• Tuberculosis and Respiratory Diseases
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• 제79권4호
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• pp.257-266
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• 2016

Background: Idiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to $NAD^+$ by various quinones and thereby elevates the intracellular $NAD^+$ levels. In this study, we examined the effect of increase in cellular $NAD^+$ levels on bleomycin-induced lung fibrosis in mice. Methods: C57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with ${\beta}$-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor ${\beta}1$ (TGF-${\beta}1$) and ${\beta}$-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT). Results: ${\beta}$-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-${\beta}1$, ${\alpha}$-smooth muscle actin accumulation. In addition, ${\beta}$-lapachone showed a protective role in TGF-${\beta}1$-induced ECM expression and EMT in A549 cells. Conclusion: Our results suggest that ${\beta}$-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-${\beta}1$-induced EMT in vitro, by elevating the $NAD^+$/NADH ratio through NQO1 activation.

• 한국동물위생학회지
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• 제30권3호
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• pp.291-304
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• 2007

Mutants of an obligate aerobic bacterium, Vitreoscilla, that have deficiency in heat-labile catalase-peroxidase hydroperoxidase I (HPI) were created by EMS treatment. The catalase-peroxidase HPI-deficient mutant showed substantially lower peroxidase activity in exponential and mid-stationary phase compared with the wild type strain. In late stationary phase, the mutant exhibited no peroxidase activity. Peroxidase deficiency in the mutant was revealed by polyacrylamide gels stained for peroxidase activity. Characteristically, catalase levels in the mutant increased about 14- and 8-fold during growth in the exponential and stationary phases, respectively, compared to those in the wild type, suggesting a compensatory effect for protection from $H_2O_2$ toxicity. The mutant showed differences in physiology from the wild type: retardation in growth rate and decrease in oxygen consumption. Both the wild type and the catalase-peroxidase HPI-deficient mutant of Vitreoscilla had lower growth rates in media containing increasing $H_2O_2$ concentrations. However, the mutant exhibited an additionally decreased growth rate after 6 to 8 h of growth compared to the wild type. The wild type was resistent up to 20 mM $H_2O_2$, whereas the mutant was very sensitive to high concentrations of exogenous $H_2O_2$. Although elevated catalase levels would provide protection of the bacteria from the deleterious effect of $H_2O_2$, it did not appear to be complete. Cell-free extracts of the mutant showed decreased NADH oxidation rates and higher accumulation of $H_2O_2$ during this oxidation. These results may account for the impaired growth and earlier onset of death phase by the catalase-peroxidase HPI-deficient mutant of Vitreoscilla.

• 한국미생물·생명공학회지
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• 제26권2호
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• pp.89-95
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• 1998

R. sphaereides K7 및 E15-1은 혐기적 광합성조건에서 glucose를 탄소원으로 하여 배양초기 24시간 동안은 수소가스를 계속 생산하였으나, 그 이후에는 배양액에 축적된 acetic acid및 formic acid가 배양액의 pH를 4.2-4.8로 저하시켜 수소를 거의 생산하지 못하였다. 또한 배양 6일 후에도 R. sphaeroides K7 및 E15-1의 glucose의 이용율은 각각 43% 및 74%에 불과하였다. 그러나 배양액의 pH를 6.8-7.0으로 유지하면서 배양한 결과 R. sphaeroides K7및 E15-1 두 균주 모두의 수소생산율과 glucose의 이용율이 증가되어, 수소생산은 배양 10일까지도 계속 증가되었으며, glucose도 두 균주 각각 배양후 2.5일 및 4.5일 후에 완전 소비하였다. 뿐만 아니라 균체 배양액의 pH를 중성으로 유지하면서 R. sphaeroides K7 및 E15-1을 배양할 경우 균체의 표백현상이 제거되어 배양 7일 후에는 각각 균체의 bacteriochlorophyll 함량이 약 44배 및 9배 증가되었으며, 이때 균체의 농도는 각각 약 10배 및 2.4배 증가되었다. R. sphaeroides K7 및 E15-1은 혐기적 광합성조건에서 acetic, lactic, butyric 및 malic acid로 부터도, 비록 그 양이 glucose로 부터보다는 적으나, 수소를 생산하였다. 본 실험 결과로 미루어 혐기적 광합성 조건에서 R. sphaeroides K7 및 E15-1은 glucose로부터 수소를 생성할 때 NADH 산화 및 hydrogenase가 관여한 대사가 우선적으로 일어나고, 2차적으로는 이때 생성된 유기산을 전자 공여체로 광합성 작용에 의해 질소원이 존재하지 않을 때 nirogenase에 의해서 양성자(H$^{+}$)가 환원되어 수소(H$_2$)가 생성되는 것으로 생각된다.

• 한국균학회지
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• 제17권3호
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• pp.161-168
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• 1989

표고버섯(L. edodes) 중의 mitochondria는 설탕밀도단계기울기법에 따라 분리정제 하였다. 앞서 보고한 바와 같이, 각 파장별 빛조사(400-700nm)에 따른 mitochondrial ATPase와 ATP synthase의 활성도는 680nm와 470nm에서 각각 활성화되었다. 본 연구에서, 400nm 이하의 파장별 빛조사에 따른 mitochondrial ATPase 및 ATP synthase의 활성도는 380nm와 330nm에서 각각 활성화되었으며, 330nm 및 350에서 각각 억제되었다. FAD의 존재하에서, mitochondrial ATP synthase는 활성화 파장 및 억제 파장의 조사에 의하여 활성도가 각각 증가된 반면, mitochondrial ATPase의 활성도는 감소되었다. 그러나, NADH의 존재하에서, 이들 파장의 조사에 의한 효소의 활성도는 변화가 없었다. 또한, 두 효소는 각각의 활성화 파장 및 억제 파장이 조사됨에 따라 $FADH_2$를 FAD로 산화시키는 spectrum을 보였다. 이로써, 이 두 효소는 빛 조사에 의하여 생체내의 산화 환원반응의 산화제로 작용하였으며, 특히 mitochondrial ATP synthase의 활성화에 따른 광유발물질은 mitochondria 중에 존재하는 flavin 또는 flavoprotein으로 추정된다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1120-1128
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• 2004

The SCBFC was composed of bilayered cathode, the outside of which was modified with $Fe^{3+}$ (graphite-Fe(III) cathode) and the inside of which was porcelain membrane, and of an anode which was modified with $Mn^{4+}$ (graphite­Mn(lV) anode). The graphite-Fe(III), graphite-Mn(IV), and porcelain membrane were designed to have micropores. The outside of the cathode was exposed to the atmosphere and the inside was contacted with porcelain membrane. In all SCBFCS the graphite-Fe(III) was used as a cathode, and graphite-Mn(IV) and normal graphite were used as anodes, for comparison of the function between normal graphite and graphite-Mn(IV) anode. The potential difference between graphite-Mn(IV) anode and graphite-Fe(III) cathode was about 0.3 volt, which is the source for the electron driving force from anode to cathode. In chemical fuel cells composed of the graphite-Mn(IV) anode and graphite-Fe(III) cathode, a current of maximal 13 mA was produced coupled to oxidation of NADH to $NAD^{+}$ the current was not produced in SCBFC with normal graphite anode. When growing and resting cells of E. coli were applied to the SCBFC with graphite-Mn(IV) anode, the electricity production and substrate consumption were 6 to 7 times higher than in the SCBFC with normal graphite anode, and when we applied anaerobic sewage sludge to SCBFC with graphite-Mn(IV) anode, the electricity production and substrate consumption were 3 to 5 times higher than in the SCBFC with normal graphite anode. These results suggest that useful electric energy might possibly be produced from SCBFC without electron mediators, electrode-active bacteria, and extra energy consumption for the aeration of catholyte, but with wastewater as a fuel.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.16-16
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• 2001

Apoptosis로 일컬어지는 예정된 세포사멸(programmed cell death)은 개별 세포의 입장에서는 곧바로 사멸을 의미하지만, 정상적인 고등 생물의 입장에서는 개체의 발생과 분화하는데 프로그램된 과정이다. 자발적 세포사멸은 다른 조직에 비해 생식 조직인 난소나 정소에서 복잡한 apoptosis 기작들을 가지리라 사료된다. 본 연구는 Bcl-2 family중 apoptotic protein인 Bax에 대해 suppression하는 유전자를 yeast system을 활용하여 돼지 정소와 난소로부터 각각 cDNA library를 구축한 후 탐색하였다. 탐색에 활용된 cDNA library는 돼지의 정소와 난소로부터 mRNA를 분리하여 yeast vector인 pAD-GAL4-2.1에 구축하였고, 마우스 bax 유전자는 gal 1 promoter의 조절 하에 glucose 배지에서는 유도되지 않고, galactose 배지에서만 선택적으로 Bax를 발현할 수 있는 효모 vector(pL19-bax)를 구축하였다. Bax에 의한 apoptosis suppressor를 탐색하기 위해 우선 효모 W303에 pL19-bax를 transform하여 glucose 배지에서 Bax의 발현을 억제하였다. pL19-bax를 가진 효모에 정소와 난소로부터 구축된 cDNA library를 transform 시키고, transform된 효모는 각각 Bax에 의한 toxicity를 저해하는 유전자를 찾기 위해 스크린되었다. 이러한 방법으로 정소 cDNA library 탐색에서는 5 $\times$ $10^{6}$ transformant중 39개, 난소cDNA library 탐색에서는 2 $\times$ $10^{6}$ transformant중 26개의 콜로니가 생존하였다. 이들 콜로니로부터 유전자를 분리하여 분석해 본 결과 여러 그룹으로 분류할 수 있었다. 각 그룹의 관련 유전자는 protein synthesis/degradation 12종, oxidation/reductation 5종, detoxin/ cell cycle promoter 3종, signal transduction/growth factor 5종, 그리고 알려지지 않은 유전자 9종이었다. 그 중, bax-toxicity inhibition에 강력한 survival phenotype을 가지는 유전자(pSEDL)를 동정하였다. 이것은 T3-4-1 콜로니로부터 분리하였는데 140개 아미노산으로 이루어진 인간 SEDL(GenBank, XM_013096) 유전자와 매우 유사한 homology를 가지며, bax와 관련된 기능은 밝혀져 있지 않다. 이외에도 분리된 유전자에는 NADH, thioreduction, 그리고 cytochrome oxidase와 같은 positive 유전자 군이 크로닝되어, Bax를 이용한 효모에서 apoptosis suppressor에 관련된 유전자를 손쉽게 스크린하는 것이 가능하고, 분리된 유전자의 기능을 예측할 수 있어 지금까지 보고된 유전자 크로닝법 보다는 강력한 수단으로 활용될 수 있다는 사실을 시사하였다. 그러나, ORF에 관계없이 Bax 발현에 저항하는 유전자군이 선발된다든지 하는 문제점은 금후 검토가 필요하리라 사료된다.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.298-303
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• 2007

호염성세균 H. subglaciescola DH-1은 염화나트륨이 없거나 0.8 M 이하로 존재하는 환경에서 생장하지 못한다. 이 호혐성세균은 2.0 M의 염화나트륨이 존재하는 조건에서는 최적온도($30^{\circ}C$)보다 높은 $40^{\circ}C$에서 생장이 가능하였으나, 0.8 M의 염화나트륨이 존재하는 조건에서는 생장이 크게 저하되었다. 세포추출물을 염화나트륨이 존재하는 조건에서 $50^{\circ}C$로 1시간 동안 열처리하였을 때 세포내 효소의 활성이 유지되었으나, 염화나트륨이 없는 조건에서 열처리하였을 때 효소의 활성은 유지되지 않았다. 반면, 대장균의 세포추출물의 효소활성은 1.0 M이상의 염화나트륨이 존재할 때 온도 또는 pH와 관계없이 측정되지 않았다. H. subglaciescola DH-1은 pH $7{\sim}10$의 범위에서 생장하였고, 생장을 위한 최적 pH는 8이었다. 이러한 생리적인 특성으로부터 염화나트륨은 H. subglaciescola DH-1의 물질대사를 위한 필수적인 무기영양소라는 사실을 유추할 수 있다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2007년도 Proceedings of The Convention
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• pp.57-64
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• 2007

Metabolic interactions of the three major cannabinoids, ${\Delta}^9$-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) with steroid hormones were investigated. These cannabioids concentration-dependently inhibited $3{\beta}$-hydroxysteroid dehydrogenase and $17{\alpha}$-hydroxylase in rat adrenal and testis microsomes. CBD and CBN were the most potent inhibitors of $3{\beta}$-phydroxysteroid dehydrogenase and progesterone $17{\alpha}$-hydroxylase, respectively, in rat testis microsomes. Three cannabinoids highly attenuated hCG-stimulated testosterone production in rat testicular interstitial cells. These cannabinoids also decreased in levels of mRNA and protein of StAR in the rat testis cells. These results indicate that the cannabinoids could interact with steroid hormones, and exert their modulatory effects on endocrine and testicular functions. Metabolic interaction of a THC metabolite, $7{\beta}$-hydroxy-${\Delta}^8$-THC with steroids is also investigated. Monkey liver microsomes catalyzed the stereoselective oxidation of $7{\beta}$-hydroxy-${\Delta}^8$-THC to 7-oxo-${\Delta}^8$-THC, so-called microsomal alcohol oxygenase (MALCO). The reaction is catalyzed by CYP3A8 in the monkey liver microsomes, and required NADH as well as NADPH as an efficient cofactor, and its activity is stimulated by some steroids such as testosterone and progesterone. Kinetic analyses revealed that MALCO-catalyze reaction showed positive cooperativity. In order to explain the metabolic interaction between the cannabinoid metabolite and testosterone, we propose a novel kinetic model involving at least three binding sites for mechanism of the metabolic interactions.

• The Korean Journal of Physiology
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• 제17권2호
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.