• The Korean Journal of Physiology
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• 제23권2호
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• pp.291-299
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• 1989

Na and Ca effects on contracture were studied in order to estimate Na/Ca exchange ratio in the isolated atrial muscle of the rabbit. All experiments were performed in tris-buffered Tyrode solution which was being aerated with 100% $O_2\;and\;kept\;at\;37^{circ}C$. To load intracellular $Na^+,\;10{-6}M$ ouabain or K-free solution were used. Contractures were induced by brier exposure of atrial muscle to Tyrode solution containing various concentrations of Ca or of Na. The results obtained were as follows: 1 ) Increasing the extracellular Ca concentration, the amplitude of contracture also increased and was maximum at 8 mM Ca-Tyrode solution. 2) The relationship between extracellular Ca concentrations and relative amplitude of the contractures showed hyperbolic pattern. By using Hill plot, the line has the slope of 1 12 which means the number of Ca binding sites of the carrier in the cell membrane. 3) The amplitude of the contracture was maximum in 0 mM Na-Tyrode solution and decreased in dose dependent manner when the Na concentration increased. 4) When the relationship between extracellular Na concentrations and the amplitude of contractures was expressed as dose-response curve, the curve showed sigmoid pattern. The line with the slope of 2.82 was obtained by using Hill plot. 5) From above all the results, it is suggested that exchange ratio of Na and Ca via Na/ca exchange system in the atrial muscle of rabbit could be 3:1 approximately.

• Bulletin of the Korean Chemical Society
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• 제33권10호
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• pp.3248-3252
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• 2012

Vibrio extracellular metalloprotease (vEP), secreted from Vibrio vulnificus, shows various proteolytic function such as prothrombin activation and fibrinolytic activities. Premature form of vEP has an N-terminal (nPP) and a C-terminal (C-ter100) region. The nPP and C-ter100 regions are autocleaved for the matured metalloprotease activity. It has been proposed that two regions play a key role in regulating enzymatic activity of vEP. Especially, C-ter100 has a regulatory function on proteolytic activity of vEP. C-ter100 domain has been cloned into the E. coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using gel-filtration chromatography followed by affinity chromatography. To understand how C-ter100 modulates proteolytic activity of vEP, structural studies were performed by heteronuclar multi-dimensional NMR spectroscopy. Backbone $^1H$, $^{15}N$ and $^{13}C$ resonances were assigned by data from standard triple resonance and HCCH-TOCSY experiments. The secondary structures of vEP C-ter100 were determined by TALOS+ and CSI software based on hydrogen/deuterium exchange. NMR data show that C-ter100 of vEP forms a ${\beta}$-barrel structure consisting of eight ${\beta}$-strands.

• 유기물자원화
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• 제5권1호
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• pp.25-32
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• 1997

토양에서 분리한 광합성 세균인 Rhodospirillum rubrum N-1 균주에 의한 ${\delta}$-aminolevulinic acid (ALA) 생산에 있어서 levulinic acid (LA) 및 ALA 생합성 경로의 첨가효과를 검토하였다. Glutamate를 제외한 Lascelles의 기본배지에 LA를 배양초기에 10mM, 이후 대수기 중기에 30 mM을 첨가한 결과 균체외의 ALA 생산성은 최고 45 mg/l 도달, LA 미첨가 대비 23배 증가하였다. ALA 생합성의 전구물질인 glycine-succinate ($C_4$) 및 glutamate ($C_5$)의 feeding 효과는 배양초기/대수기 중기에 LA 10/30 mM을, glutamate 30 mM/30 mM을 각각 첨가하는 한편, 대수기 중기에 10 mM 농도의 $C_4$ 전구물질을 별도로 첨가배양함으로써 미첨가 대비의 ALA 생산성이 약 40배 (75 mg/l) 증가하였다.

• The Korean Journal of Physiology and Pharmacology
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• 제6권1호
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• pp.9-14
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• 2002

In the present study, we used the microdialysis technique combined with high performance liquid chromatography (HPLC) and electrochemical detection to measure the extracellular levels of norepinephrine (NE) in the posterior hypothalamus in vivo, and to examine the effects of various drugs, affecting central noradrenergic transmission, on the extracellular concentration of NE in the posterior hypothalamus. Microdialysis probes were implanted stereotaxically into the posterior hypothalamus (coordinates: posterior 4.3 mm, lateral 0.5 mm, ventral 8 mm, relative to bregma and the brain surface, respectively) of rats, and dialysate collection began 2 hr after the implantation. The baseline level of monoamines in the dialysates were determined to be: NE $0.17{\pm}0.01,$ 3,4-dihydroxyphenylacetic acid (DOPAC) $0.94{\pm}0.07,$ homovanillic acid (HVA) $0.57{\pm}0.05$ pmol/sample (n=8). When the posterior hypothalamus was perfused with 90 mM potassium, maximum 555% increase of NE output was observed. Concomitantly, this treatment significantly decreased the output of DOPAC and HVA by 35% and 28%, respectively. Local application of imipramine $(50\;{\mu}M)$ enhanced the level of NE in the posterior hypothalamus (maximum 200%) compared to preperfusion control values. But, DOPAC and HVA outputs remained unchanged. Pargyline, an irreversible monoamine oxidase inhibitor, i.p. administered at a dose of 75 mg/kg, increased NE output (maximum 165%), while decreased DOPAC and HVA outputs (maximum 13 and 12%, respectively). These results indicate that NE in dialysate from the rat posterior hypothalamus were neuronal origin, and that manipulations which profoundly affected the levels of extracellular neurotransmitter had also effects on metabolite levels.

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.817-824
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• 1997

Extracellular ATP elicits various biological responses and plays a significant role in physiological regulation. Recently, ATP-induced growth inhibitions were reported in some tumor cell lines, but these effects and mechanisms are not well hewn. This study was conducted to investigate ATP-induced growth inhibition in mouse $leukemic(P388D_1)$ cells. ATP inhibited cell growth in a dose-dependent manner as analyzed by MTS assay$(IC_{50}: 33.1\;{\mu}M)$. Nucleotides other than ATP, such as ADP$(37.5;{\mu}M)$ and AMP$(33.2;{\mu}M)$ had the same effects as ATP but adenosine$(57.8;{\mu}M)$ showed less effect than ATP. ATP attenuated the cells in $G_0/G_l\;and\;G_2/M$ phases but increased those in S phase in flow cytometric analysis. Hypodiploid cells$(A_0)$, the presumptive findings of apoptosis, were found among the ATP-treated cells. ATP induced DNA fragmentation into $180{\mu}200\;bps $as measured by electrophoresis. some apoptotic cells were stained by TUNEL method. ATP increased the intracellular free $Ca^{++}$concentration$([Ca^{++}]_i)$ and the increment of $([Ca^{++}]_i)$ was caused by influx from the extracellular space. These results suggest that extracellular ATP induces growth inhibition through apoptosis.

• KSBB Journal
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• 제21권5호
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• pp.336-340
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• 2006

본 연구에서는 참당귀 현탁세포배양에 의한 면역 증강성 ECP의 생산을 증진시키기 위하여 배지교환식 배양을 수행하였으며, ECP의 분비를 촉진하는 초음파 처리와 Pluronic F-68의 영향을 조사하였다. 참당귀의 현탁세포배양시, 최대 세포생장은 6일째에 16.8 g DCW/L였고, ECP의 생산은 세포생장과 함께 증가하다가 8일째에 최고 0.9 g/L가 생산되었다. 식물세포 고농도배양법인 배지교환식 배양을 적용해, 초기 당 농토를 높이고 접종 후 5일부터 연속적으로 배지교환을 해주어 23.8 gDCW/L의 고농도의 현탁세포를 얻을 수 있었다. 초음파 처리 및 Pluronic F-68의 첨가는 참당귀 세포의 세포막의 투과성을 증진시켜 ECP의 생산성을 높임과 동시에 세포 내의 다당의 배지로의 배출을 유도한 것으로 판단된다.

• BMB Reports
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• 제53권2호
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• pp.118-123
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• 2020

Cardiac regeneration with adult stem-cell (ASC) therapy is a promising field to address advanced cardiovascular diseases. In addition, extracellular vesicles (EVs) from ASCs have been implicated in acting as paracrine factors to improve cardiac functions in ASC therapy. In our work, we isolated human cardiac mesenchymal stromal cells (h-CMSCs) by means of three-dimensional organ culture (3D culture) during ex vivo expansion of cardiac tissue, to compare the functional efficacy with human bone-marrow derived mesenchymal stem cells (h-BM-MSCs), one of the actively studied ASCs. We characterized the h-CMSCs as CD90^low, c-kit^negative, CD105^positive phenotype and these cells express NANOG, SOX2, and GATA4. To identify the more effective type of EVs for angiogenesis among the different sources of ASCs, we isolated EVs which were derived from CMSCs with either normoxic or hypoxic condition and BM-MSCs. Our in vitro tube-formation results demonstrated that the angiogenic effects of EVs from hypoxia-treated CMSCs (CMSC-Hpx EVs) were greater than the well-known effects of EVs from BM-MSCs (BM-MSC EVs), and these were even comparable to human vascular endothelial growth factor (hVEGF), a potent angiogenic factor. Therefore, we present here that CD90^lowc-kit^negativeCD105^positive CMSCs under hypoxic conditions secrete functionally superior EVs for in vitro angiogenesis. Our findings will allow more insights on understanding myocardial repair.

• 한국어병학회지
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• 제10권1호
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• pp.31-38
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• 1997

잉어로부터 분리한 Aeromonas hydrophila는 세포 밖으로 여러 종류의 proteases를 생산한다. A. hydrophila의 배양 상층액을 이용한 inhibitor assay를 통하여, 주된 활성을 나타내는 metallopretease와 약한 활성을 나타내는 serine protease가 있음을 알게 되었다. Gelatin SDS-PAGE를 통하여 두 개의 활성 band가 관찰되었으며, 이 들 중 넓게 퍼진 band는 metalloprotease에 특이하게 작용하는 inhibitor인 EDTA에 의해 활성이 상실되었고 따라서 metalloprotease임을 알 수 있었다. 다른 하나는 serine protease에 특이하게 반응하는 inhibitor인 PMSF에 의해 저해되어 serine protease임을 알 수 있었다. 이러한 두 extracellular protease의 활성은 $75^{\circ}C$에서 30 분간 열을 가한 후에도 Gelatin SDS-PAGE상에 남아있었다. 그런데, 주된 metalloprotease는 Sephadex G-75를 이용한 column chromatography를 거친 후 Gelatin Gel상에서 두 개의 band로 분리되었다.

• The Korean Journal of Physiology
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• 제28권2호
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• pp.159-167
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• 1994

The contraction of rabbit basilar artery was examined as a function of changes in the $Na^+$ electrochemical gradient in order to determine the contribution of $Na^+/Ca^{2+}$ exchange to the modulation of contractility. Ouabain $(10^{-5}\;M)$ or $K^+-free$ Tyrode solution caused an increase in tonic tension even in the presence of a $Ca^{2+}$ channel blocker $(10^{-6}\;M\;verapamil)$ and an ${\alpha}-receptor$ blocker $(10^{-5}\;M\;phentolamine)$. After treatment with ouabain $(10^{-5}\;M)$, contractions were augmented by reduction of external $Na^+$ concentration. The longer the treatment with ouabain $(10^{-5}\;M)$ was, the larger the amplitude of $Na^+-free$ contracture was. $Na^+-free$ contracture wag induced by either substitution of equimolar Tris for $Na^+$ or substitution of equimolar $Li^+\;for\;Na^+$. The competition between $Na^+\;and\;Ca^{2+}$ for the $Na^+/Ca^{2+}$ exchange carrier would exist, because it was observed that contractility was dependent on the $Na^+$ electrochemical gradient or the extracellular $Ca^{2+}$ concentration (2 mM, 4 mM). Ryanodine $(10^{-7}\;M)$, the blocker of intracellular $Ca^{2+}$ release from the sarcoplasmic reticulum, did not suppress the development of $Na^+-free$ contracture. The contractile response to norepinephrine $(10^{-6}\;M)$ was augmented by reducing the extracellular $Na^+$ concentration. The relaxation rate from caffeine-induced contraction was dependent on the extracellular $Na^+$ concentration (0 mM, 140 mM). From the above results, it could be suggested that $Na^+/Ca^{2+}$ exchange can move $Ca^{2+}$ either into or out of rabbit basilar arterial smooth muscle. $Ca^{2+}$ entry or extrusion is dependent upon the $Na^+$ electrochemical gradient. $Na^+/Ca^{2+}$ exchange plays a significant role in the regulation of contractility in rabbit basilar arterial smooth muscle.

• 한국균학회지
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• 제27권2호통권89호
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• pp.100-106
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• 1999

Agrocybe cylindracea의 배양방법별 균사체 및 다당류 생산수율비교 실험에서는 모두 진탕배양이 효과적이었다. 다당류의 정제는 조단백다당류를 이용하여 DEAE-cellulose column에 의한 1차 정제를 행하였고, 최종으로 Sepharose 2B를 이용한 2차 정제를 실시하여 최종적으로 균사체내다당류(ACIPDG, ACIPAG)와 균사체외다당류(ACEPDG, ACEPAG)를 얻었다. ACEPDG는 총당 75.8%, ACEPAG는 총당 65.4%를 함유하였으며, ACIPDG는 총당 89.2%, ACIPAG는 총당 54.2%의 조성을 보였다. 다당류 분획물들의 분자량을 측정한 결과, 모두 10만이 넘는 거대분자로 ACEPDG 300KDa에서 ACIPAG 600KDa까지 나타났다. 다당류의 조성을 HPLC로 분석한 결과, ACEPDG분획물은 glucose, inositol이 검출되었으며 ACIPDG, ACEPAG, ACIPAG 분획물은 glucose, fructose, inositol이 검출되었다.