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沈阳药科大学学报 2008年第S1期25卷 94-95页

作者： tsuko Minobe masaki kameyama Department of Physiology Graduate School of Medical and Dental SciencesKagoshima University

Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac ***-down was induced by the inside-out patch ***(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion ***(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac *** The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).

关键词： calpastatin calmodulin calcium channel regulation

The regulations of L-type Ca2+ channels by calmodulin-dependent protein kinaseⅡin guinea-pig cardiac myocytes

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The regulations of L-type Ca2+ channels by calmodulin-depend...

中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： masaki kameyama Department of Physiology School of Medicine and Dental Sciences Kagoshima University

L-type Ca channels are regulated by various second messenger *** recent study has found that the run-down of L-type Ca channel was reversed by calmodulin(CaM) + *** study was to investigate the possible roles of CaM-dependent kinaseⅡ(CaMKⅡ) in the regulation of L-type Ca channel basal activity in guinea-pig ventricular myocytes with patch-clamp *** found that CaMKⅡinhibitors suppressed the basal activity in cell-attached patches and the Ca channel activity induced by cytoplasmic factors in inside-out *** in the presence of 3 mmol/L ATP recovered the channel activity to 92.5%±20.1%and 28.2%±11.1% of that in the cell-attached configuration at 1 and 5 min after run-down respectively,showing significant time-dependent ***,in the presence of CaMKⅡ,the attenuation was *** pull-down assay experiments,CaMKⅡphosphorylation enhanced the binding of CaM to the *** findings suggested that CaMKⅡactivation was required to maintain the basal activity of L-type Ca channels.

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The roles and relations of calpastatin,calmodulin and an undefined cytoplasmic factor in the regulation of cardiac L-type Ca2+ channels

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The roles and relations of calpastatin,calmodulin and an und...

两岸三地药理学与临床药理学术会议

作者： tsuko Minobe masaki kameyama Department of Physiology Graduate School of Medical and Dental SciencesKagoshima University

Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca channel activity after"run-down".The factors include ATP,calpastatin and H fraction (a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca channel activities were recorded with patch clamp tech- nique in guinea-pig cardiac ***-down was induced by the inside-out patch *** (CS),calmodulin(CaM) and three GST-fusion fragment peptides derived from the C-terminal tail of guinea- pig Cav1.2,CT-1 (amino acids number 1509-1791),CT-2 (1777-2003) and CT-3 (1944-2169) were pro- duced as GST fusion *** (1)CaM+ATP or CS+ATP restored the channels after run- down;however,the CaM or CS’s effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase (CaMKII) produced Ca channel activity to only 2-10 % of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished. (3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac *** The re- sults show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca channels,and that there might be cross talks among the four factors (CS,CaM,CaMKII and the unde- fined cytoplasmic factor).

关键词： calpastatin calmodulin calcium channel regulation

Concentration-and calcium-dependent effects of calmodulin and its mutants on L-type Ca2+channel in guinea pig ventricular myocytes

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Concentration-and calcium-dependent effects of calmodulin an...

中国药理学会第十次全国学术会议

作者： Kazuto Yazawa Etsuko Minobe masaki kameyama Department of Physiology Graduate School of Medical & Dental SciencesKagoshima University.8-35-1 SakuragaokaKagoshima 890-8544Japan

Background:Voltage-gated L-type Ca channel has been suggested to be involved in many essential processes by the Ca-dependent regulation,including muscle contraction,gene expression and hormone secretion. When Ca ions flow through the pore of an individual Ca channel,both positive and negative feedback mechanisms: Ca-dependent facilitation(CDF) that enhances calcium entry and Ca -dependent inactivation(CDI) that limits calcium entry,respectively,have been indicated in those ***(CaM) is an important regulator of ion channels,serving as a Ca sensor for Ca1.2 calcium *** binding of Ca to CaM, bound to the amino-terminal(N-terminus) and carboxy-terminal(C-terminus) tail of the pore-formingα1 subunit can enhance channel opening and accelerate channel closing on the Ca1.2 *** the other hand,CaM is a protein formed by two lobes,each comprising two Ca binding *** mutated at Ca binding sites 1 and 2 in the amino-terminal(N-lobe,CaM12) and CaM mutated at Ca binding sites 3 and 4 in the carboxy-terminal lobe (C-lobe,CaM34) have been produced to demonstrate Ca-dependent regulation of Ga1.2 *** previous findings have highlighted that both N-lobe and C-lobe of CaM are of critical importance in the modulation of Ca1.2 calcium ***,the detailed mechanisms of how Ca and CaM modulate Ca2+ channel are not yet *** In an attempt to further explore the detailed roles of CaM and its mutants(CaM12 and CaM34) in the concentration and calcium regulation of Ca1.2 channel,we set out to apply CaM and its mutants to investigate the facilitation and inactivation changes of Ca1.2 channel by inside-out patch clamp technique in guinea pig ventricular *** and Methods:The point mutations for CaM12 and CaM34 were introduced into the Ca binding sites in CaM by means of a Quickchange TM site-directed mutagenesis *** CaM12,Glu was changed to Ala at two conserved calcium ligands in the first(E31 A-CaM) and second(E67A-Ca

关键词： Calcium channel Calmodulin Calcium signaling Cardiac myocyte Patch clamp

克隆人神经母细胞瘤细胞株鲀毒素抵抗型钠通道的序列分析

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中国药理学与毒理学杂志 2005年第3期19卷 186-193页

作者：欧绍武龟山亚砂子郝丽英龟山正树李金鸣中国医科大学附属第一医院神经外科辽宁沈阳110001 鹿儿岛大学医学部第二生理教研室中国医科大学基础医学院药理学教研室辽宁沈阳110001

目的　为了弄清人神经母细胞瘤(NB 1)细胞上的毒素抵抗型(TTX R)电压依赖性钠通道的编码基因及分子特性。方法　使用逆转录聚合酶链反应方法,对NB 1细胞TTX R钠通道α亚单位进行克隆。结果　该克隆命名为hNbR1,其开放阅读框架(ORF)为2... 详细信息

目的　为了弄清人神经母细胞瘤(NB 1)细胞上的毒素抵抗型(TTX R)电压依赖性钠通道的编码基因及分子特性。方法　使用逆转录聚合酶链反应方法,对NB 1细胞TTX R钠通道α亚单位进行克隆。结果　该克隆命名为hNbR1,其开放阅读框架(ORF)为2 0 16个氨基酸残基。序列分析显示,hNbR1与心肌Nav1.5 /SCN5A氨基酸相似性达99.1% ,并具有电压依赖性钠通道的结构特点。4个相似的跨膜结构域(DⅠ～DⅣ) ,其中包括6个推定的α螺旋跨膜片段(S1～S6 ) ,每个结构域的电压感受器跨膜片段(S4 )均有正电荷残基;在DⅠ的P 环存在的半胱氨酸残基(C373)提示该通道为TTX R钠通道。DⅠS3～S4一个位于第3号染色体的新的外显子(第6A外显子)编码了该通道α亚单位。另外,一个删除了DⅡ～DⅢ第18外显子的选择性剪切体(hNbR1 2 )被发现。结论Nav1.5 /SCN5A比以往所知具有更加广泛的分布,而且发现一个新的外显子在神经肿瘤组织中编码了这个TTX R钠通道的α亚单位。

关键词：神经母细胞瘤细胞,培养的钠通道基因克隆,分子

克隆的人神经母细胞瘤细胞株鲀毒素抵抗型钠通道的功能分析

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中国药理学与毒理学杂志 2005年第4期19卷 284-288页

目的作者已经克隆了人神经母细胞瘤(NB-1)细胞株毒素抵抗型(TTX-R)钠通道α亚单位及其选择性剪切体,分别命名为hNbR1和hNbR1-2,本实验观察这两个钠通道同型异构体的电生理特性。方法使用全细胞膜片钳技术在转染了hNbR1和hNbR1-2的人... 详细信息

目的作者已经克隆了人神经母细胞瘤(NB-1)细胞株毒素抵抗型(TTX-R)钠通道α亚单位及其选择性剪切体,分别命名为hNbR1和hNbR1-2,本实验观察这两个钠通道同型异构体的电生理特性。方法使用全细胞膜片钳技术在转染了hNbR1和hNbR1-2的人胚肾细胞(HEK-293)上记录这两个钠通道的钠电流。结果这两个变构体在稳态激活和失活方面略微不同,hNbR1和hNbR1-2的半数激活电压分别为-(37.8±1.3)和-(43.5±1.7)mV,半数失活电压分别为-(74.4±1.1)和-(81.5±1.1)mV,hNbR1-2的稳态激活和稳态失活曲线较hNbR1分别负向移位5.7mV和7.1mV。TTX阻断hNbR1和hNbR1-2的IC50分别为(7.9±2.3)和(8.3±2.5)μmol·L-1,二者对TTX的敏感性无显著性差异。结论两个钠通道变构体hNbR1和hNbR1-2均可在HEK-293细胞上表达并产生钠电流,二者在稳态激活和失活方面略微不同,对TTX的敏感性无显著性差异。

关键词：神经母细胞瘤钠通道膜片钳技术,全细胞

Different properties and cooperative effects of calmodulin N-and Clobes in regulations of Cav1.2 channel

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Different properties and cooperative effects of calmodulin N...

第十一届全国钙信号和细胞功能研讨会

作者： Dongxue Shao Jianjun Xu Meimi Zhao Etsuko Minobe Jingyang Su Yi Du Jiayao Sun Mo Li Zijian Tang masaki kameyama Liying Hao Department of Pharmaceutical Toxicology School of Pharmacy China Medical University Department of Physiology Graduate School of Medical and Dental Sciences Kagoshima University

Voltage-gated Ca channels(Cavs) are regulated by Ca/calmodulin(Ca M) through two opposing feedback mechanisms, Ca-dependent facilitation(CDF) and inactivation(CDI). A Ca M molecule consists of two connected lobes, the N-terminal lobe(N-lobe) and the C-terminal lobe(C-lobe). It is currently quite controversial whether the two lobes of Ca M mediate the opposing feedback systems independently. By using Ca M N-and C-lobe fragments, we confirmed that the N-lobe was bound to the IQ motif, a Ca M-binding site on the C-terminus of Cav1.2 in free or low Ca conditions, revealing that N-lobe played a more important role when intracellular Ca concentration is free or low. While, neither individual N-lobe nor C-lobe but equimolar mixture of N-and C-lobe(N&C) could induce CDI under our experimental condition, indicating that CDI is a process that starts by the binding of the single N-lobe to the channel and ends with the co-existence of N-and C-lobes. The binding affinities of the individual lobes were much lower than that of the full-structured Ca M, implying that the linker may contribute to the effective cooperation between the N-and C-lobes. We propose a model wherein the CDI process is akin to double touching magic locks controlled by N-and C-lobe bindings, which provides an important perspective to understand the regulatory mechanisms of Cavs and the control of intracellular Ca homeostasis.

关键词：

Impact of obesity on the surgical outcome following repeat hepatic resection in Japanese patients with recurrent hepatocellular carcinoma

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World Journal of Gastroenterology 2008年第10期14卷 1553-1558页

作者： Tohru Utsunomiya Masahiro Okamoto Toshihumi kameyama Ayumi Matsuyama Manabu Yamamoto Megumu Fujiwara masaki Mori Shiomi Aimitsu Teruyoshi Ishida Department of Surgery Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital Hiroshima Japan Department of Pathology Hiroshima RedCross Hospital and Atomic Bomb Survivors Hospital Hiroshima730-8619 Japan Department of Radiology Hiroshima Red CrossHospital and Atomic Bomb Survivors Hospital Hiroshima730-8619 Japan Department of Internal Medicine of the Liver Hiroshima Red Cross Hospital and Atomic Bomb SurvivorsHospital Hiroshima 730-8619 Japan

AIM: To evaluate the impact of obesity on the posto- perative outcome after hepatic resection in patients with hepatocellular carcinoma (HCC). METHODS: Data from 328 consecutive patients with primary HCC and 60 patients with recurrent HCC were studied. We compared the surgical outcomes between the non-obese group (body mass index: BMI < 25 kg/m2) and the obese group (BMI ≥ 25 kg/m2). RESULTS: Following curative hepatectomy in patients with primary HCC, the incidence of postoperative complications and the long-term prognosis in the non- obese group (n = 240) were comparable to those in the obese group (n = 88). Among patients with recurrent HCC, the incidence of postoperative complications after repeat hepatectomy was not significantly different between the non-obese group (n = 44) and the obese group (n = 16). However, patients in the obese group showed a significantly poorer long-term prognosis than those in the non-obese group (P < 0.05, five-yearsurvival rate; 51.9% and 92.0%, respectively). CONCLUSION: Obesity alone may not have an adverse effect on the surgical outcomes of patients with primary HCC. However, greater caution seems to be required when planning a repeat hepatectomy for obese patients with recurrent HCC.

关键词： Body mass index Hepatocellular carcinoma Hepatectomy Prognosis