Methods in molecular biology最新文献_第9页

Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry. 液相色谱-耦合质谱法测定细胞腺嘌呤核苷酸。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4284-9_1

Ashley J Ovens, Dingyi Yu, Toby A Dite, Bruce E Kemp, Jonathan S Oakhill

{"title":"Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry.","authors":"Ashley J Ovens, Dingyi Yu, Toby A Dite, Bruce E Kemp, Jonathan S Oakhill","doi":"10.1007/978-1-0716-4284-9_1","DOIUrl":"10.1007/978-1-0716-4284-9_1","url":null,"abstract":"Adenine nucleotides (AXPs, also referred to as adenosines or adenylates) are a group of organic molecules including adenosine 5'- mono-, di-, and tri-phosphate (AMP, ADP, and ATP, respectively) that, combined, resembles an electrochemical storage cell to facilitate cellular energy storage and transfer. ATP, generated from ADP by photosynthesis, anaerobic respiration, and oxidative phosphorylation, powers many energy-requiring processes in the cell through hydrolysis of its terminal (γ) phosphate, whereas ADP is equilibrated with AMP and ATP by the adenylate kinase reaction. AXPs are major signaling molecules that regulate a wide range of anabolic and catabolic enzymes including AMP-activated protein kinase (AMPK), phosphofructokinase, and pyruvate dehydrogenase.Methods to determine concentrations of AXPs from cells and biological samples have historically relied on high-performance liquid chromatography (HPLC)/capillary electrophoresis techniques to measure [ATP] and [ADP]. However, due to its low basal concentrations, these techniques lack sufficient sensitivity to directly measure [AMP], which must be extrapolated using assumptions of adenylate kinase equilibrium that neglect AMP degradation and synthesis pathways. Here, we describe a detailed protocol to accurately measure [AXP] from cells by liquid chromatography-coupled mass spectrometry (LC/MS), applicable to a wide range of fields including our specific interest in AMPK-dependent metabolic regulation.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2882 ","pages":"3-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Picrotoxin-Induced Epileptogenic Hippocampal Organotypic Slice Cultures (hOTCs). picrotoxin诱导的癫痫性海马器官型切片培养（hotc）。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4386-0_23

Ane Rodríguez-Bodero, Paolo Bonifazi, Jan Tønnesen, Juan Manuel Encinas-Pérez

引用次数: 0

Engineered Hydrogels for 3D Cell Culture and Bioprinting of Human Induced Pluripotent Stem Cell-Derived Neuroepithelial Stem Cells. 人类诱导多能干细胞衍生神经上皮干细胞的3D细胞培养和生物打印工程水凝胶。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4530-7_16

Daniel Aili, Anna Herland

引用次数: 0

Using Zymography to Assess Circulating Matrix Metalloproteinase (MMP)-2 and MMP-9 in Clinical Samples. 应用酶谱法评估临床样品中循环基质金属蛋白酶(MMP)-2和MMP-9。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4482-9_14

Sandra O Conde-Tella, Raquel F Gerlach, Jose E Tanus-Santos

{"title":"Using Zymography to Assess Circulating Matrix Metalloproteinase (MMP)-2 and MMP-9 in Clinical Samples.","authors":"Sandra O Conde-Tella, Raquel F Gerlach, Jose E Tanus-Santos","doi":"10.1007/978-1-0716-4482-9_14","DOIUrl":"https://doi.org/10.1007/978-1-0716-4482-9_14","url":null,"abstract":"Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that cleave a variety of different substrates and play key roles in both physiological and pathological processes. They are involved in the pathophysiology of many disease conditions, and for this reason, many clinical studies have been carried out to examine whether circulating MMP concentrations reflect the severity of disease conditions. This article presents detailed information that is critical to assess circulating MMP-2 and MMP-9 activity in clinical samples by using zymography, which is a powerful technique that has been used for more than three decades to detect picogram quantities of MMPs. Important methodological issues have been addressed in the past regarding this technique, and some analytical and preanalytical details may severely affect the results and thus hamper the diagnostic and prognostic value of such measurements. While fasting or storing plasma samples for 1 month at -20 °C or at -70 °C does not significantly affect plasma MMP-2 or MMP-9 activity, repeating freeze-thaw cycles of plasma samples decrease MMP-9 activity after seven cycles. Importantly, the appropriate volume to assess MMP-2 is significantly lower than the appropriate volume to assess MMP-9 activity. Finally, special concerns exist with respect to circulating MMP-9 (but not MMP-2) measurements, which are found at artificially higher concentrations in serum than in plasma (EDTA, citrate or heparin) samples, even though significant correlations exist between MMP-9 levels assessed in plasma and in serum samples.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2918 ","pages":"177-186"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Psoralen Crosslinking-Chromatin Endogenous Cleavage Assay to Examine Histone DNA Interactions of Active and Inactive rRNA Genes. 补骨脂素交联-染色质内源性切割试验检测活性和非活性rRNA基因的组蛋白DNA相互作用。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4486-7_8

Alexia Muguet, Thomas Gardrat, Antonio Conconi, Audrey Paillé

{"title":"Psoralen Crosslinking-Chromatin Endogenous Cleavage Assay to Examine Histone DNA Interactions of Active and Inactive rRNA Genes.","authors":"Alexia Muguet, Thomas Gardrat, Antonio Conconi, Audrey Paillé","doi":"10.1007/978-1-0716-4486-7_8","DOIUrl":"https://doi.org/10.1007/978-1-0716-4486-7_8","url":null,"abstract":"In the nucleoli of eukaryotic cells, the multiple copies of ribosomal RNA genes (rRNA genes) coexist in two different forms that have distinct characteristics: transcribed (active) and non-transcribed (inactive) units. \"Active\" rRNA genes are loaded with RNA polymerase I and are largely depleted of nucleosomes, whereas \"inactive\" rRNA genes are covered with two copies of the four histone proteins that are folded in nucleosomes. A third form of chromatin is observed in Saccharomyces cerevisiae (here called as yeast) arrested in the G1 phase of the cell cycle. In yeast synchronized before DNA replication, nucleosomes are also absent in the non-transcribed rRNA genes, which are described as \"open\" units.The presence of two distinct groups of rRNA genes compromises the interpretation of standard biochemical assays that are employed to study the structure of chromatin during DNA transcription, DNA replication, and DNA repair. This chapter describes protocols to investigate the association of histone proteins with rRNA genes in yeast. In addition, it provides a comprehensive list of studies that applied psoralen photo-crosslinking to follow the structure of rRNA gene chromatin in a variety of high eukaryotic cells.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2919 ","pages":"133-154"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Kinetics of Protease Thermal Inactivation. 蛋白酶热失活动力学。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4478-2_21

Natalija Andrejević, Natalija Polović, Jelica Milošević

引用次数: 0

Zymography for the Detection of Bacterial Proteases. 酶谱法检测细菌蛋白酶。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4482-9_16

Mary E Marquart

引用次数: 0

ELISA for Detection of Total IgG Against YF 17DD. ELISA检测YF - 17DD总IgG。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4458-4_3

Ana Carolina R A Cajaraville

引用次数: 0

An Improved Two-Dimensional HPLC Method for Endogenous 2'-Deoxy-ADPR and 2'-Deoxy-NAD. 内源性2’-脱氧- adpr和2’-脱氧- nad的改进二维高效液相色谱法

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4414-0_6

Feng Gu, Ralf Fliegert, Andreas Bauche, Andreas H Guse

引用次数: 0

Yellow Fluorescent Protein Quenching Assay for Analyzing Odorant Receptor Activity. 黄色荧光蛋白猝灭法分析气味受体活性。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4466-9_11

JiWoo Choi, JaeHyung Koo

引用次数: 0