• Biomolecules & Therapeutics
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• 제26권6호
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• pp.546-552
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• 2018

A comprehensive collection of proteins senses local changes in intracellular $Ca^{2+}$ concentrations ($[Ca^{2+}]_i$) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular $Ca^{2+}$ concentrations in cat esophageal smooth muscle cells. To measure $[Ca^{2+}]_i$ levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in $[Ca^{2+}]_i$ in the cells. Pretreatment with EGTA, an extracellular $Ca^{2+}$ chelator, decreased the S1P-induced increase in $[Ca^{2+}]_i$, and an L-type $Ca^{2+}$-channel blocker, nimodipine, decreased the effect of S1P. This indicates that $Ca^{2+}$ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an $InsP_3$ receptor blocker, the S1P-evoked increase in $[Ca^{2+}]_i$ was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of $G_i$-protein, suppressed the increase in $[Ca^{2+}]_i$ evoked by S1P. These results suggest that the S1P-induced increase in $[Ca^{2+}]_i$ in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of $Ca^{2+}$ from the $InsP_3$-sensitive $Ca^{2+}$ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular $Ca^{2+}$ via the L type $Ca^{2+}$ channel, which was dependent on activation of the $S1P_4$ receptor coupled to PTX-sensitive $G_i$ protein, via phospholipase C-mediated $Ca^{2+}$ release from the $InsP_3$-sensitive $Ca^{2+}$ pool in cat esophageal smooth muscle cells.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.173-181
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• 2012

Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with $1{\mu}M$ of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.

• The Korean Journal of Physiology and Pharmacology
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• 제17권3호
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.300-306
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• 2000

The rate of apparent nitric oxide (NO) release, as measured in the exhaust gas of green alga, Scenedesmus obliquus, depended on the light intensity and pH. It doubled after lowering the temperature from $25^{\circ}C{\;}to{\;}15^{\circ}C$ and strongly decreased from $35^{\circ}C{\;}to{\;}42^{\circ}C$. The Scenedesmus cells, deficient in nitrogen or phosphorus, demonstrated a significant increase in NO production following their transfer to nitrate- and phosphate-rich media. The addition of herbicides (DCMU and glyphosate) or toxic concentrations of $Cu^{2+}{\;}or{\;}Fe^{3+}$ produced strong NO peaks, resembling those that occurred after sudden darkening. An increase in the $Ni^{2+}$ concentration to 20 ppm resulted in a gradual increase of NO release from the initial ~1.5 ppbv to>20 ppbv, whereas $Cd^{2+}$ instantaneously suppressed the NO by the cultures of Scenedesmus was not altered by L-NNA, an inhibitor of nitric oxide synthase (NOS), or by its substrate, L-arginine. This seems to exclude the role of NOS in the NO formation under study. Accordingly, it can be assumed that the rate of NO formation is mainly a function of dynamic nitrite pool sizes and environmental factors significantly affect the NO production in algae.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.717-724
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• 2016

Methane (CH₄) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH₄ can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH₄; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30℃, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl₂ as a methanol dehydrogenase inhibitor, 50% CH₄ concentration, 24 h of incubation, and 9 mg of dry cell mass ml^-1 inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH₄.

• 한국식품영양과학회지
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• 제27권3호
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• pp.367-375
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• 1998

The physicochemical properties of the $\alpha$-amylase inhibitors from black bean and naked barley is Korea were investigated. Preincubation time for maximum inhibition was 30min and no activity change was seen after that time. Optimum pH of the $\alpha$-amylase inhibitors from the black bean and naked barley was pH 7.0 and the inhibitory activities were stable in the range of pH 6.0~8.0 in both phosphate and Tris-HCI buffer solutions. Both inhibitors maintained more than 50% of activity after incubation for 17 min at 7$0^{\circ}C$. The inhibitors from the black bean and naked barley maintained more than 50% of activities after treatment for 40 min and 30 min with pepsin, and 30 min and 50 min with trypsin, respectively. Both inhibitors functioned via a noncompetitive mechanism and were active against porcine pancreatic and human salivary $\alpha$-amylases. The activities of both inhibitors were linear for the ionic stength ranging from 0 to 0.9. The addition of 70 mM maltose to the reaction mixture caused a maximum increase in the relative activities of both inhibitors, but it did not affect the dissociation of the EI complex. The activities of both inhibitors were significantly enhanced by adding 1mM of K+ or Mg2+.

• Biomolecules & Therapeutics
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• 제19권1호
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• pp.134-139
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• 2011

Sphingosine kinase (SPHK) has a central role to control cell death and cell proliferation, which is suggested as a sphingolipid rheostat by regulating the levels between ceramide and sphingosine 1-phosphate (S1P). Therefore, physiological regulators of SPHK will be a good candidate to develop a new targeted drug. For this purpose, a series of synthetic pseudoceramides were tested by SPHK assay either cell-based or cell-free system. K10PC-5 strongly inhibited SPHK, while K6PC-5 activated SPHK in cell-free system. Specifically, K6PC-5 activated SPHK under the co-treatment with $50\;{\mu}M$ dimethylsphingosine (DMS), a SPHK inhibitor. Collectively, we developed a simple SPHK assay system to find SPHK regulatory pseudoceramide compounds, K10PC-5 and K6PC-5 which may be useful to cancer treatment or immune regulation like FTY720, a synthetic sphingolipid mimetic compound.

• Food Science and Biotechnology
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• 제15권4호
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• pp.516-522
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• 2006

This study was to assess the antimicrobial action of 2-hydroxyethyl ${\beta}$-undecenate purified from cumin (Cuminum cymium L.) seed against the oral anaerobe, Streptococcus mutans, which is associated with gingivitis, specifically focusing on the catabolic effect. 2-Hydroxyethyl ${\beta}$-undecenate inhibited the acid production and growth of S. mutans after 30 hr incubation at 50 mM. The glycolysis of S. mutans with glucose as substrate was similarly sensitive to 2-hydroxyethyl ${\beta}$-undecenate, with 70% inhibition of glucose utilization at 5 mM and 90% inhibition at 50 mM. In addition, this substance potently inhibited the glycolysis enzyme, glyceraldehyde-3-phosphate dehydrogenase (GADP); the phosphoenolpyruvate, glucose phosphotransferase (Glucose-PTS); and membrane ATPase, in a concentration dependent manner. The $IC_{50}$ values for inhibition of GADP, Glucose-PTS, and ATPase were 1, 0.9, and 5 mM, respectively. Furthermore, 2-hydroxyethyl ${\beta}$-undecenate inhibited teeth calcium ion elution by 80% at 50 mM. These results suggest that 2-hydroxyethyl ${\beta}$-undecenate is a potent inhibitor of carbohydrate metabolism and the growth of S. mutans JC-2.

• Radiation Oncology Journal
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• 제28권4호
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• pp.219-223
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• 2010

목적: 국내에서 합성된 히스톤 탈아세틸 효소 억제제인 SK-7041의 생체내 방사선 감수성 증진 효과를 확인하고자 하였다. 대상 및 방법: 6주령의 웅형 C3H 마우스에 RIF-1 세포를 주입한 후 대조군 약물군 방사선군, 방사선 및 약물군으로 분류하였다. 약물군과 방사선 및 약물군에는 SK-7041 4 mg/kg을 12시간 간격으로, 6회에 걸쳐 복강 내 투여하였다. 대조군 및 방사선군에는 동일 용량의 phosphate buffered saline (PBS)를 같은 방법으로 투여하였다. 방사선군과 방사선 및 약물군에는 4번째 투약 후 6시간 후에 5 Gy를 1회 조사하였다. 이후 2~3일 간격으로 종양의 크기를 측정하여 종양 성장 연기 곡선을 산출하였다. 결과: 접종된 종양이 $1,500mm^3$까지 자라는 데 소요된 기간은 대조군, 약물군, 방사선군, 방사선 및 약물군에서 각각 10일, 9일, 9일, 12일이고, 14일째의 체적은 각각 $276.7mm^3$, $279.9mm^3$, $292.5mm^3$, $185.5mm^3$로, 각 군간의 차이는 통계학적으로 유의하였다(p=0.0004). 아울러, 대조군과 약물군의 차이와 방사선군과 방사선 및 약물군의 차이를 비교하였을 때, 통계학적으로 유의한 경향을 보였다(p=0.0650). 결론: 히스톤 탈아세틸 효소 억제제인 SK-7041 의 RIF-1 세포주에 대한 마우스내 방사선 감수성 증진 효과를 저농도에서 확인할 수 있었으며, 방사선과 SK-7041의 상호작용은 상승 작용일 가능성을 시사하였다. 향후 환자에서의 적용 가능성에 대한 연구가 필요할 것으로 생각한다.

• 생명과학회지
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• 제21권2호
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• pp.183-193
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• 2011

중간엽 줄기세포의 이동과 분화는 손상된 조직의 재생을 위해 필수적이다. Sphingosine-1-phosphate (S1P)는 세포성장, 생존, 분화, 이동성 등 여러 가지 생명현상에 중요한 역할을 하는 생리활성 지질이다. 본 연구에서는 인체 골수유래 중간엽 줄기세포의 이동과 세포분화에 대한 S1P의 영향을 조사하였다. S1P는 중간엽 줄기세포의 이동을 증가시켰으며 pertussis toxin의 전처리는 S1P에 의한 세포이동을 억제하였다. 본 결과는 S1P에 의한 세포 이동과정에 Gi에 연결된 수용체가 관여함을 제시한다. $S1P_1$과 $S1P_3$ 수용체에 대한 길항제인 VPC23019의 전처리나 siRNA를 이용한 $S1P_1$ 수용체의 발현억제는 S1P에 의한 세포 내 칼슘 증가와 중간엽 줄기세포의 이동을 저해 하였다. 또한, S1P의 처리는 중간엽 줄기세포에서 평활근세포의 표지유전자인 $\alpha$-smooth muscle actin ($\alpha$-SMA)의 발현을 증가시켰으며 VPC23019의 전처리는 S1P에 의한 $\alpha$-SMA의 발현증가를 저해하였다. S1P는 중간엽 줄기세포에서 p38 mitogen-activated protein kinase (p38 MAPK)의 인산화를 촉진하였으며 p38 MAPK의 저해제인 SB202190의 전처리 또는 p38 MAPK의 dominant negative mutant의 과발현은 S1P에 의한 중간엽 줄기세포의 이동 $\alpha$-SMA 발현증가를 억제하였다. 본 연구결과는 S1P가 $S1P_1$-p38 MAPK 신호전달기전을 통해 중간엽 줄기세포의 이동과 평활근세포로의 분화를 촉진함으로써 중간엽 줄기세포를 이용한 조직재생에의 활용 가능성을 제시한다.