• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2004년도 Annual Meeting of the Korean Society ofApplied Pharmacology
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• pp.124-128
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• 2004

We have assessed amyloid ${\beta}-peptide$ $(A{\beta})-induced$ neurotoxicity in primary neurons and organotypic hippocampal slice cultures (OHC) in rat. Exposing cultured hippocampal and cerebellar granule neurons to $A{\beta}$ resulted in a decrease of MTT reduction, and in destruction of neuronal integrity. Treatment of these neurons with tunicamycin, an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), also decreased MTT reduction in these neurons. S-allyl-L-cysteine (SAC), an active organosulfur compound in aged garlic extract, protected hippocampal but not cerebellar granule neurons against $A{\beta}$- or tunicamycin-induced toxicity. In the hippocampal neurons, protein expressions of casapse-12 and GRP 78 were significantly increased after $A{\beta}_{25-35}$ or tunicamycin treatment. The increase in the expression of caspase-12 was suppressed by simultaneously adding $1{\mu}M$ SAC in these neurons. In contrast, in the cerebellar granule neurons, the expression of caspase-12 was extremely lower than that in the hippocampal neurons, and an increase in the expression by $A{\beta}_{25-35}$ or tunicamycin was not detected. In OHC, ibotenic acid (IBO), a NMDA receptor agonist, induced concentration-dependent neuronal death. When $A{\beta}$ was combined with IBO, there was more intense cell death than with IBO alone. SAC protected neurons in the CA3 area and the dentate gyrus (DG) from the cell death induced by IBO in combination with $A{\beta}$, although there was no change in the CA1 area. Although protein expression of casapse-12 in the CA3 area and the DG was significantly increased after the simultaneous treatment of AI3 and IBO, no increase in the expression was observed in the CA1 area. These results suggest that SAC could protect against the neuronal cell death induced by the activation of caspase-12 in primary cultures and OHC. It is also suggested that multiple mechanisms may be involved in neuronal death induced by AI3 and AI3 in combination with IBO.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.32-32
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• 2003

소 난자의 체외성숙 과정에서 세포질 내 단백질의 생산과 축적의 변화는 핵 및 세포질 성숙과 밀접한 관계가 있다는 보고가 있지만, 난자의 성숙과 관련된 특정 단백질의 종류에 대한 보고는 없었다. 따라서 본 연구는 한우 난자의 체외성숙과 관련된 단백질의 생산 및 축적의 변화와 그 종류에 대해서 검토하였다. 체외성숙 시간(4.5, 9, 13.5, 18 및 24시간)에 따른 배양액 내의 단백질 합성의 변화는 2D gel electrophoresis를 이용하였고, 단백질 spot에 대해서는 peptide mass fingerprinting(PMF) 방법을 이용하였다. 또한 단백질 측정 시간에 신선 체외성숙 배양액으로 교환 후 난포란의 핵성숙과 배발달율을 검토하였다. 그 결과 한우 난포란의 체외성숙 시간에 따라 배양액에서 단백질의 양 및 질적인 변화를 확인하였다. 그리고 총 296개 단백질 spot들을 확인하였고, 그 중 30개 spot에서 유의적인 변화가 인정되었다. 또한 유의적인 변화를 보인 spot에 대한 PMF 분석을 통하여 Apolipoprotein A-1 precursor, Alpha enolase, Aldose reductase, 43kDa collectin precursor, Heat shock 27kDa protein, Plasminogen activator inhibitor-1 precursor, Thrombospondin 1 및 Transitional endoplasmic reticulum ATPase가 동정되었다 그리고 총 단백질 합성 경향은 0∼4.5 시간에는 감소하였고, 13.5∼18시간에 증가 한 후 다시 감소하는 경향을 나타내었으며, 단백질의 종류도 시간대별로 현저한 변화가 있었다. 한편 단백질을 측정하는 시기에 신선 체외성숙 배양액으로 교환한 후 난포란의 핵성숙 및 배발달율을 검토한 결과 18시간 체외성숙군에서 9시간째의 교환이 유의적으로 높은 핵성숙을 나타내었으나, 배발달율에서는 유의성이 인정되지 않았다. 그러나 24시간 체외성숙군에서 18시간째의 배양액 교환은 8세포기 및 배반포 발달율이 유의적(P<0.05)으로 높았다. 연구 결과로부터 소 난자의 체외성숙 시간에 따른 단백질 합성 경향의 차이를 확인하였고, 유의적인 변화를 나타낸 8가지의 단백질을 분리할 수 있었으며, 향후 이들 작용기전에 대한 연구가 필요하다고 사료된다.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.

• 대한방사선치료학회지
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• 제14권1호
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• pp.165-174
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• 2002

혈관내피세포 성장 인자(Vascular endothelial growth factor, VEGF)는 혈관내피세포 특이하게 성장요인으로 작용하는 물질로 알려져 있다. 전리방사선에 대한 혈관내피세포의 효과는 정상조직에 대한 반응에 있어 아주 중요한 요소일 것으로 생각된다. 본 연구는 방사선 조사에 의해 배양시킨 혈관내피세포에서 apoptosis가 유도되는지, 유도가 된다면 VEGF에 의해 apoptosis가 억제되는지 그리고 apoptosis의 억제가 어떤 경로를 경유하는지를 실험하였다. 혈관내피세포에 방사선를 조사한 결과, 대조군에 비하여 선량이 증가함에 따라 apoptosis가 증가하였다. 같은 조건하에서 VEGF는 농도 의존적으로 apoptosis를 억제하였다. Antiapoptotic factor로서 VEGF가 어떤 신호 과정을 경유하는지를 밝히고자, 혈관내피세포에 방사선을 조사하여 apoptosis를 유도하면서 Flt-1과 Flk-1/KDR receptor를 처리하였다. 그결과 VEGF에 유도된 apoptosis 억제효과가 차단되었다. Phosphatidylinositol 3'-kinase(PI3-kinase) 특정 억제 물질인 Wortmanin과 LY294002를 방사선 조사한 혈관내피에 VEGF와 함께 처리했을 때 VEGF에 의해 유도된 apoptosis를 억제하였다. 이같은 결과는 VEGF가 방사선 조사로 일어나는 세포 치사를 억제하는 중요한 역할을 담당하며, In Vivo의 실험이 더 이루어져야 할 것으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제9권6호
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• pp.353-361
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• 2005

The interaction of cyclosporine A (CsA), an immunosuppressant, with rat brain Kv1.5 (Kv1.5) channels, which were stably expressed in Chinese hamster ovary cells, was investigated using the whole-cell patch-clamp technique. CsA reversibly blocked Kv1.5 currents at +50 mV in a reversible concentrationdependent manner with an apparent $IC_{50}$ of 1.0μM. Other calcineurin inhibitors (cypermethrin, autoinhibitory peptide) had no effect on Kv1.5 and did not prevent the inhibitory effect of CsA. Fast application of CsA led to a rapid and reversible block of Kv1.5, and the onset time constants of the CsA-induced block were decreased in a concentration-dependent manner. The CsA-induced block of Kv1.5 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the block exhibited voltage independence over the voltage range in which channels were fully activated. The rate constants for association and dissociation of CsA were $7.0{\mu}M{-1}s^{-1}$ and $8.1s^{-1}$, respectively. CsA slowed the deactivation time course, resulting in a tail crossover phenomenon. Block of Kv1.5 by CsA was use-dependent. CsA also blocked Kv1.3 currents at +50 mV in a reversible concentration-dependent manner with an apparent $IC_{50}$ of $1.1{\mu}M$. The same effects of CsA on Kv1.3 were also observed in excised inside-out patches when applied to the internal surface of the membrane. The present results suggest that CsA acts directly on Kv1.5 currents as an open-channel blocker, independently of the effects of CsA on calcineurin activity.

• 약학회지
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• 제41권2호
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• pp.247-254
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• 1997

The proteinase-activated receptor (PAR-2) belongs to the family of seven transmembrane region receptors, like the thrombin receptor, it is activated by specific proteolytic clea vage of its extracellular amino terminus and a synthetic peptide (SLIGRL). The earthworm protein fraction (EPF) extracted from Lumbricus rubellus elicted dose- and endothelium-dependent relaxations in phenylephrine-contracted rat thoracic aorta, whereas heat inactivated EPF (0.5 ${\mu}g$ /ml) had no effect. In the presence of the nitric oxide synthase inhibitor NG-methyl-L-arginine (1.8 micro M), EPF (0.5 ${\mu}g$ /ml)-induced relaxations were partially inhibited. Furthermore, EPF (0.5 ${\mu}g$ /ml) dramatically caused relaxation of thrombin-desenstized rat thoracic aorta. These results indicate that EPF activates PAR-2 in vascular endothelial cell. Intravenous injection of EPF (20 mg/kg, bolus) into anesthetized rats produced a marked depressor response. EPF (0 ~ 80 ${\mu}g$ /ml, gradient) was very effective on increasing of perfusion volume in rabbit ear vessel preparations. These results imply the usefulness of EPF as a vascular smooth muscle relaxant and indicate that the activation of PAR-2 may be a mechanism of EPF on hemokinetic improvement.

• 대한의생명과학회지
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• 제11권3호
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• pp.365-373
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• 2005

The aim of this study was to investigate the alterations in meningeal blood flow by stimulation of trigeminovascular system. An open cranial window was prepared on the right parietal bone of male Sprague-Dawley rats. Trigeminovascular system was stimulated by electrical stimulation of trigeminal ganglion (ETS), somatosensory (whisker) stimulation, or topical applications of capsaicin and neuropeptides including substance P and calcitonin gene-related peptide (CGRP). Neonatal capsaicin pretreatment was performed with subcutaneous administration of capsaicin (50 mg/kg) within the first 24 hours after birth. Changes in regional blood flow of dural artery (rDBF) and pial artery (rPBF) were continuously measured through the cranial window by laser-Doppler flowmetry. Both ETS and capsaicin caused a chain of alterations in rPBF and rDBF responses, i.e., an immediate transient decrease followed by rapid and marked increase in rPBF, which were significantly attenuated not only by pretreatments with L-733,060, a $NK_1$ receptor blocker, $CGRP_{8-37}$, a $CGRP_1$ receptor blocker, and 7-nitroindazole monosodium salt (7-NINA), a neuronal nitric oxide synthase inhibitor but also by neonatal capsaicin treatment. Exogenous neuropeptides including substance P and CGRP increased the meningeal blood flow, which was significantly attenuated not only by pretreatment with L-733,060 and $CGRP_{8-37}$, respectively, but also by pretreatment with 7-NINA. The rPBF response to whisker stimulation was significantly attenuated not only by trigeminovascular system injuries including nasociliary nerve denervation and neonatal capsaicin treatment but also by pretreatments with L-733,060, $CGRP_{8-37}$ and 7-NINA. These results suggest that the stimulation of trigeminovascular system causes prominent alterations in meningeal blood flow, and that neuropeptides as well as nitric oxide in the trigeminovascular system are importantly implicated in the regulation of meningeal blood flow.

• 한국수산과학회지
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• 제50권1호
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• pp.85-91
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• 2017

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of insidious onset that causes gradual loss of memory and cognitive function, and it is the most common form of dementia in the elderly. AD is characterized by neuritic plaques and neurofibrillary tangles in the brain, together with loss of neuronal cells. The major neuropathological hallmark of AD is the accumulation of extracellular neurotoxic ${\beta}-amyloid$ ($A{\beta}$) peptides, such as $A{\beta}1-42$, in the brain. In the present study, we investigated the effect of sargachromenol (SCM), sargaquinoic acid (SQA) and sargahydroquinoic acid (SHQA) isolated from Sargassum serratifoilum ethanol extract (SSE) on $A{\beta}$ production in vitro using APP751-transfected Chinese hamster ovary cells (CHO-751). CHO-751 cells were treated with various concentrations of SSE, SCM, SQA and SHQA, and the level of extracellular $A{\beta}1-42$ was evaluated by enzyme-linked immunosorbent assay. SSE and SHQA reduced the production of $A{\beta}1-42$ in CHO-751 cells. Therefore, SHQA isolated from S. serratifolium has potential as an inhibitor of neurotoxic $A{\beta}$ peptide production.

• BMB Reports
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• 제48권7호
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• pp.407-412
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• 2015

The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression. [BMB Reports 2015; 48(7): 407-412]

• 한국균학회지
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• 제36권2호
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• pp.178-182
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• 2008

콘텍트렌즈의 착용이 증가하면서 알칸사아메바에 의한 각막염이 급증하지만 아직도 이병을 치료하기가 매우 어려운 실정이다. 이 아메바에서 나오는 serine 단백질분해효소는 각막염의 유발에 중요한 역할을 하고 있다고 알려졌다. 본 연구에서는 A. castellanii의 세포외 serine 단백질분해효소의 활성을 저해하는 능력이 우수한 추출물을 230종 버섯의 메탄올 추출물로부터 선별하였다. 버섯을 메탄올과 물($65^{\circ}C$)로 추출한 후 여과.농축하여 이 추출물을 0.3 mg/ml의 농도로 효소와 반응시킨 후 기질 N-succinylala-ala-pro-phe p-anilide을 넣고 효소의 활성을 측정하여 우수한 저해능을 가진 추출물을 선별하였다. 이렇게 선별된 추출물을 가지고 성질을 연구하였는데, 한 추출물로 활성이 완전하게 억제되지 않았으나 두 추출물을 조합하여 사용하면 활성을 완전하게 억제할 수 있었다. 이 추출물은 다른 serine 단백질분해효소인 인간과 소의 트롬빈과 일반적인 단백질분해효소인 Protease K에는 영향을 미치지 않았다.