Xenopus oocyte에서 애기장대 AtPIP2-1 활성측정을 위한 발현 최적화 조건 규명

김현미,이석찬,조수현,김범기,황현식 한국응용생명화학회 2010 Journal of Applied Biological Chemistry (J. Appl. Vol.53 No.4

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus β-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1. Xenopus oocyte을 이용하여 식물 aquaporin 단백질의 물 흡수 활성을 측정하기 위한 최적의 조건을 확립하기 위하여 애기 장대의 AtPIP2-1유전자를 클로닝하여 cRNA 제작용 vector, buffer osmolarity, hypoosmotic shock 처리 시간, 발현 단백질의 localization등을 검토한 결과, Xenopus β-globin 유전자의 5' 과 3' UTR(untranlation region)염기서열을 갖고 있는 pGEMHE vector가 단백질 생산에 더욱 효과적이며, 이 vector를 사용하였을 경우 hypoosomotic stress는 1/2ND buffer에서 6분간 처리시 가장 큰 차이를 볼 수 있었으며, 애기장대 AtPIP2-1단백질과 GFP를 결합시켜 발현시킬 경우 GFP가 plasmamembrane에 위치하는 것을 보아 올바른 subcelluar localization이 이루어지는 것을 확인할 수 있었다.

Calcium Influx Factor (CIF) as a diffusible Messenger for the Activation of Capacitative Calcium Entry in Xenopus Oocyte

Kim, Hak Yong,Hanley, Michael R. Korean Society of Molecular Biology 1999 Molecules and cells Vol.9 No.3

Effect of Cysteine Modifying Agents on Cystic Fibrosis Transmembrane Regulator in Rat Tracheal Epithelia

KIM, Dong-Jin,CHO, Se-Heon,HWANG, Tae-Ho,SUH, Duk-Joon 동아대학교의과대학 1997 동아의대학술지 Vol.9 No.1

Expression and Characterization of G Protein-activated Inward Rectifier $K^+$ Channels in Xenopus Oocytes

Kim, Han-Seop,Lee, Chang-Ho,Min, Churl K. The Korean Society for Integrative Biology 1998 Korean journal of biological sciences Vol.2 No.4

The G protein-activated inwardly rectifying $K^+$ channel (GIRK1) was coex-pressed in Xenopus oocytes along with the $5-HT_{1A}$ receptor, a 7-helix receptor known to be coupled to $K^+$ channels in many neural tissues. Thus, the activation of the $5-HT_{1A}$ receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5-HT in the presence of various external potassium concentrations $[K^+]_0$. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. $K^{+}$ was the major ion carrier as evidenced by about 44㎷ voltage shift corresponding to a 10-fold external 〔$K^+$〕 change. 5-HT induced a concentration-dependent inward $K^+$ current ($EC_{50}{\equation omitted}10.7nM$) which was blocked by $Ba^{2+}$. Pertussis toxin (PTX) pre-treatment reduced the $K^+$ current by as much as about 70%, suggesting that PTX-sensitive G protein ($G_i or G_o$ type) are involved in the $5-HT_{1A}$ receptor-GIRK1 coupling in Xenopus oocytes.

Inhibitory effects of Metergoline on Nav1.2 voltage-dependent sodium channel

Junho Lee 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. In the present study, we investigated the effects of metergoline on neuronal Nav1.2 voltage-dependent sodium channel activity. The two-microelectrode voltage clamp technique was used to study the regulation of metergoline on Na+ current in Xenopus oocytes expressing cRNA-encoding Nav1.2 α and β1 subunits in the rat brain. In oocytes that expressed neuronal Na+ channels, metergoline induced inhibitory effects on the peak of Na+ currents. The metergoline-induced tonic inhibitions of peak Na+ currents were voltage- and concentration-dependent and reversible. In addition, metergoline produced a use-dependent blockade of the Na+ channel after high-frequency stimulation, indicating that metergoline could exert an inhibitory effect on the open state of the Na+ channel. The metergoline produced a use-dependent blockade of the Na+ channel after high-frequency stimulation, indicating that metergoline could exert an inhibitory effect on the open state of the Na+ channel. Taken together, these results indicate that metergoline might regulate neuronal Nav1.2 voltage-dependent channels that are expressed in Xenopus oocytes. Taken together, these results indicate that metergoline might regulate neuronal Nav1.2 voltage-dependent channels that are expressed in Xenopus oocytes. Our study further suggests that metergoline can be an important pharmacological target as a potent inhibitor of neuronal Nav1.2 channels.

Effects of triterpenoid Alisol-F on human 5-hydroxytryptamine 3A and nicotinic acetylcholine receptor channel activity

Junho Lee 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Alisma Rhizome is a known tradition medication, which has been used for its diuretic, hypolipidemic, anti-diabetic, and anti-inflammatory purposes for thousands of years. The primary compounds of Alisma Rhizome are protostane type triterpenes, such as Alisols A, B or C. We previously demonstrated that Alisol derivatives (Alisols A, B, and C) have inhibitory effects on 5-hydroxytryptamine 3A (5-HT3A) currents.1 In this study, we tested the effects of a new triterpene, Alisol-F, on human 5-HT3A and nicotinic acetylcholine (nACh) receptor channel currents by using Xenopus oocytes expressing these channels. Co-application of Alisol-F inhibited 5-HT3A and nACh receptor-mediated inward peak currents. The inhibitory effect of Alisol-F on 5-HT and ACh-induced inward peak currents occurred in a reversible and concentration-dependent manner. The half maximal inhibitory concentrations (IC50) of Alisol-F were 79.4 ± 11.0 and 21.2 ± 6.0 uM for the 5-HT3A and nACh receptors, respectively. In addition, the inhibition of I5-HT and IACh by Alisol-F occurred noncompetitive and voltage insensitive manner. Taken together, these results show that Alisol-F may regulate 5-HT3A and nACh receptors channel expressed in Xenopus oocytes.

Effect of Tea Polyphenols on Conversion of Nicotine to Cotinine

( Dong Hee Lee ),( Ha Won Kim ) 한국응용약물학회 2003 Biomolecules & Therapeutics(구 응용약물학회지) Vol.11 No.4

N/A Nicotine is one of the major hazardous components in cigarette smoke. Nicotine deals a harmful effect to smokers and passive smokers due to its rapid conversion to various carcinogenic metabolites. Nitro-samine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) is believed to cause lung cancers among the nicotine-derived carcinogens. Recent studies report that NNK synthesis can be inhibited by the metabolism pathway to produce a stable metabolite cotinine from nicotine. Tea polyphenols have been known to contain factors to prevent cancers and to retard progression of cancers. This study aims to correlate tea polyphenol`s potential for cancer prevention with an accelerated formation of cotinine. The conversion from nicotine to cotinine in the presence of tea extracts or three polyphenols (Catechin, epicatechin gallate, epigallocatechin gallate) was measured in established cell lines and in Xenopus oocytes. Among three lines of cell used, PLC/PRF5 and HEK293 cells showed a fast turnover from nicotine to cotinine while HepG2 cell line showed a marginal difference between groups treated and non-treated with tea polyphenols. When Xenopus oocytes were microinjected with nicotine, tea polyphenols appear to accelerate the conversion of nicotine to cotinine. Among the polyphenols tested in this study, (+)-catechin showed the best efficiency overall in accelerating conversion from nicotine to cotinine both in the cell lines and in the oocytes. In summary, the present study indicated that tea polyphenols have a positive effect on conversion of nicotine to cotinine.

Propofol이 Xenopus Oocyte에 발현된 제3형 글루탐산염염 운반자의 활성도에 미치는 효과 : Protein Kinase C의 역할 The Role of Protein Kinase C

도상환,김진희,함병문,zhiyi zuo 대한정맥마취학회 2003 정맥마취 Vol.7 No.4

Comparison of Membrane Currents in Xenopus Oocytes in Response to Injection of Calcium Influx Factor(CIF) and Depletion of Intracellular Calcium Stores

Kim,Hak Yong,Hanely,Michel R. The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.3

The depletion of intracellular calcium stores by thapsigargin treatment evoked extracellular calcium-dependent membrane currents in Xenopus laevis oocytes. These currents have been compared to those evoked by microinjection of a calcium influx factor (CIF) purified from Jurkat T lymphocytes. The membrane currents elicited by thapsigargin treatment (peak current, 163±60 nA) or CIF injection (peak current, 897±188 nA) were both dependent on calcium entry, based o their eradication by the removal of extracellular calcium. The currents were, in both cases, attributed primarily to well-characterized Ca²+-dependent Cl- currents, based on their similar reversal potentials (-24 mV vs. -28 mV) and their inhibition by niflumic acid (a Cl²+ channel blocker). Currents induced by either thapsigargin treatment or CIF injection exhibited an identical pattern of inhibitory sensitivity to a panel of lanthanides, suggesting that thapsigargin treatment or CIF injection evoked Cl currents by stimulating calcium influx through pharmacologically identical calcium channels. These results indicate that CIF acts on the same calcium entry pathway activated by the depletion of calcium stores and most lanthanides are novel pharmacological tools for the study of calcium entry in Xenopus oocytes.

Effect of Calmodulin on Ginseng Saponin-Induced $Ca^{2+}$-Activated $Cl^{-}$ Channel Activation in Xenopus laevis Oocytes

Lee Jun-Ho,Jeong Sang-Min,Lee Byung-Hwan,Kim Jong-Hoon,Ko Sung-Ryong,Kim Seung-Hwan,Lee Sang-Mok,Nah Seung-Yeol The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.4

We previously demonstrated the ability of ginseng saponins (active ingredients of Panax ginseng) to enhance $Ca^{2+}$-activated $Cl^{-}$ current. The mechanism for this ginseng saponin-induced enhancement was proposed to be the release of $Ca^{2+}$ from $IP_{3}-sensitive$ intracellular stores through the activation of PTX-insensitive $G\alpha_{q/11}$ proteins and PLC pathway. Recent studies have shown that calmodulin (CaM) regulates $IP_{3}$ receptor-mediated $Ca^{2+}$ release in both $Ca^{2+}-dependent$ and -independent manner. In the present study, we have investigated the effects of CaM on ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current responses in Xenopus oocytes. Intraoocyte injection of CaM inhibited ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement, whereas co-injection of calmidazolium, a CaM antagonist, with CaM blocked CaM action. The inhibitory effect of CaM on ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement was dose- and time-dependent, with an $IC_{50} of 14.9\pm3.5 {\mu}M$. The inhibitory effect of CaM on saponin's activity was maximal after 6 h of intraoocyte injection of CaM, and after 48 h the activity of saponin recovered to control level. The half-recovery time was calculated to be $16.7\pm4.3 h$. Intraoocyte injection of CaM inhibited $Ca^{2+}$-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement and also attenuated $IP_{3}$-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement. $Ca^{2+}$/CaM kinase II inhibitor did not inhibit CaM-caused attenuation of ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement. These results suggest that CaM regulates ginseng saponin effect on $Ca^{2+}$-activated $Cl^{-}$ current enhancement via $Ca^{2+}$-independent manner.