这项工作是由美国心脏协会14FTF20370058（DMA）和美国国立卫生研究院T32 HL007101-35（DMA）的支持。

1.电导导管准备和压力校准<ol><li>电导导管连接至血流动力学导管模块。电子校准压力和体积测量通过记录预设的压力和体积的导管模块上设置。记录0毫米汞柱和25毫米汞柱（图1A）一个跟踪和分配的电压到两个压力描记（图1B 和1C）。同样地，记录5 RVU和25 RVU（图1D）的体积跟踪和分配的电压到两个体积描记（1E和1F）。 </li><li>确认与手动压力校准电子压力校准，使用水银柱式血压计。装上血压计袖带端口与3路阀。填3端口止血阀系统，通常用于冠状血管成形术，在使用侧端口RT水。 <ol><li>放置一个电导导管的尖端放入充满流体的止血阀，轻轻地固定机智豪特扭结导管。止血阀连接到血压计和膨胀到200毫米汞柱，并锁定3路阀。检查是否所测量的压力对应于充气的血压计的压力。 </li></ol></li><li>在盐水代替导管加热到37℃，即在操作场和测量压力的水平。调整压力控制，直到录制的压力是零。 </li></ol> 2.麻醉/插管<ol><li>施用氯胺酮/甲苯噻嗪（80〜100/10毫克千克-1），为腹膜内注射。 注意：替代麻醉剂都可以使用。在以前的这种技术11,12的评语是提供麻醉剂的详细列表。适当的麻醉可以通过温和的尾巴捏确认。 </li><li>一旦麻醉，剃颈部和胸部剪及地方上的加热垫。保持鼠标肛温在36.5-37.5ºC。低体温会导致抑郁症心脏率。适用软膏的眼睛，以防止干燥</li><li>做一个中线切口在颈部和解剖气管肌肉掉，露出气管。放置气管内管通过口，同时可视化气管以确保插管，并连接到呼吸器。 </li><li>在手术过程中保持鼠标呼吸机上。根据动物体重设置呼吸机参数设置如前面所述11。潮气量（毫升）= 6.2×（动物体重以公斤为单位），1.01和呼吸率= 53.5×（动物体重以公斤为单位）-0.26。 </li></ol> 3.放置电导导管的LV室<ol><li>颈动脉的方法<ol><li>以确保无菌性，两套无菌手术器械的使用 - 是一个用于初始皮肤切口，一个在开胸进行操作。仪器应使用动物的干燥灭菌个人外科会话期间进行消毒次高压灭菌在每个外科一天结束。 </li><li>后皮肤已经被净化了洗必泰+酒精擦洗皮肤（0.5％洗必泰/ 70％的酒精溶液）的三个周期，做一个切口在右颈从下颌到胸骨。解剖周围组织以暴露右颈，并切断它运行邻近于颈动脉的迷走神经。 </li><li>周围放置的远端的颈动脉（远离胸部）中的无菌6-0丝线缝合，领带和安全的。放置在颈动脉近端下方两个附加​​缝合线（接近胸部）到第一缝合，松散捆扎中间缝合。轻轻拉动近端缝合，并通过将其夹紧在皮肤上固定。确保颈动脉已在继续之前被夹住两个近端和远端。 </li><li>作一小切口在R.颈动脉，接近第一缝合，并纵向朝胸部延伸。 </li><li>将电导导管尖端，先前在温盐水浸泡30分钟，进容器通过切口并使用中间缝合固定导管。 </li><li>轻轻推进导管中通过颈动脉左心室，一边看压力 - 容积环追踪，以确保正确放置。 注意：在导管应产生显示矩形压力-体积环的最优放置（ 见图 2）。如果导管的通过任何遇到阻力，轻轻地拉了回来，并用温和的压力再次前进。导管轻柔旋转可以帮助置入左心室。迫使电导导管可导致严重的心血管并发症或损坏导管。 </li><li>记录基线压力-体积环的导管放置后〜10分钟，获得一个稳定的状态（ 图2）。 </li></ol></li><li>心尖​​方法<ol><li>在麻醉和通风的鼠标，做一个切口FR嗡剑突，并通过胸壁横向直至隔膜是可见的切割。 </li><li>切，虽然隔膜和可视化的心脏顶点。 </li><li>将电导导管进入左心室的穿过针刺伤口（使用25-30g的针）的先端，直到近侧电极只是心室内侧。 </li><li>记录基线压力 - 体积环的导管放置后〜10分钟，达到稳定状态。 </li></ol></li></ol> 4.变后负荷使用瞬态主动脉阻断<ol><li>要进行短暂的主动脉阻塞，做一个小的水平切口，在胸腔上部和解剖周围组织揭露横向主动脉。 </li><li>将横向主动脉下6-0丝线结扎。经过压力容积环已经恢复到基线，扣住缝线的两端用针钳，轻轻地，慢慢抬高缝合过1-2秒，慢慢释放压力。 </LI&gt; <li>重复此过程，直到三个独立的最佳记录被从同一动物制成。 注：最佳录音应至少有5个压力容量循环周期和紧张的缝合（ 图3A和3C）的应用过程中稳步上升，收缩末期压力。 </li></ol> 5.变预紧使用瞬态下腔静脉阻塞（IVC） <ol><li>要进行短暂的下腔静脉阻塞，使下面的剑突水平切口，隔膜暴露下腔静脉下方。 </li><li>放置IVC下6-0丝线结扎。后压力 - 容积环已经回到基线，​​扣用针夹具的缝合线的两端;轻轻地，慢慢抬高缝合过1-2秒，慢慢释放压力。 IVC闭塞，也可以通过压力使用棉花温柔应用程序执行倾斜拭子。 </li><li>重复这个过程，直到三个独立的最佳录音m个从相同的动物ADE。 注意：最佳记录应至少有5个压力体积循环周期和张力对缝线施加期间中左心室舒张末期压力稳定下降（图4A 和4B）。 </li></ol> 6.盐水校准<ol><li>在研究结束时，一个并联电导（Ⅴp）的值可以是获得由通过颈静脉注射高渗盐水（15％）到动物的10微升丸药（图5A 和5B）。 注：此丸会导致体积的压力没有变化的明显增加。这在体积明显的变化是在血池电导，而不是一个改变的结果，由于在卷的实际增加。在DP / dt的最大瞬时下降可以看出，由于高渗盐水有负性肌力作用13。计算出的V P能够沿着进入压力容积环分析软件与试管校准参数和从转换的RVU以微升。 </li></ol> 7.试管校准<ol><li>要执行校准试管，放置具有在加热垫上或水浴提供由制造商已知直径的孔中的试管加热到37ºC。从小鼠进行血流动力学评估填充第4-5孔新鲜温暖的肝素的血液。 注：试管校准允许使用鼠标血左心室的血池准确的评估，并允许从的RVU微升的转换数据量。 </li><li>将电导导管进入第一口井，直到所有的电极被淹没。轻柔地将导管孔，这将产生不同的RVU。 </li><li>记录中的RVU音量通道的电导的变化。选择最高RVU用于校准。 注：各孔的体积既可以由1使用等式为volum计算）Ë圆柱体，其中所述半径是试管井和长度的是基于内部的两个感测电极或2）在制造商的说明检查之间的长度。电导输出可以与已知体积相关，以开发出将数据从进入的RVU微升11将校准方程。 </li></ol> 8.安乐死<ol><li>在协议结束时，将小鼠用颈椎脱臼而麻醉下安乐死。 </li><li>为了确保死亡，已经历颈脱位小鼠必须经过安乐死的辅助方法。我们使用放血麻醉下，与在麻醉下采集心脏组织的用于实验，或双边开胸。 </li></ol> 9.使用数据分析压力容积环分析软件<ol><li>计算V p从盐水校准<ol><li>选择压力和VOLU我回路高渗盐水的喷射期间获得（图5A和5B） </li><li>出口循环到压力体积分析软件。选择选项盐水校准（图5C） </li><li>记录计算出的V P值 （图5D）。 注：购买V P值是通过识别1）舒张末期容积与从盐校准收缩末期容积的交集和2）舒张末期容积=收缩末期容积线计算。这些线的交叉点提供在V p，是由压力容积环的分析软件进行计算。 </li></ol></li><li>输入传导到体积（RVU）的体积通道选项的关系（ 图1F） </li><li>测量基线压力 - 体积循环关系<ol><li>从压力和体积渠道8-10个心动周期，一旦稳定状态已经实现（ 图2A），并出口到分析软件洁具。确定5-6呼气末环（ 图2B） </li><li>使用V P值来校正平行电导。选择“稳态”选项，并产生一个血液动力学汇总表（图2C） </li></ol></li><li>在主动脉缩窄测量压力容量环的关系<ol><li>选择从压力和体积沟道8-10个心动周期对应于之前的舒张末期压力（ 图3A）和出口到分析软件的增加主动脉缩窄。识别5-6呼气末回路（图3C） </li><li>选择“收缩”的分析（ 图3B），这将计算结束收缩的压力容积关系（ESPVR） </li></ol></li><li>在IVC缩窄测量压力容量环的关系<ol><li>从对应于下腔静脉收缩压力和体积渠道8-10个心动周期（FIGURË4A），并出口到分析软件。确定5-6呼气末环（ 图4B） </li><li>选择“收缩”的分析（图3B），这将计算预压Recruitable冲程工作（PRSW）（图4C），最大上升/ dt的VS EDV（图4D），以及ESPVR和最终舒张压容积关系（图4E） </li></ol></li></ol>

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+correction+for+parallel+conductance,+GP,+and+gain+factor,+alpha,+in+invasive+murine+left+ventricular+volume+measurements.">Dynamic correction for parallel conductance, GP, and gain factor, alpha, in invasive murine left ventricular volume measurements.</a> J Appl Physiol (1985). 107 (6), 1693-1703 (2009).</li></ol>

作者什么都没有透露。

我们描述了使用小鼠电导导管，以获得两个心脏收缩和舒张的综合分析perfoming压力 - 容积环分析的方法。菅佐川急便和他 ​​的同事利用压力容积循环，以确定心脏收缩的措施，在ESPVR专门的坡度，或收缩末期弹性（E ES）和E 最大 。顺应性，通过压力的比率定义为体积（P / V），上变化收缩的持续时间。在每个心脏收缩，瞬时弹性度是依赖于心脏速率和心肌收缩力，但在很大程度...

心脏压力容积环分析提供心脏功能的详细信息，其中的金标准功能评估1。虽然诸如超声心动图或心脏MRI成像技术提供的功能的措施，这些措施在很大程度上取决于负载条件。心脏收缩和舒张的负载独立的措施需要心室压力和体积的关系在一定范围内的前后负荷的动态测量。压力容积关系的这种认识源于佐川急便和同事2,3的开创性工作。他们在体外灌注犬心脏表明，压力容积环衍生收缩措施是独立的负载条件4。 在这些分析的体内应用成为可能的传导导管在20世纪80年代的发展。这使得嘉的技术进步SS和他的同事进行的人类5,6压力-容积环分析。电导导管和改进手术技术在上世纪90年代7小型化由啮齿类动物心脏功能分析是可行的，允许遗传和药理学研究被执行。这一进步此后引发广泛使用的压力容积环分析，并产生了大量的洞察哺乳动物心脏生理。 在使用电导导管，并从中获得的数据的解释的一个关键概念是体积和电导之间的关系。电导是负相关的电压，这是使用与近侧放置电极，通常放在下面的主动脉瓣，和远侧，在LV尖8的导管测量。被改变电压或电导的变化电流从近端到远端电极的测量。虽然血池贡献š显著电导，心室壁的贡献，称为并联电导（V p）的 ，以测得的电导必须减去，以获得绝对的LV体积测量。 所述的方法来执行此校正，称为盐水校准，在下面的协议进行了讨论。电导和体积之间的数学关系，由班和同事描述的是体积= 1 /α; （ρL 2）（GG p），其中 α=均匀的场校正系数，ρ=电阻率血液，L为电极之间的距离，G =电导和 G p值=非血液电导9。值得注意的是，在小鼠的统一场校正系数接近1.0，由于小室容积10。再加上压力传感器，电导导管提供实时同步的压力和体积数据。 心脏PRESSU再卷分析呈现特别优势的心脏功能的其他措施，因为它们允许用于心室功能独立的负载条件和心脏速率的测量。收缩的具体负载无关的心脏指标包括：收缩末期压力容积关系（ESPVR），D P / D T最大末端舒张末期容积关系，最大顺应性（E max）和预加载recruitable工作行程（PRSW）。舒张功能的负载无关的指标是舒张末期压力容积关系（EDPVR）11。以下协议描述心脏的压力容积环分析的行为，同时使用一个颈动脉和顶部的方法。虽然进行这些研究的方法进行了详细先前8,11进行了说明，我们将审查的关键步骤，以获得精确的压力容积测量，包括盐水和试管校准修正，并提供THES的可视化演示Ë程序。研究与开展这项研究的动物按批准的方案和杜克大学医学中心的机构动物护理和使用委员会的动物福利法规进行处理。

<table border="0" cellpadding="0" cellspacing="0">
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			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
			<td>Anesthetic</td>
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			<td>440842</td>
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			<td>VIP3000</td>
			<td>Matrx Medical Inc.</td>
			<td></td>
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			<td>Model 683</td>
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			<td>5628</td>
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			<td>14041-10</td>
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			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
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			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
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			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
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			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
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			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
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			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
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			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
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			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
		</tr>
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			<td>Sphygmomanometer</td>
			<td>Baumanometer</td>
			<td>320</td>
			<td>Hemodynamic equipment</td>
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			<td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td>
			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
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			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
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			<td>ADInstruments Inc.</td>
			<td>ML866/P</td>
			<td>Hemodynamic software</td>
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			<td>animal clipper</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
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			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
		</tr>
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			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
		</tr>
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			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
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			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
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			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
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			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
	</tbody>
</table>

cardiac pressure-volume loop analysis using conductance catheters in mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

压力-容积环分析可以用于测量在基因修饰的小鼠14,15或小鼠接受药物研究16心脏功能。代表压力容积环是由以前发表的著作16提供的调查β-arrestin的效果偏向AT1受体配体，TRV120023。为了测试TRV120023是否影响在体内的心脏功能，在接收到传统的和新颖的血管紧张素受体 ​​阻滞剂的野生型小鼠中进行压力-容积环分析。 TRV120023静脉滴注增强心肌收缩显著（</...

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

心脏压力容量环分析小鼠使用电导导管

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K 次观看.  Duke University Medical Center. Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. 

视频: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

科研

生物学

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

测量压力容量环中的鼠标

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...