| Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
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Author:
Date:
2017-06-05
[Abstract] The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of ...
[摘要] 消除HIV-1患者的主要障碍是后整合延迟（Finzi等人，1999）。抗逆转录病毒治疗仅针对主动复制病毒，而具有低转录活性或无转录活性的潜伏感染仍未得到治疗（Sedaghat等人，2007）。为了消除病毒性水库，一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期（Deeks，2012）。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制，并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近，许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端（BET）家族的成员的治疗潜力（Filippakopoulos等人，2010; Dawson等人＆ / em>，2011; Delmore等人，2011），其包括BRD2，BRB3，BRD4和BRDT。小分子BET抑制剂，例如JQ1（Filippakopoulos et al。，2010; Delmore等人，2011），I-BET（Nicodeme等人＆lt; / ...
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| Cell Surface Protein Detection to Assess Receptor Internalization
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Author:
Date:
2016-10-20
[Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. ...
[摘要] 膜受体在暴露于不同的刺激剂/抑制剂时的迁移是非常重要的。其中，膜受体的内化影响其对配体的可及性和对环境线索的细胞应答性。在实验上，受体内化可用作其活化的量度。在我们的研究中，我们采用这种方法来探讨神经肽Y（NPY）的七个跨膜结构域受体，Y5R和脑源性神经营养因子（BDNF），TrkB的酪氨酸激酶受体之间的串扰。为此，我们测量了用TrkB配体BDNF刺激后Y5R的内化。在用BDNF处理后，将细胞暴露于选择性标记表面蛋白的不透膜的生物素化试剂。随后，生物素化的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化，并通过蛋白质印迹分析。存在于对照和配体处理的细胞的细胞表面上的受体部分的差异用作其内化和对特定刺激的反应的量度。
[Backg 回合] 可以使用两种主要策略 - 显微镜和生物化学来测量响应外部刺激的细胞膜受体内化。最常见的方法是使用显微镜 - ...
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| 3H-Penciclovir (3H-PCV) Uptake Assay
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Author:
Date:
2013-09-05
[Abstract] Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to ...
[摘要] 来自人单纯疱疹病毒1型（HSV1-TK）的胸苷激酶与特定底物前药核苷酸类似物更昔洛韦（GCV）的组合已广泛用作癌症基因治疗的自杀性治疗基因。将具有改善的底物特异性的HSV1和其突变体（ HSV1-sr39TK ）用作小动物中各种生物学功能的PET成像的报告基因，通过与放射性标记的底物例如18 F-FHBG和124 I-FIAU。 H-Penciclovir（PCV）摄取试验是一种选择用于测定哺乳动物细胞和组织中HSV1-TK表达水平的方法。 HSV1-TK磷酸化PCV并导致形成penciclovir单磷酸，并且其随后通过细胞TK的磷酸化导致形成选择性地捕获在表达HSV-TK的细胞中的三磷酸喷昔洛韦。通过诸如闪烁计数的放射性方法，可以检测到更昔洛韦对哺乳动物细胞和组织的摄取。在这里我们描述在哺乳动物细胞中进行 H - Penciclovir摄取的方案。
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