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L-Glutamine (200 mM)

L-谷氨酰胺200mM(100x)

Company: Thermo Fisher Scientific
Catalog#: 25030081
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Analysis of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines
Author:
Date:
2018-09-05
[Abstract]  Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. We studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived RK13 cells and African green monkey (Cercopithecus aethiops) kidney-derived Vero cells. Our data further demonstrated that RuV used cellular sphingomyelin and cholesterol for its binding to cells and membrane fusion at the step of virus entry. Detailed protocols of our assays, which assess the effects of SMase treatment on RuV infectivity in RK13 and Vero cells, are described. [摘要]  风疹是一种轻度传染性疾病,其特征是低风热和由风疹病毒(RuV)引起的麻疹样皮疹。 病毒通常使用细胞磷脂进行感染。 我们研究了细胞鞘磷脂在RuV感染中的作用。 用鞘磷脂酶(SMase)处理细胞抑制兔肾衍生的RK13细胞和非洲绿猴( Cercopithecus aethiops )肾源性Vero细胞中的RuV感染。 我们的数据进一步证明RuV在病毒进入的步骤中使用细胞鞘磷脂和胆固醇来结合细胞和膜融合。 描述了我们的测定的详细方案,其评估了SMase处理对RK13和Vero细胞中RuV感染性的影响。

【背景】风疹病毒(RuV)是一种正链RNA病毒,属于 Togaviridae 家族中的 Rubivirus 属。该家族有两个属, Rubivirus 和 Alphavirus 。风疹病毒是属的鞋底构件的风疹病毒,而许多病毒,例如塞姆利基森林病毒(SFV)和辛德毕斯病毒(SINV),是归类于甲病毒属。 RuV是风疹和先天性风疹综合征(CRS)的致病因子。风疹的特征是低烧,麻疹样皮疹和淋巴结肿大。它通常是一种轻微的疾病。然而,CRS是一种严重的疾病。 CRS导致在怀孕早期患有风疹的母亲所生的新生儿出现多器官缺陷。白内障,感音神经性听力损失和心血管缺陷在CRS中很常见。

以前的研究表明,细胞膜脂质作为RuV感染的结合或进入因子(Mastromarino ...

Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism
Author:
Date:
2018-09-05
[Abstract]  Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery.

The ...
[摘要]  使用气相色谱质谱(GC-MS)分析柠檬酸循环(CAC)和相关中间体(如谷氨酸,谷氨酰胺,GABA和天冬氨酸)是一种分析方法,用于识别明显相关和不相关的能量途径之间的意外相关性代谢。通过使用已知量的样品添加的参考标准,中间体可以表示为它们的绝对浓度或相对比例。 GC-MS还可以区分重标记分子( 2 H-或 13 C-标记的)和天然存在的最丰富的分子。使用示踪剂的应用还可以评估在各种生理和病理条件下以及用于途径发现的特定代谢池的周转。

以下方案是一种相对简单的方法,不仅对小浓度的代谢中间体敏感,而且还可以 in vivo 或 in vitro 用于确定各种新陈代谢的完整性途径,如特定代谢物池内的通量变化。我们使用该协议来确定磷酸烯醇丙酮酸羧激酶1 ( Pck1 )基因在小鼠巨噬细胞中的作用,以确定 13 C将葡萄糖标记为特定的CAC代谢物库

【背景】随着细胞和小鼠中基因表达改变的发展,需要了解这些缺失或过表达的基因如何影响代谢途径的调节。在该方案中,我们使用稳定同位素来确定进入CAC的葡萄糖通量如何改变葡萄糖对柠檬酸盐,琥珀酸盐和苹果酸盐的贡献。使用稳定同位素和目标分析代谢只是在细胞培养中使用稳定同位素的一个好处。

本方案中描述的用于细胞内代谢物功能定量的方法是通过在U- 13 ...

CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
Author:
Date:
2018-08-20
[Abstract]  Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are ... [摘要]  大型DNA病毒(例如非洲猪瘟病毒(ASFV))的基因编辑传统上依赖于由报道盒组成的供体质粒与周围同源病毒DNA的同源重组。然而,这种导致所需修饰病毒的同源重组是罕见的事件。我们最近报道了使用CRISPR / Cas9编辑ASFV。使用CRISPR / Cas9修饰非洲猪瘟病毒基因组导致了引入遗传变化的快速且相对简单的方法。为了实现这一目标,我们首先用田间分离株ASFV-G感染原代猪巨噬细胞,并用CRISPR / Cas9供体质粒转染质粒,该质粒将表达靶向我们基因的特异性gRNA被删除。通过插入报告盒,我们能够通过有限稀释和噬菌斑纯化从亲本中纯化我们的重组病毒。我们以前曾报道将传统的同源重组方法与CRISPR / Cas9进行比较,结果导致重组增加超过4个对数。

【背景】 非洲猪瘟(ASF)是一种由ASF病毒(ASFV)引起的高度致命的猪传染性病毒性疾病。 ASFV的基因组由大约180-190千碱基对的双链DNA基因组组成。 ASFV引起一系列疾病,从高度致命到亚临床,取决于宿主特征和病毒株(Tulman et al。,2009)。 ASFV没有商业疫苗;实验上,2007年格鲁吉亚爆发的唯一能够抵御目前流行病毒株的疫苗(ASFV-G)是含有一个或多个病毒基因组缺失的减毒活疫苗,例如:( O'Donnell et ...

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