• 한국식품위생안전성학회:학술대회논문집
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• 한국식품위생안전성학회 2002년도 춘계학술발표대회 및 심포지움
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• pp.215-215
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• 2002

Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major funational domains such as dihydrplipoamide adetyltransferase(E2)-binding domain, regulatory subunit of PDP(PDP)r-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase(rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPc binds to the inner lipoyl domain (L2) of E2 of ppyruvate dehydrogenase complex (PDC) in the presence of Ca+2, not under EGTA. PDPc was limited-proteolysed by typsin, chymotypsin, Arg-C, and elastase at pH 7.0 and 30C and N-terminal analysis of the fragments was done. Chymotrypsin, trypsin, and elastase made two major fragments: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx.10 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• 약학회지
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• 제33권1호
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• pp.39-45
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• 1989

Effects of dammarane glycosides of Panax ginseng on primary cultured chicken embryonic brain cells were studied by microscopic observation and determination of the activity of pyruvate dehydrogenase complex (PDHC). Brain cells were prepared from the brain of 10-day-old chicken embryo and cultured with either a standard medium consisted of 85% Dulbecco's Modified Eagle Medium (DMEM), 10% horse serum and 5% chicken embryonic extracts or a deficient medium consisted of 90% DMEM and 10% horse serum. It was observed that dammarane glycosides of Panax ginseng seemed to show the tendency to stimulate the neurite outgrowth of brain cells which were cultured with a deficient medium under microscopic observation. The activity of PDHC in brain cells cultured with a deficient medium was increased by dammarane glycosides of Panax ginseng.

• 생약학회지
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• 제20권1호
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• pp.32-36
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• 1989

Effects of Lycium chinensis, Epimedium koreanum and tuguaconitine which is isolated from Aconitum sibiricum on primary culture chicken embryonic brain cells were studied by microscopic observation and determined of the activity of pyruvate dehydrogenase complex(PDHC). Brain cells were prepared from the brain of 10-day-old chicken embryo and cultured with a medicine consisted of 90% Dulbecco's Modified Eagle Medium(DMEM) and 10% horse serum. It was observed that all substances studied seemed to show the tendency to stimulate the neurite outgrowth of brain cells which were cultured with a deficient medium under microscopic observation. The activity of PDHC in brain cells cultured with a deficient medium was increased by Lysium chinensis and Epimedium koreanum. However, tuguaconitine had not influence on the activity of PDHC.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.260-260
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• 1994

Mammalian pyruvate dehydrogenase complex(PDC) enzyme consists of multiple oopies of three major oligomeric enzymes-El, E2 E3. And protein X is one of the enzymatic constituents which is tightly bound to E2 subunit This complex enzyme is responsible for the oxidative decarboxylation of pyruvate producing of acetyl CoA which is a key intermediate for the entry of carbohydrates into the TCA cycle for its complete metabolic conversion to CO$_2$. And the overall activity of the complex enzyme is regulated via covalent nodification of El subunit by a El specific phosphatase ad kinase. Protein X has lipoyl moiety that undergoes reduction and acetylation during ezymatic reaction and has been known h be involved in the binding of E3 subunit to E2 core and in the regulatory activity of kinase. The purification of protein X has not been achieved majorly because of its tight binding to E2 subunit The E2-protein X subcomplex was obtained by the established methods and the detachment of protein X from E2 was accomplished in the 0.1M borate buffer containing 150mM NaCl. During the storage of the subcomplex in frozen state at -70$^{\circ}C$, the E2 subunit was precipitated and the dissociated protein X was obtained by cntrifegation into the supernatant The verification of protein X was accomplished by (1)the migration on SDS-PAGE, (2)acetylation by 〔2$\^$-l4/C〕 pyruvate, and (3)internal amino acid sequence analysis of tryptic digested enzyme.

• Archives of Pharmacal Research
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• 제14권4호
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• pp.325-329
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• 1991

Effects of Lycii Fructus on primary cultured chicken embryonic brain cells were studied by microscopic observation, determination of the activity of pyruvate dehydrogenase complex (PDHC), and syntheses of protein, RNA and DNA. The brain cells were prepared from the brains or 10-day-old chicken embryos and cultured with a deficient medium. The activity of PDHC in the brain cells cultured with a deficient medium was increased to 1.8 times by the addition of $30\;{\mu}g/ml$ of the total methanol extract of Lycii Fructus. To seek the active fraction, total methanol extract was further fractionated by the polarity. The survival rate of neuronal cells was significantly increased by the addition of $100\;{\mu}g/ml$ of the buthanol or aqueous fraction. At this concentration, the significant increase of the syntheses of protein and RNA, but not of DNA, indicates that the fractions may act on the neuronal cells which are known to be non-dividing cells.

• The Korean Journal of Physiology and Pharmacology
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• 제9권4호
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• pp.195-201
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• 2005

High extracellular glucose concentration was reported to suppress intracellular $Ca^{2+}$ clearing through altered sarcoplasmic reticulum (SR) function. In the present study, we attempted to elucidate the effects of pyruvate and fatty acid on SR function and reveal the mechanistic link with glucose-induced SR dysfunction. For this purpose, SR $Ca^{2+}$-uptake rate was measured in digitonin-permeabilized H9c2 cardiomyocytes cultured in various conditions. Exposure of these cells to 5 mM pyruvate for 2 days induced a significant suppression of SR $Ca^{2+}$-uptake, which was comparable to the effects of high glucose. These effects were accompanied with decreased glucose utilization. However, pyruvate could not further suppress SR $Ca^{2+}$-uptake in cells cultured in high glucose condition. Enhanced entry of pyruvate into mitochondria by dichloroacetate, an activator of pyruvate dehydrogenase complex, also induced suppression of SR $Ca^{2+}$-uptake, indicating that mitochondrial uptake of pyruvate is required in the SR dysfunction induced by pyruvate or glucose. On the other hand, augmentation of fatty acid supply by adding 0.2 to 0.8 mM oleic acid resulted in a dose-dependent suppression of SR $Ca^{2+}$-uptake. However, these effects were attenuated in high glucose-cultured cells, with no significant changes by oleic acid concentrations lower than 0.4 mM. These results demonstrate that (1) increased pyruvate oxidation is the key mechanism in the SR dysfunction observed in high glucose-cultured cardiomyocytes; (2) exogenous fatty acid also suppresses SR $Ca^{2+}$-uptake, presumably through a mechanism shared by glucose.

• Molecules and Cells
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• 제45권7호
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• pp.454-464
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• 2022

DJ-1 is one of the causative genes of early-onset familial Parkinson's disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting our findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.

• KSBB Journal
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• 제15권5호
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• pp.482-488
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• 2000

유전자 재조합 대장균을 이용하여 pyruvate dehydrogenase complex-E2 특이성 인간 모노클론 항체의 Fab부분이 생산되 었다. 재조합 대장균의 고밀도 배양과 이를 통한 재조합 인 간 항체의 고생산성을 확보하기 위한 최적 전략을 개발하기 위해 포도당과 초산의 영향에 대해 조사하였다. 포도당의 농 도가 높을수록 세포의 성장속도는 빨라지고 포도당의 소모속 도도 빨라짐을 알 수 있었다, 이때 포도당의 소모속도가 빨 라질수록 초산의 대사 부산물로서의 생성속도도 빨라졌다. 그러나 포도당이 고갈되면 일단 축적된 초산은 에너지원으로 소모되기 시작하고 이때의 소모속도는 배지내의 포도당의 농 도에 의존함을 알 수 있었다. 즉 초기 포도당의 농도가 어느 정도 높아 잔존 포도당의 농도가 높은 경우, 초산의 생성 속 도는 높고 에너지원으로 소모되는 속도는 느려져서 초산의 축적현상이 기하급수적으로 증가한다. 이렇게 높은 포도당 농도에 따라 축적된 초산은 항체 생산을 저해하였고, 저해를 위한 임계 초산 농도는$0.6g/\ell$이었다. 발현에 의해 항체를 생산하는 시기에서는, 포도당 농도가 높을수록 세포성장이 증대되나 초산에 의 한 저해 현상 및 catabolic repression의 영 향으로 항체 생산이 감소하였다. 따라서 발현 후 항체의 생산을 증진시키기 위해서는 포도당과 초산의 농도를 가능한 한 낮게 유지하는 것이 중요하다. 그러므로 고말도 배양과 재조합 인간 항체의 높은 생산성을 확보하기 위해서는 배양 액내의 포도당과 초산의 농도를 정밀하게 조절하는 것이 절실히 요구된다.

• 한국체육학회지인문사회과학편
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• 제55권3호
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• pp.649-658
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• 2016

본 연구는 지구성 트레이닝과 thiamine(thiamine tetrahydrofurfuryl disulfide: TTFD)의 투여가 골격근 내 글리코겐과 PDH(Pyruvate dehydrogenase), 그리고 PDH 활성에 관여하는 효소의 단백질인 PDK4(Pyruvate dehydrogenase kinase 4)와 PDP1(PDH phosphatase 1)의 발현에 어떠한 영향을 미치는지 알아보는 것을 목적으로 하였다. 6주령의 ICR 마우스를 대상으로 비운동집단(Sedentary; CON, TH), 운동집단(Exercise; EX, THEX)으로 나누어 4주간의 지구성 트레이닝과 체중 kg 당 50 mg의 thiamine을 경구투여 하였다. 4주간의 지구성 트레이닝은 간과 근육 내 glycogen의 저장량에 유의한 증가가 나타났지만 thiamine 투여에 따르는 차이는 나타나지 않았다. 마찬가지로 골격근 내 PDH와 PDH 조절에 관련한 PDK4, PDP1의 단백질 발현을 측정한 결과 4주간의 지구성 트레이닝에 따르는 효과는 관찰되었지만, thiamine 투여에 집단간 유의한 효과는 나타나지 않았다. 이러한 결과는 장기간 지구성 트레이닝에 따른 골격근의 적응으로 인하여 thiamine 투여에 따른 시너지 효과가 나타나지 않은 것으로 보인다. 따라서 추후 연구에서는 지구성 트레이닝의 기간을 고려한 thiamine의 섭취 타이밍 그리고 탄수화물의 복합투여에 따른 PDH와 관련 단백질의 분석이 필요할 것으로 보인다.

• KSBB Journal
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• 제15권5호
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• pp.489-496
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• 2000

고농도 유전자 재조합 대장균을 이용하여 pyruvate dehy-drogenase complex-E2 특이성 인간 모노클론 항체의 Fab 부분을 효율적으로 생산하기 위해 회분식, 이단 연속식, 반 유 가식, two-stage cyclic fed-batch 동 여러 가지 배양 방법이 조사되었다. 먼저 플라스미드 안정성 문제를 극복하기 위해 growth stage와 production stage를 분리하는 two-phase 회분식 배양과 이단 연속식 시스템을 시도하였다 그 결과 two-phase 회분식 배양보다는 이단 연속식 배양에서의 세포농도와 항체 생산성이 우수하였다 또한 이단 연속식 배양에서의 세포 성 장과 항처l 생산성은 용존산소를 제어한 경우가 그렇지 않은 경우보다 월등하게 높았다. 그리고 plasmid 안정성에 있어서 는 실험기간 내에 거의 100%를 유지하여 높은 안정도를 보 여주었다. 유가식 공정에 적합한 공급 배지로 변형된 M9 배 지가 최적배지로 선정되었고 이 배지 중 최적의 CjN 비율을 조사한 결파 2:3으로 결정되었다. 반 유가식 시스템에서 constant feeding 전략을 사용할 경우 최적 공급속도는 $0.6g/\ell/hr$이었다. 또한 pulse에 의해 공급배지를 공급할 경우에는 총 공급 량이 같을 경우 소량으로 자주 공급해 주는 것이 공급배지를 한꺼번에 많은 양을 공급해주는 것 보다 바람직하였다. 여러 가지 feeding 전략을 조사해 본 결과 linear feeding 방법이 가장 효과적이었다. 하지만 linear feeding 방법마저도 고농도 세포배양에 한계가 있었기 때문에 pH-stat 방법을 이용한 two-stage cyclic fed-batch 시스템을 시도하여 $54 g/\ell$의 세포 를 얻을 수 있었다. 따라서 이 방법이 일단 생산성 향상을 위한 세포의 고농도 배양에는 조사한 여러 배양 시스템 중에 가장 효율적인 시스템임올 알 수 있었다 하지만 이 시스템 에서 포도당을 낮은 level로 유지할 수 있었으나, 초산의 과도한 축적으로 항체 생산성의 향상은 예상에 비해 크지 않았다.