart muscle and urinary 4-pyridoxic acid (4-PA) excretion were compared. Fasting resulted in a significant increase in PLP concentration in the plasma, liver and heart muscle of rats fed the -B6 diet. Skeletal muscle PLP concentration was significantly decreased in +B6 rats but not in -B6 rats. Following vitamin B6 repletion, PLP concentration in the plasma, liver and heart muscle in previously -B6 rats was similar to the respective concentration in +B6 rats while PLP concentration in the skeletal muscle of previously -B6 rats increased, but it was not reached to that of +B6 rats. At day 1 and 2 of the fast, urinary 4-PT excretion increased in both +B6 and -B6 rats although there was no supply of vitamin B6 due to fasting. These results suggest that vitami B6 is redistributed as PLP when there is a caloric deficit and PLP is supplied by an endogenous source, possibly PLP bound to skeletal muscle glycogen phosphorylase.

• Journal of Nutrition and Health
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• v.35 no.3
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• pp.322-331
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• 2002

This study was carried out to evaluate the effect of vitamin $B_{6}$ intake by normal term delivery pregnant women on the concentration of vitamin $B_{6}$ in the material plasma, the umbilical cord plasma, and the placental tissue. Dietary intake data were obtained from a semi-quantitative frequency questionnaire. The daily mean energy and protein intakes were 2189.5 kcal (93.2% of RDA) and 79.3 g (113.3% of RDA), respectively. The average daily vitamin $B_{6}$ intake was 1.7 mg (91.4% of RDA) for the pregnant women. Their main sources of vitamin $B_{6}$ were cereal & starch (50%), and vegetables & fruits (33%). The pyridoxal phosphate (PLP) concentration of the maternal plasma, the umbilical cord plasma, and the placenta were 16.7 $\pm$ 4.1 nmol/1, 61.3 $\pm$ 19.8 nmol/l and 898.6 $\pm$ 159.2 ng/g, respectively. The PLP level was the highest in the placenta. The PLP level of the maternal plasma was significantly lower than the of the umbilical cord plasma (p < 0.001). The PLP level of maternal plasma correlated positively with that of the placenta (p < 0.0001) and the umbilical cord plasma (p < 0.05). Also the PLP level of the placenta correlated positively with that of the umbilical cord plasma (p < 0.05). These findings indicate that the vitamin $B_{6}$ nutritional status of the fetus is affected by placental vitamin $B_{6}$ levels, and that the placental vitamin B$_{6}$ levels reflect the maternal vitamin $B_{6}$ status. The umbilical cord plasma PLP level showed a positive correlation with the gestational length (p < 0.05). A negative association was observed between the PLP level showed of the umbilical cord plasma and the pregnancy weight gain (p < 0.03). The results suggest that the transfer of PLP from maternal plasma to the placental tissue could be an active transport, white the transfer of PLP from the placenta to the fetus is by means of simple diffusion. Thus, neonatal vitamin $B_{6}$ nutrition is influenced by the maternal nutritional status.

• BMB Reports
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• v.28 no.2
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• pp.124-128
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• 1995

Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.