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no-repeat 6px;background-position:center;margin-top:10px;min-width:55px;display:none;} @media (min-width:768px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .expand_btn{display:none;}} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-text{-webkit-flex:9;-ms-flex:9;flex:9;text-align:left;padding:21px 37px 10px 0px;font-size:16px;font-weight:700;} @media (min-width:768px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-text{padding:19px 18px 10px 18px;}} @media (min-width:992px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-text{padding:19px 21px 10px 21px;}} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-partial-url{-webkit-flex:1;-ms-flex:1;flex:1;background:url('data:image/svg+xml;base64,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') no-repeat 6px;background-position:center;padding:16px 0 16px 0;min-width:62px;max-width:62px;} @media (min-width:768px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-partial-url{padding:16px 0 17px 0;min-width:52px;max-width:52px;}} @media (min-width:992px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-partial-url{padding:15px 0 17px 0;min-width:60px;max-width:60px;}} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption .caption-partial-url:hover{-webkit-text-decoration:none;text-decoration:none;} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption-text{font-size:14px;padding:0px 18px 17px 22px;} @media (min-width:768px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption-text{padding:0px 15px 15px 18px;}} @media (min-width:992px){.ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption-text{padding:0px 18px 19px 21px;}} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption-text figcaption{margin:0;padding:0;} .ejdfnh .floats-partial-section .floats-partial-footer .floats-partial-caption-text svg{max-width:100%;} .ejdfnh ol{font-size:16px;padding-left:32px;font-family:STIXGeneral-Regular;line-height:24px;margin:15px 0;} .ejdfnh ol li{margin:10px 0;padding-left:10px;word-break:normal;} .ejdfnh ol li a{word-break:break-all;} .ejdfnh ol li .reflinks{display:block;} .ejdfnh ol li .reflinks a{color:#4D8A17;-webkit-text-decoration:none;text-decoration:none;cursor:pointer;} .ejdfnh ol li .reflinks a:hover{-webkit-text-decoration:underline;text-decoration:underline;} .ejdfnh ol li .reflinks .sep{color:#000000;} .ejdfnh .table-wrapper{width:auto;overflow-x:auto;} .ejdfnh .table-wrapper::-webkit-scrollbar{-webkit-appearance:none;width:5px;height:5px;} .ejdfnh .table-wrapper::-webkit-scrollbar-thumb{border-radius:5px;background-color:rgba(0,0,0,.5);-webkit-box-shadow:0 0 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svg{fill:#007B8B;} .ejdfnh .floats-partial-middle--thumbs .partial-carousel-dot .nowrap{white-space:nowrap;} .ejdfnh .partial-carousel-dot{text-align:center;cursor:pointer;border:0;width:100px;padding-bottom:20px;background-color:#fff;display:table-cell;} .ejdfnh .partial-carousel-dot:focus-visible{outline-offset:-3px;} .ejdfnh .partial-carousel-dot strong{font-size:11px;padding-top:12px;} @media (min-width:767.5px){.ejdfnh .partial-carousel-dot strong{font-size:16px;}} .ejdfnh .partial-carousel-dot img{width:90px;height:90px;border:1px solid #979797;margin:10px 0;} .ejdfnh .partial-carousel-wrapper .floats-partial-middle{border-top:1px solid #e3e3e3;display:block;margin-bottom:50px;padding-top:1px;} @media (min-width:767.5px){.ejdfnh .partial-carousel-wrapper .floats-partial-middle{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;margin-bottom:0;}} .ejdfnh .partial-carousel--single-image{overflow:hidden;text-align:center;} .ejdfnh 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href="//www.newsama.com/journals/omcl/" aria-label="Oxidative Medicine and Cellular Longevity">氧化医学和细胞寿命</a>/<a class="sc-htpNat bUhGXt link sc-eitiEO jXeALb breadCrumb" href="//www.newsama.com/journals/omcl/contents/year/2011/" aria-label="2011">2011年</a>/<年代pan class="sc-bhlBdH UVThf">文章</年代pan> </div> <div class="threeColumn__ThreeColumnWrapper-sc-1bf4078-0 dDyxFF threeColumnWrapper"> <div class="threeColumn__LeftWrapper-sc-1bf4078-1 fkVFlF leftWrapper"> <button type="button" class="sc-eKZiaR dzZrgS section__button " id="sectionButton"><span class="sc-htoDjs diutvq icon__dots dots__detector icon-colored _3n-7Ho7DlfjMlZbrRr9Q4a icon-themeable" title="" font-size="22"></span>文章部分</button> <div class="sc-bEjcJn jLUVhb static_contents__section_links links__hidden "> <div id="leftSectionMenu" class="sc-cbkKFq prcY sc-bGbJRg dVCLgr left_section_menu"> <p>在这一页上</p> <span class="sc-likbZx bBmZJZ grey" property="background-color" color="#D8D8D8"><a class="sc-htpNat bUhGXt link sc-ePZHVD 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</div> <div class="sc-LKuAh lcOiDF article_full_citation"> <div class="sc-hzNEM gsmOCv isHide"> <p>(Norihiro Ryuman Nobuo渡边,Arai高雄, ”<年代pan class="adjust-article-svg-size">S-Nitrosation没有捐赠者鼠胚胎成纤维细胞的细胞蛋白3日元细胞:S-Nitrosation影响因素</年代pan>”,<i>氧化医学和细胞寿命</我>, 卷。2011年, 文章的ID450317年, 8   页面, 2011年。 https://doi.org/10.1155/2011/450317</p> </div> <button class="sc-hSdWYo etauus sc-iBEsjs kPshFw" color="">显示引用</button> </div> <h1 class="sc-iRbamj jZOxOO sc-iSDuPN kmVdUp article_title adjust-article-svg-size heading-title">S-Nitrosation没有捐赠者鼠胚胎成纤维细胞的细胞蛋白3日元细胞:S-Nitrosation影响因素</h1> </div> <div class="sc-jrIrqw eHMFfo article_authors"> <hr class="sc-cEvuZC XkrFe hr__author"> <span class="">(Norihiro Ryuman,<年代up>1</年代up></span> <span class=""><b>田中伸男(Nobuo渡边</b><a class="sc-htpNat bUhGXt link" href="mailto:" aria-label="Mail Option"><span class="sc-htoDjs kNoiZu sc-hjRWVT irNWJI icon-colored _2zt-1j0LmaF8UjTQAM413G icon-themeable" title="" font-size="16"></span></a>,<年代up>1</年代up> 和</年代pan> <span class="">高雄Arai<年代up>1</年代up></span> <div class="sc-fHxwqH dMuGGA isHide"> <p><sup>1</年代up><span>应用生物科学,科学和技术学院,东京大学的科学,2641山崎,野田佳彦千叶市278 - 8510年,日本</年代pan></p> </div> <button class="sc-hSdWYo etauus sc-iQtOjA kfDwZY" color="">显示更多</button> </div> <div class="articleContent__EditorWrapper-sc-2yl9jy-3 evMkvU"> <div class="articleContent__TextWrapper-sc-2yl9jy-8 fYrLtk"> <b>学术编辑器:</b>赵中庄</div> <div class="articleContent__PublicationTimeLineWrapper-sc-2yl9jy-4 fjviok"> <div class="articleContent__PublcationCol-sc-2yl9jy-5 hgKieC notPublished"> <span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">收到了</年代pan> <span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2011年5月18日</年代pan> </div> <div class="articleContent__PublcationCol-sc-2yl9jy-5 hgKieC notPublished"> <span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">接受</年代pan> <span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2011年6月20日</年代pan> </div> <div class="articleContent__PublcationCol-sc-2yl9jy-5 hgKieC isPublished"> <span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">发表</年代pan> <span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2011年8月17日</年代pan> </div> </div> </div> <article class="sc-jtRlXQ ejdfnh article_body "> <div> <div class="xml-content"> <h4 class="header" id="abstract">文摘</h4> <p>细胞内蛋白质的机制S-nitrosation并不完全理解。使用老鼠3日元细胞,我们解决这个问题。在S-nitrosothiols和捐助者测试中,只有S-nitrosocysteine (CysNO)诱导S-nitrosation当暴露在汉克斯平衡盐溶液(hbs),而不是在普通培养基血清。在哈佛商学院,没有释放CysNO几乎完全被隔绝金属离子与金属螯合剂在不影响细胞S-nitrosation。相比之下,L-leucine,底物l型氨基酸转运蛋白(背阔肌),显著抑制S-nitrosation。缺乏与CysNO S-nitrosation一般培养基结果不仅从竞争与介质中的氨基酸半胱氨酸残基的背阔肌,还有从transnitrosation血清白蛋白。总的来说,这些结果表明,简单的缓冲盐,CysNO-dependent S-nitrosation通过细胞incorporation-dependent机制发生,但如果它发生在一般文化媒体,它可能是通过一氧化氮机制。</p> <span class="end-abs"></span> <h4 id="introduction">1。介绍</h4> <p>一氧化氮(NO)在生理过程中扮演不同的角色,如血管舒张、宿主防御感染和神经调节,其中一些是由激活的鸟苷酸环化酶(GS) / cGMP通路(<a href="#B1">1</a>,<a href="#B2">2</a>]。越来越多的证据表明,没有还可以作为信号分子通过S-nitrosothiols在蛋白质的形成。S-nitros (yl)是一种翻译后修饰的蛋白质和低分子量硫醇nitrosonium阳离子高度硫醇盐阴离子的半胱氨酸残基在蛋白质净反应(<a href="#B2">2</a>,<a href="#B3">3</a>]。蛋白质S-nitrosation已被证明监管还存在各种蛋白质的功能包括(<a href="#B3">3</a>,<a href="#B4">4</a>]。然而,过度S-nitrosation某些蛋白质已被建议作为某些疾病的诱发事件(<a href="#B5">5</a>- - - - - -<a href="#B7">7</a>]。超过100种蛋白质已确定接受S-nitrosation [<a href="#B3">3</a>]。</p> <p>孵化的cysteine-containing蛋白质或低分子量硫醇没有捐赠者的一个简单的水溶液在有氧条件下收益率S-nitrosothiols。的主要机制提出了由三氧化二氮(N<年代ub>2</年代ub>O<年代ub>3</年代ub>)形成如下<a href="#B8">8</a>]:<年代pan class="equation" id="EEq1"> <svg height="99.125" id="M1" preserveaspectratio="xMaxYMin" style="vertical-align:-0pt;width:700.9375px;" version="1.1" viewbox="0 0 700.9375 99.125" width="700.9375" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,99.125)"> <g transform="translate(72,7.3)"> <text transform="matrix(1,0,0,-1,117.59,63.2)"> <tspan style="font-size: 12.50px; " x="0" y="0"> 2</t年代pan> <tspan style="font-size: 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</text> </g> </g> </svg></span>以防S-nitrosation不完整的细胞,类似的机制被认为发生在蛋白质的疏水室内或者等离子体膜<a href="#B9">9</a>,<a href="#B10">10</a>];然而,最近的研究涉及的参与低分子量胺,尿酸盐等的催化剂N之间的反应<年代ub>2</年代ub>O<年代ub>3</年代ub>和半胱氨酸<a href="#B11">11</a>,<a href="#B12">12</a>]。另外,建议dinitrosyl铁复合物(DNIC),这是由不,胞质chelatable铁,硫醇负责S-nitrosation细胞蛋白(<a href="#B13">13</a>]。同样,几种机制提出了S-nitrosation细胞暴露于细胞外S-nitrosothiols。特别是,S-nitroso-L-cysteine (CysNO)被广泛用作“捐赠”(<a href="#B5">5</a>,<a href="#B6">6</a>,<a href="#B14">14</a>,<a href="#B15">15</a>因为它在水溶液释放没有自发分解。然而,CysNO建议通过l型氨基酸转运蛋白进入细胞(背阔肌)<a href="#B16">16</a>- - - - - -<a href="#B19">19</a>),从而导致S-nitrosation细胞蛋白质。在某些类型的细胞,细胞surface-associated蛋白二硫化物异构酶(PDI)已被证明参与合并的亚硝基的半个或没有从CysNO或其他S-nitrosothiols进入细胞(<a href="#B20">20.</a>- - - - - -<a href="#B22">22</a>]。此外,最近的研究也提出了一个新颖的机制从S-nitrosated白蛋白亚硝基的根细胞的转移通过小窝(<a href="#B23">23</a>]。</p> <p>除了模棱两可的机制,S-nitrosation的程度,可以检测到常用的化验,如biotin-switch化验或2,3-diaminonaphthalene(丹)试验<a href="#B24">24</a>,<a href="#B25">25</a>),不一致在不同细胞类型和试验条件。例如,尽管激活没有合成酶或接触外源性没有源自纯粹的捐助者,如DETA NONOate,会导致S-nitrosation 30分钟内的细胞蛋白质在某些细胞类型(<a href="#B14">14</a>,<a href="#B26">26</a>在其他细胞类型这些没有捐赠者未能诱导S-nitrosation在这么短的时间内(<a href="#B27">27</a>]。同样,简短(< 30分钟)接触CysNO,但不是GSNO,导致各种各样的细胞类型(S-nitrosation<a href="#B18">18</a>,<a href="#B28">28</a>];然而,在HEK293细胞中,这是非常受欢迎的分子生物学研究由于他们的友善与外源基因转染,GSNO会导致S-nitrosation在这样短的时间(<a href="#B14">14</a>,<a href="#B27">27</a>,<a href="#B29">29日</a>]。此外,在脊髓神经元的情况下,S-nitrosation GSNO引起的,而不是由CysNO [<a href="#B27">27</a>]。</p> <p>这些混杂的情况下促使我们调查因素负责S-nitrosation观察培养细胞的不一致,以及S-nitrosation机制。我们在这里展示,在潜在nitrosating代理检查,只有CysNO可以诱导S-nitrosation鼠胚胎成纤维细胞3日元细胞治疗在缓冲盐。这种机制是独立解放的行动没有或transnitrosation细胞表面的蛋白质,但依赖于其背阔肌细胞吸收。然而,在细胞培养基,S-nitrosating CysNO活动不仅是抑制竞争与氨基酸反应的介质,也由transnitrosation反应在血清白蛋白与半胱氨酸残基。</p> <h4 id="materials-and-methods">2。材料和方法</h4> <h5 id="sec2.1">2.1。化学物质</h5> <p>5、5′-Dithiobis (2-nitrobenzoic酸)(DTNB)和氯化汞(二)(HgCl<年代ub>2</年代ub>)购买和光化学物质从kouichi(日本大阪)。半胱氨酸,diethylenetriaminepentaacetic酸(DETAPAC)、乙二胺四乙酸(EDTA),汉克斯L-leucine平衡盐(hbs),甲基methanethiosulfonate,和N-ethylmaleimide (NEM)从Sigma-Aldrich购买(圣路易斯,密苏里州)。Biotin-N - [6-Biotinamido)己基]3′- (2′-pyridyldithio)丙酰胺是购自热科学(伊利诺伊州)。2,3-diaminonaphthalene(丹),(±)——(E) 4-ethyl-2 - [(E) -hydroxyimino] 5-nitro-3-hexenamide (NOR-3)和3-morpholinosydnonimine (SIN-1)从Dojindo购买(熊本、日本)。辣根过氧化物酶(合)共轭antibiotin抗体购自Bethyl实验室(蒙哥马利,特克斯)。D-cysteine购买从东京透有限公司(日本东京)。所有其他化学物质和盐纯度最高的可用。</p> <p>CysNO和S-nitrosoglutathione (GSNO)合成了混合0.2纳米<年代ub>2</年代ub>与同等浓度的盐酸的各自的硫醇,其次是与氢氧化钠中和<a href="#B30">30.</a>]。确定各自的浓度nitrosothiol spectrometrically使用消光系数<年代vg height="14.75" id="M4" style="vertical-align:-3.25793pt;width:67.550003px;" version="1.1" viewbox="0 0 67.550003 14.75" width="67.550003" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,14.75)"> <g transform="translate(72,-60.2)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </t年代pan> </text> <text transform="matrix(1,0,0,-1,-65.9,60.37)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 3</t年代pan> <tspan style="font-size: 8.75px; " x="4.3759999" y="0"> 3</t年代pan> <tspan style="font-size: 8.75px; " x="8.7519999" y="0"> 8</t年代pan> </text> <text transform="matrix(1,0,0,-1,-48.8,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> =</t年代pan> <tspan style="font-size: 12.50px; " x="12.027886" y="0"> 9</t年代pan> <tspan style="font-size: 12.50px; " x="18.279387" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="24.530886" y="0"> 0</t年代pan> </text> </g> </g> </svg>米<年代up>−1</年代up>厘米<年代up>−1</年代up>和<年代vg height="14.75" id="M5" style="vertical-align:-3.25793pt;width:67.550003px;" version="1.1" viewbox="0 0 67.550003 14.75" width="67.550003" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,14.75)"> <g transform="translate(72,-60.2)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </t年代pan> </text> <text transform="matrix(1,0,0,-1,-65.9,60.37)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 3</t年代pan> <tspan style="font-size: 8.75px; " x="4.3759999" y="0"> 3</t年代pan> <tspan style="font-size: 8.75px; " x="8.7519999" y="0"> 5</t年代pan> </text> <text transform="matrix(1,0,0,-1,-48.8,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> =</t年代pan> <tspan style="font-size: 12.50px; " x="12.027886" y="0"> 9</t年代pan> <tspan style="font-size: 12.50px; " x="18.279387" y="0"> 2</t年代pan> <tspan style="font-size: 12.50px; " x="24.530886" y="0"> 0</t年代pan> </text> </g> </g> </svg>米<年代up>−1</年代up>厘米<年代up>−1</年代up>分别为CysNO GSNO。在这种情况下,每个nitrosothiol的收益率超过90%。</p> <h5 id="sec2.2">2.2。细胞和治疗</h5> <p>鼠胚胎成纤维细胞3日元细胞(<a href="#B31">31日</a>)经常在DMEM培养基培养和光041 - 29775)(kouichi补充10% (v / v) FCS, 100 U /毫升青霉素G, 100<我>μ</我>g / ml链霉素在湿润的气氛中含有5%的股份有限公司<年代ub>2</年代ub>在37°C。实验中使用细胞融合6-well盘子菜肴或60毫米。暴露在各自S-nitrosating代理之前,细胞培养基被新的取代10% FCS / DMEM培养基或哈佛商学院,产物和细胞培养在一个有限公司<年代ub>2</年代ub>30分钟的孵化器。(接下来,集中测试还有亚硝基硫醇分解得到50 - 250倍最终浓度)或没有添加到各自的培养基,捐赠者的细胞保持有限公司指定的时间段<年代ub>2</年代ub>孵化器。</p> <h5 id="sec2.3">2.3。丹化验</h5> <p>荧光检测细胞S-nitrosothiols执行根据Kostka所使用的方法和公园(<a href="#B25">25</a>]。短暂治疗后,细胞被洗PBS和细胞溶解裂解缓冲(0.1% (w / v) SDS, 0.5% (v / v)特里同x - 100, 0.5毫米EDTA,和5毫米NEM PBS)。200年的溶解产物被分成两个部分<我>μ</我>l, 10<我>μ</我>l丹在0.8 N HCl是添加到两个部分。接下来,一个部分,1<我>μ</我>HgCl l(100毫米<年代ub>2</年代ub>溶解在二甲基甲酰胺(DMF)和添加到其他只有DMF补充道。孵化后在室温下15分钟,10<我>μ</我>l 2 N氢氧化钠添加到每个部分,和200 -<我>μ</我>l整除的井被转移到一个白色荧光测定板。Naphthotrizole fluoresecence测量使用荧光板读者(氟计数,帕卡德)的激发和发射波长380 nm和460 nm,分别。Mercury-dependent荧光被转换为S-nitrosothiol浓度标准曲线的连续稀释NaNO<年代ub>2</年代ub>在同一个没有HgCl裂解缓冲<年代ub>2</年代ub>治疗。</p> <h5 id="sec2.4">2.4。Biotin-Switch化验</h5> <p>蛋白S-nitrosation biotin-switch试验检测到的如前所述[<a href="#B24">24</a>]。短暂治疗后,细胞组成的细胞溶解在裂解缓冲HNE缓冲区(250毫米玫瑰(pH值7.8),1毫米EDTA,和0.1毫米neocuproine), 10% (w / v) SDS,和10% (v / v) mmt在DMF比9:1:0.2,分别。样本然后孵化在50°C和偶尔的涡流屏蔽任何自由硫醇。蛋白质和70%丙酮沉淀−20°C,收集沉淀,在8000×g离心5分钟。球洗在类似的方式有70%丙酮三倍。母鸡颗粒溶解在缓冲含0.25毫克/毫升biotin-HPDP和1% SDS,暂时用,然后与抗坏血酸钠混合获得最后一个1毫米的浓度。1小时后在室温下孵化、蛋白质沉淀被丙酮洗降水方法如上所述。球被溶解在0.1% SDS在PBS。等量的生物素化的蛋白质被解决通过sds - page使用10%或6% - -16.5%梯度丙烯酰胺凝胶,然后转移到一个聚乙二烯二氟化物膜(Immobirone,微孔)。屏蔽膜后1% (w / v)在PBS脱脂牛奶含有0.05% (v / v)渐变20,细胞膜是探测HRP-conjugated antibiotin抗体。 The biotinylated proteins were visualized using an ECL reagent (GE Healthcare), and the signal was recorded using LAS3000 image analyzer (Fuji Film Co., Japan).</p> <h5 id="sec2.5">2.5。其他化验</h5> <p>媒体没有释放nitrosothiol降解耗氧测量使用一个没有电极(1000年阿波罗,世界精密仪器)37°C。该设备是校准根据制造商的指示。</p> <p>S-alkylation BSA的半胱氨酸残基是使用NEM执行。脱脂BSA在PBS(30毫克/毫升)与NEM孵化(15毫米)在室温下1 h,和过度NEM被透析对PBS彻底删除。NEM治疗前后BSA的硫醇含量由DTNB法使用谷胱甘肽作为评估标准。NEM治疗后,自由硫醇从0.37下降到小于0.02 SH / BSA分子。</p> <p>蛋白质含量是由BCA法(皮尔斯生物技术)与BSA作为标准。</p> <h5 id="sec2.6">2.6。统计数据</h5> <p>数据表示为±SEM。学生的<我>t</我>以及或单向方差分析其次是图基的测试是用于适当的统计分析。</p> <h4 id="results-and-discussion">3所示。结果与讨论</h4> <h5 id="sec3.1">3.1。所有潜在S-Nitrosating代理3日元S-Nitrosation细胞溶解产物引起的</h5> <p>在水溶液中,S-nitrosation硫醇可引起各种没有捐赠者和S-nitrosothiols,以及条件,没有<年代vg height="14.6" id="M6" style="vertical-align:-3.13504pt;width:25.612499px;" version="1.1" viewbox="0 0 25.612499 14.6" width="25.612499" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,14.6)"> <g transform="translate(72,-60.32)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> O</t年代pan> </text> <text transform="matrix(1,0,0,-1,-62.92,60.37)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 2</t年代pan> <tspan style="font-size: 8.75px; " x="4.8699999" y="-8.3000002"> −</t年代pan> </text> </g> </g> </svg>生成在一起(<a href="#B32">32</a>]。这些潜在的能力S-nitrosating代理/条件S-nitorosate蛋白质3日元细胞溶解产物进行了比较。CysNO和GSNO用作测试S-nitrosothiols, NOR-3作为没有捐助,SIN-1 NO /<年代vg height="14.6" id="M7" style="vertical-align:-3.13504pt;width:25.612499px;" version="1.1" viewbox="0 0 25.612499 14.6" width="25.612499" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,14.6)"> <g transform="translate(72,-60.32)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> O</t年代pan> </text> <text transform="matrix(1,0,0,-1,-62.92,60.37)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 2</t年代pan> <tspan style="font-size: 8.75px; " x="4.8699999" y="-8.3000002"> −</t年代pan> </text> </g> </g> </svg>热电联供装置。在中性pH值和37°C, NOR-3和SIN-1都有一个半衰期约30分钟(<a href="#B33">33</a>,<a href="#B34">34</a>]。细胞溶解产物与CysNO孵化(200<我>μ</我>GSNO(200米)<我>μ</我>NOR-3(200米)<我>μ</我>米),或者SIN-1(1毫米)30分钟37°C,和S-nitrosated蛋白质biotin-switch试验检测到的。如图<a href="//www.newsama.com/journals/omcl/2011/450317/fig1/" target="_blank">1(一)</a>,GSNO CysNO强有力地S-nitrosated几乎相同的蛋白质阵列类似的程度。NOR-3和SIN-1(测试时更高浓度的1毫米)也可能导致S-nitrosation许多蛋白质。然而,要么S-nitrosation由于复合的程度远远小于GSNO或CysNO所造成的。biotin-switch S-nitrosated蛋白质的特异性检测的试验证实。没有生物素酰化步骤中抗坏血酸盐,S-nitrosated蛋白质乐队的数量下降到几乎未经处理的控制水平,表明我们biotin-switch化验检测S-nitrosation(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig1/" target="_blank">1 (b)</a>)。</p> <div class="floats-partial-section partial-carousel-wrapper" data-section="fig1"> <div class="floats-partial-content fig-content-fig1"> <div class="partial-carousel--single-image"> <img alt="(一)”name="450317.fig.001a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.001a.jpg"> <br> <strong>(一)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(b)”name="450317.fig.001b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.001b.jpg"> <br> <strong>(b)</年代trong> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs fig-thumbs-fig1"> <button class="partial-carousel-dot" aria-label="(a)"><img alt="(一)”name="450317.fig.001a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.001a.jpg"><br><strong>(一)</年代trong></button> <button class="partial-carousel-dot" aria-label="(b)"><img alt="(b)”name="450317.fig.001b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.001b.jpg"><br><strong>(b)</年代trong></button> </div> <div class="partial-carousel-next-prev fig-nav-fig1"> <button class="carousel-prev" aria-label="Previous"></button> <button class="carousel-next" aria-label="Next"></button> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图1</年代pan> <a aria-label="full view" class="caption-partial-url" href="//www.newsama.com/journals/omcl/2011/450317/fig1/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <i>μ</我> <i>μ</我> <i>μ</我> <i>μ</我> <table> 蛋白质S-nitrosation引起潜在S-nitrosating代理3日元细胞溶解产物。(a)检测S-nitrosated蛋白质在细胞溶解产物处理潜在S-nitrosating代理。3日元细胞溶解产物在PBS包含0.5毫米EDTA和0.5% Triton x - 100与CysNO孵化(200GSNO(200米)NOR-3(200米)米),或者SIN-1(1毫米)在37°C 30分钟。S-nitrosated biotin-switch测定蛋白质检测。(b)特异性的生物素开关试验。3日元细胞溶解产物有或没有孵化GSNO (500米),以及由此产生的S-nitrosated蛋白质被biotin-switch评估分析如上所述,但是有或没有抗坏血酸盐生物素酰化的步骤。10%的凝胶用于确认实验。因此,解决S-nitrosated蛋白质乐队不同于在其他实验中6.0% - -16.5%梯度凝胶。</table> </div> </div> </div> <h5 id="sec3.2">3.2。只有CysNO S-Nitrosate 3日元细胞生活在哈佛商学院但不是在培养基</h5> <p>接下来,蛋白质的程度S-nitorosation发生在完整细胞暴露于每个S-nitrosating代理检查。(即减少反应和/或交互。,transnitrosation) of the nitrosating agents with media components and to evaluate the primary effects of the test agents, exposure time was set to 30 min. When cells maintained in HBSS were incubated with CysNO (200 <我>μ</我>GSNO(200米)<我>μ</我>NOR-3(200米)<我>μ</我>米),或者SIN-1(1毫米),只有CysNO导致S-nitrosation各种蛋白质在这些细胞(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2(一个)</a>)。在这些治疗条件下,1毫米CysNO导致最大S-nitrosation而200<我>μ</我>M CysNO导致大约50%的最高水平(数据未显示)。然而,当执行同样的待遇一般培养基(10% FCS / DMEM),这些药物导致这些细胞S-nitrosation(数据没有显示)。在10% FCS / DMEM,甚至1毫米CysNO无法诱导蛋白质S-nitrosation(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2 (b)</a>)。此外,1毫米GSNO未能诱导S-nitrosation不管媒体(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2 (b)</a>)。丹分析可以检测不仅S-nitrosated蛋白质还胞质S-nitrosated GSNO等低分子量硫醇。当使用丹化验检查,突出S-nitrosating CysNO能力在观察GSNO哈佛商学院(每个1毫米),但不是10% FCS / DMEM(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2 (c)</a>)。这些结果表明,S-nitrosation完好无损的效率3日元细胞文化不仅取决于nitrosating代理还在孵化的媒介。</p> <div class="floats-partial-section partial-carousel-wrapper" data-section="fig2"> <div class="floats-partial-content fig-content-fig2"> <div class="partial-carousel--single-image"> <img alt="(一)”name="450317.fig.002a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002a.jpg"> <br> <strong>(一)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(b)”name="450317.fig.002b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002b.jpg"> <br> <strong>(b)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(c)”name="450317.fig.002c" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002c.jpg"> <br> <strong>(c)</年代trong> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs fig-thumbs-fig2"> <button class="partial-carousel-dot" aria-label="(a)"><img alt="(一)”name="450317.fig.002a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002a.jpg"><br><strong>(一)</年代trong></button> <button class="partial-carousel-dot" aria-label="(b)"><img alt="(b)”name="450317.fig.002b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002b.jpg"><br><strong>(b)</年代trong></button> <button class="partial-carousel-dot" aria-label="(c)"><img alt="(c)”name="450317.fig.002c" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.002c.jpg"><br><strong>(c)</年代trong></button> </div> <div class="partial-carousel-next-prev fig-nav-fig2"> <button class="carousel-prev" aria-label="Previous"></button> <button class="carousel-next" aria-label="Next"></button> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图2</年代pan> <a aria-label="full view" class="caption-partial-url" href="//www.newsama.com/journals/omcl/2011/450317/fig2/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <i>μ</我> <i>μ</我> <i>μ</我> <table> 暴露于潜在的引起的蛋白质S-nitrosation S-nitrosating代理完整3日元细胞。(a)比较效率的潜在S-nitrosating特工S-nitrosate 3日元细胞。细胞保持在哈佛商学院接受CysNO (200GSNO(200米)NOR-3(200米)米),或者SIN-1(1毫米)30分钟。S-nitrosated biotin-switch测定蛋白质检测。(b和c)媒体对S-nitrosation S-nitrosothiols效率的影响。细胞培养在10% FCS / DMEM和哈佛商学院接受GSNO(1毫米)或CysNO 30分钟(1毫米),(b)和S-nitrosated蛋白质进行检测biotin-switch试验和(c)丹化验。所示的屁股(a)和(b)是代表从几个实验结果得到了类似的结果。(c)中的数据显示为±SEM三到四个独立的化验。</table> </div> </div> </div> <h5 id="sec3.3">3.3。CysNO细胞吸收,而不是没有解放,负责S-Nitrosation完整3日元细胞</h5> <p>接下来,S-nitrosating CysNO能力3日元细胞研究确定S-nitrosation CysNO公布的是由于没有或其他机制。因此,我们测量的浓度没有,源自于自发退化CysNO (200<我>μ</我>米)在媒体上使用一个电极。当稀释与哈佛商学院在37°C, CysNO立即解放没有稀释后达到了顶峰3分钟,然后逐步下降(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3(一个)</a>)。相比之下,只有边际量不相同的条件下从GSNO获释。中可观察到类似的显著差异CysNO和在哈佛商学院GSNO Hg的稳定<年代up>2 +</年代up>可分裂的SNO半个被Saville-Griess测量分析(<a href="#B35">35</a>];CysNO衰变的半衰期10分钟,而基本上没有衰变后观察GSNO 2 h(数据没有显示)。CysNO在解决方案的稳定性取决于过渡金属离子杂质的数量,如铁(<a href="#B36">36</a>]。检查的参与任何过渡金属离子,金属螯合剂的作用DETAPAC没有释放测量。从CysNO DETAPAC存在显著抑制没有释放,表明金属催化分解反应负责没有解放CysNO在哈佛商学院在试验条件下使用。</p> <div class="floats-partial-section partial-carousel-wrapper" data-section="fig3"> <div class="floats-partial-content fig-content-fig3"> <div class="partial-carousel--single-image"> <img alt="(一)”name="450317.fig.003a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003a.jpg"> <br> <strong>(一)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(b)”name="450317.fig.003b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003b.jpg"> <br> <strong>(b)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(c)”name="450317.fig.003c" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003c.jpg"> <br> <strong>(c)</年代trong> </div> <div class="partial-carousel--single-image"> <img alt="(d)”name="450317.fig.003d" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003d.jpg"> <br> <strong>(d)</年代trong> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs fig-thumbs-fig3"> <button class="partial-carousel-dot" aria-label="(a)"><img alt="(一)”name="450317.fig.003a" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003a.jpg"><br><strong>(一)</年代trong></button> <button class="partial-carousel-dot" aria-label="(b)"><img alt="(b)”name="450317.fig.003b" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003b.jpg"><br><strong>(b)</年代trong></button> <button class="partial-carousel-dot" aria-label="(c)"><img alt="(c)”name="450317.fig.003c" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003c.jpg"><br><strong>(c)</年代trong></button> <button class="partial-carousel-dot" aria-label="(d)"><img alt="(d)”name="450317.fig.003d" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.003d.jpg"><br><strong>(d)</年代trong></button> </div> <div class="partial-carousel-next-prev fig-nav-fig3"> <button class="carousel-prev" aria-label="Previous"></button> <button class="carousel-next" aria-label="Next"></button> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图3</年代pan> <a aria-label="full view" class="caption-partial-url" href="//www.newsama.com/journals/omcl/2011/450317/fig3/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <i>μ</我> <i>μ</我> <i>μ</我> <i>μ</我> <svg height="11.0625" id="M8" style="vertical-align:-0.30096pt" version="1.1" viewbox="0 0 63.962502 11.0625" width="63.962502" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.0625)"> <g transform="translate(72,-63.15)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </t年代pan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</t年代pan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</t年代pan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="44.810753" y="0"> 1</t年代pan> </text> </g> </g> </svg> <table> DETAPAC的影响,DTNB, L-leucine CysNO-induced S-nitrosation 3日元细胞。(一)不释放S-nitrosothiols在哈佛商学院。CysNO或GSNO 200年哈佛商学院最终浓度的稀释米没有或DETAPAC(0.5毫米)。没有集中在中耗氧监测在37°C没有使用一个电极。所示的跟踪是一个代表性的结果。DETAPAC (b和c)的影响,DTNB, L-leucine CysNO-induced S-nitrosation 3日元细胞。细胞在哈佛商学院接受CysNO (200米)30分钟没有或存在DETAPAC(0.5毫米),L-leucine(1毫米)和DTNB (100米),添加添加CysNO前20分钟。蛋白质S-nitrosation检测(b)使用丹biotin-switch化验分析和(c)。是一个代表的结果显示的污点。(d)的效率CysNO——D-CysNO-induced S-nitrosation,每个200米,测量了丹化验(b)。S-nitrosothiol水平仅在细胞接受CysNO(标记为“没有”(b))为100% (12±2 nmol SNO /毫克蛋白),和值表示为均值±SEM六到八个独立的实验(b)和(d)从三个独立的实验。*。</table> </div> </div> </div> <p>澄清是否没有来自在中负责S-nitrosation CysNO退化细胞的蛋白质,我们检查的影响DETAPAC CysNO S-nitrosation的细胞保持在哈佛商学院。DETAPAC的存在并不影响的程度评估的S-nitrosation丹化验(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (b)</a>)。评估个人biotin-switch蛋白质的分析也表明,DETAPAC未能抑制蛋白质S-nitrosation(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (c)</a>)。峰值以来没有水平来源于CysNO大约3分钟,有些蛋白质可能是暂时性的经历S-nitrosation [<a href="#B37">37</a>通过CysNO一氧化氮机制。然而,由于S-nitrosating能力(NOR-3)或不/<年代vg height="15.35" id="M9" style="vertical-align:-3.13504pt;width:31.3375px;" version="1.1" viewbox="0 0 31.3375 15.35" width="31.3375" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,15.35)"> <g transform="translate(72,-59.72)"> <text transform="matrix(1,0,0,-1,-71.95,62.89)"> <tspan style="font-size: 12.50px; " x="0" y="0"> O</t年代pan> </text> <text transform="matrix(1,0,0,-1,-62.92,59.77)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 2</t年代pan> <tspan style="font-size: 8.75px; " x="4.8699999" y="-8.29"> •</t年代pan> <tspan style="font-size: 8.75px; " x="9.4472961" y="-8.29"> −</t年代pan> </text> </g> </g> </svg>(SIN-1)很穷(数字<a href="//www.newsama.com/journals/omcl/2011/450317/fig1/" target="_blank">1(一)</a>和<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2 (b)</a>),S-nitrosated蛋白质的形成来源于CysNO不可能是小的数量和金额,如果任何。与此同时,对于大多数S-nitrosated蛋白质稳定后30分钟,结果到目前为止明确排除的参与没有作为一个中间细胞S-nitrosation CysNO。</p> <p>纬度(<a href="#B18">18</a>,<a href="#B28">28</a>,<a href="#B38">38</a>)和thiol-containing等离子体膜蛋白,包括细胞surface-associated PDI (<a href="#B20">20.</a>- - - - - -<a href="#B22">22</a>),建议参与S-nitrosation。添加L-leucine(1毫米),竞争基质LAT [<a href="#B39">39</a>),抑制的程度CysNO-induced S-nitrosation 60%以上,所评估的丹化验(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (b)</a>)。biotin-switch试验还演示了一个类似的抑制亮氨酸对S-nitrosation单个蛋白质的影响(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (c)</a>)。DTNB,由于其亲水的特性,只能面具细胞表面半胱氨酸巯基团体通过与thionitrobenzoic s债券形成的酸。预处理与DTNB 30分钟蒙面12 nmol半胱氨酸/毫克的蛋白质(数据未显示)。然而,预处理DTNB没有影响的程度或S-nitrosated蛋白(数字轮廓度<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (b)</a>和<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (c)</a>)。这些结果表明,CysNO-induced S-nitrosation细胞蛋白质在很大程度上是由通过LAT CysNO吸收,而不是细胞表面相互作用或transnitrosation thiol-containing蛋白质。</p> <p>进一步确定纬度的这些细胞参与S-nitrosation CysNO, S-nitrosating活动CysNO立体异构体,S-nitroso-D-cysteine (D-CysNO),评估。先前的研究已经表明,D-CysNO不能注册通过纬度<a href="#B39">39</a>]。没有释放的动力学D-CysNO在哈佛商学院,衡量使用没有电极,基本上是重合的,L-CysNO(数据没有显示)。S-nitrosation的程度由D-CysNO L-isomer引发的不到10%(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig3/" target="_blank">3 (d)</a>),从而确认的主要角色LAT CysNO-dependent S-nitrosation细胞系。</p> <h5 id="sec3.4">3.4。与氨基酸Transnitrosation BSA和竞争的LAT负责镇压CysNO-Mediated S-Nitrosation在细胞培养基</h5> <p>CysNO未能S-nitrosate细胞细胞培养基(10% FCS / DMEM;图<a href="//www.newsama.com/journals/omcl/2011/450317/fig2/" target="_blank">2</a>)。因此,我们接下来研究机制缺乏CysNO-induced S-nitrosation在细胞培养基(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig4/" target="_blank">4</a>)。CysNO——(200<我>μ</我>米)全身S-nitrosation细胞保持在哈佛商学院显著抑制的FCS(10%),表明血清抑制S-nitrosation包含因素。主要血清蛋白质的组成部分是BSA以来,大约3毫克/毫升10%浓度的血清,它测量的影响。BSA补充(3毫克/毫升)在哈佛商学院抑制CysNO-induced S-nitrosation,虽然不如FCS在这个明显的浓度。BSA有半胱氨酸残基的34个参与分子内二硫桥和剩余一半的人被一个免费的半胱氨酸通过二硫键(<a href="#B40">40</a>]。评估的作用的自由巯基BSA在S-nitrosation抑制行动,BSA NEM-treated测量的影响。NEM治疗降低了BSA的自由巯基群体数量从0.37到小于0.02(见部分<a href="#sec2">2</a>)。同样有趣的是,当浓度NEM-treated BSA在哈佛商学院的补充,没有观察到有抑制作用,表明BSA的自由巯基组织发挥作用的抑制CysNO-dependent S-nitrosation,可能通过接收transnitrosation亚硝基的反应。</p> <div class="floats-partial-section partial-carousel-wrapper" data-section="fig4"> <div class="floats-partial-content fig-content-fig4"> <div class="partial-carousel--single-image"> <img alt="" name="450317.fig.004" src="https://static-01.hindawi.com/articles/omcl/volume-2011/450317/figures/450317.fig.004.jpg"> <br> <strong></strong> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图4</年代pan> <a aria-label="full view" class="caption-partial-url" href="//www.newsama.com/journals/omcl/2011/450317/fig4/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <i>μ</我> <svg height="11.0625" id="M10" style="vertical-align:-0.30096pt" version="1.1" viewbox="0 0 63.962502 11.0625" width="63.962502" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.0625)"> <g transform="translate(72,-63.15)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </t年代pan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</t年代pan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</t年代pan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="44.810753" y="0"> 1</t年代pan> </text> </g> </g> </svg> <sup>#</年代up> <svg height="11.0625" id="M11" style="vertical-align:-0.30096pt" version="1.1" viewbox="0 0 63.962502 11.0625" width="63.962502" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.0625)"> <g transform="translate(72,-63.15)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </t年代pan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</t年代pan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</t年代pan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 0</t年代pan> <tspan style="font-size: 12.50px; " x="44.810753" y="0"> 1</t年代pan> </text> </g> </g> </svg> <table> FCS的影响,BSA, NEM-BSA CysNO-mediated S-nitrosation 3日元细胞。细胞保持在哈佛商学院、DMEM没有或存在FCS (10%)、BSA(3毫克/毫升)或NEM-treated BSA(3毫克/毫升)服用CysNO (200米)30分钟。丹S-nitrosated蛋白质的含量测定的试验,和的值细胞处理CysNO仅在哈佛商学院(没有)为100% (12±2 nmol /毫克蛋白)。值意味着±SEM三到四个独立的化验。*或无;与BSA。</table> </div> </div> </div> <p>在FCS的缺席,CysNO可以弱S-nitrosate细胞在DMEM,虽然这是不到10%的程度,在哈佛商学院(图<a href="//www.newsama.com/journals/omcl/2011/450317/fig4/" target="_blank">4</a>),这表明DMEM基础培养基还包含抑制因素。然而,当细胞接受高CysNO浓度(1毫米)的抑制效应DMEM(数据未显示)下降了50%,表明抑制竞争的本质。这些结果表明,压制在10% FCS S-nitrosation / DMEM是由于中性氨基酸的存在基础培养基中充当竞争基质LAT,除了在BSA CysNO提供免费的半胱氨酸残基之间的相互作用。</p> <h5 id="sec3.5">3.5。暗示供CysNO S-Nitrosation机制</h5> <p>CysNO被广泛使用作为一个“不”捐赠S-nitrosate细胞(<a href="#B5">5</a>,<a href="#B6">6</a>,<a href="#B14">14</a>,<a href="#B15">15</a>),虽然S-nitrosation并非与没有我们的试验条件下的作用。在各种机制为S-nitrosation细胞外S-nitrosothiols [<a href="#B17">17</a>,<a href="#B18">18</a>,<a href="#B20">20.</a>- - - - - -<a href="#B23">23</a>,<a href="#B28">28</a>,<a href="#B38">38</a>,<a href="#B41">41</a>),何克et al<我>。</我>(<a href="#B17">17</a>,<a href="#B18">18</a>,<a href="#B41">41</a>和沃顿等<我>。</我>(<a href="#B28">28</a>,<a href="#B38">38</a>)表明,在一些细胞类型,包括红细胞,内皮细胞,平滑肌细胞,上皮细胞,S-nitrosation细胞蛋白涉及LAT-mediated CysNO吸收。通过这项研究,我们加胚胎成纤维细胞3日元细胞细胞库存增长。关于这个,没有S-nitrosation GSNO LAT作为主要的角色是一致的路线S-nitrosothiol吸收细胞系。目前的结果与先前的结果上述报告的作者(<a href="#B18">18</a>,<a href="#B28">28</a>,<a href="#B38">38</a>,<a href="#B41">41</a>)警告说,谨慎的解释是必要的结果表明细胞S-nitrosation CysNO。具体来说,如果CysNO能导致一些细胞类型S-nitrosation一般细胞培养基(<a href="#B5">5</a>,<a href="#B6">6</a>,<a href="#B14">14</a>,<a href="#B15">15</a>),机制可能是一氧化氮的某些部分,如N的形成<年代ub>2</年代ub>O<年代ub>3</年代ub>和DNIC中间体,因为LAT-mediated可以抑制在这些条件下吸收。事实上,没有发布的数量从10% FCS / DMEM CysNO减毒,但它坚持可能是因为transnitrosation半胱氨酸残基的血清白蛋白(数据没有显示)。然而,当细胞S-nitrosation是一个简单的缓冲盐溶液诱导,主要机制可能很大程度上取决于LAT-mediated细胞吸收。因此,使用D-CysNO可能是更可取的,当采用CysNO作为没有捐赠者nitrosate细胞。</p> <h5 id="sec3.6">3.6。结论</h5> <p>目前的研究表明在缓冲盐只有CysNO可以导致大鼠显著水平的蛋白质S-nitrosation 3日元细胞当暴露在很短的时间内。机制的行为不受任何来自CysNO退化的媒介,但通过LAT CysNO的合并。然而,在细胞培养基,LAT-mediated CysNO吸收几乎完全阻止竞争存在的氨基酸和transnitrosation血清白蛋白。应该小心当口译CysNO在细胞的生物学效应,因为它的一些影响可能是由于没有捐赠和其他蛋白质S-nitrosation和整体效果见过在某种程度上取决于特定的细胞类型和孵化条件研究<b>。</b></p> <h4 id="acknowledgment">承认</h4> <p>这项研究部分支持科研补助金从教育部(C),科学,体育,和日本的文化。</p> </div> <div class="xml-content"> <h4 class="references" id="references">引用</h4> <ol> <li id="B1">l . j . 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Hogg&publication_year=2009" target="_blank">谷歌学术搜索</a></span></li> </ol> </div> <div class="xml-content"> <h4 class="copyright" id="copyright">版权</h4> <p>版权©2011 (Norihiro Ryuman等。这是一个开放的分布式下文章<a rel="license" href="http://creativecommons.org/licenses/by/3.0/">知识共享归属许可</a>,它允许无限制的使用、分配和复制在任何媒介,提供最初的工作是正确引用。</p> </div> </div> </article> </div> </div> </div> <div class="biblio__StyledBibblio-sc-1nou1lz-0 eSVfHT"> <div id="takeover" class="biblio__TakeoverWrapper-sc-1nou1lz-2 htjhiS"> <div id="bib--hide-takeover-element-mobile"> <div id="bib--takeover-element-mobile"> <h4 class="biblio__H4-sc-1nou1lz-4 lhUKwc">相关文章</h4> <div id="bib--rcm-takeover-element-mobile"> 对本文没有相关内容可用。</div> </div> </div> </div> </div> <div class="biblio__StyledBibblio-sc-1nou1lz-0 eSVfHT"> <div class="biblio__FooterWrapper-sc-1nou1lz-3 etZutQ mobile-widget" id="related-articles-hidden"> <div id="bib--hide-footer-element-mobile"> <hr class="biblio__HR-sc-1nou1lz-5 bkemVQ"> <h4 class="biblio__H4-sc-1nou1lz-4 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height="24"><span class="sc-dRFtgE jrsDZP">下载其他格式</年代pan><span class="sc-gkFcWv gWTCXQ">更多的</年代pan></span> <span class="sc-htoDjs bMdQcY sc-gbOuXE jGPvPL icon-colored _1CwL9R82TPl2HxDleeYGDR icon-themeable" title="" font-size="11"></span> </div> <div class="sc-fnwBNb etzuBr"> <a class="sc-htpNat bUhGXt link sc-iNhVCk gWgCNb open_panel" href="//www.newsama.com/downloads/journals/omcl/2011/450317.epub" aria-label="ePub" property="background-color" color="#D8D8D8"> <div class="sc-eAKXzc WHkfb"> ePub</div><img alt="" class="sc-EHOje jOLhQl sc-bfYoXt cGuwDn" title="" role="presentation" 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height="24"><span class="sc-dRFtgE jrsDZP">订单打印副本</年代pan><span class="sc-gkFcWv gWTCXQ">订单</年代pan></span><span class="sc-htoDjs kNoiZu sc-gbOuXE jGPvPL right__icon icon-colored _32KP5uexH-Bgs7Kj8Gk1Tz icon-themeable" title="" font-size="16"></span></a> <div class="sc-hBbWxd kdRRST"> <div class="sc-dyGzUR cHatnS"> <div> 的观点<年代pan>1886年</年代pan> </div> <div> 下载<年代pan>1032年</年代pan> </div> </div> <div> <div class="sc-drKuOJ kxdGAZ"> <span class="__dimensions_badge_embed__" data-doi="10.1155/2011/450317" data-style="small_circle"></span>引用</div> </div> </div> <nav class="sc-bRBYWo fXBKFa sc-hUfwpO jHPgUP" id="toolbar__iconNav"> <h3 class="sc-Rmtcm hNPrIr">分享文章</h3> <ul class="sc-hzDkRC bnbfTS"> <li class="iconBorder"><a class="sc-htpNat bUhGXt link" href="https://twitter.com/share?url=https://doi.org/10.1155/2011/450317&via=Hindawi" aria-label="Sharing on Twitter" rel="noopener noreferrer" target="_blank"><img alt="" class="sc-EHOje jOLhQl socialTwitter" title="" role="presentation" 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