Abstract:
:We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic system for targeting the homodimerization of histidine kinases essential for the bacterial two-component signal transduction. By using these methods, we have been able to identify various types of inhibitors that block the autophosphorylation of histidine kinases with different modes of actions.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Okada A,Gotoh Y,Watanabe T,Furuta E,Yamamoto K,Utsumi Rdoi
10.1016/S0076-6879(06)22019-6subject
Has Abstractpub_date
2007-01-01 00:00:00pages
386-95eissn
0076-6879issn
1557-7988pii
S0076-6879(06)22019-6journal_volume
422pub_type
杂志文章abstract::Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer p...
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abstract::Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in t...
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abstract::Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric o...
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abstract::Organ-specific delivery of biofunctional agents is thought to enhance their activity and to reduce their side effects. Liposomes have been used as drug carriers in cancer chemotherapy, since they accumulate passively in tumor tissues due to an enhanced permeability and retention (EPR) effect. In addition, modification...
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abstract::After cell lysis, the most often used second step in a protein purification procedure is some sort of a rapid, bulk precipitation step. This is commonly accomplished by altering the solvent conditions and taking advantage of the changes in solubility of your protein of interest relative to those of many of the other p...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(09)63020-2
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abstract::An overview of resolution enhancement of conventional, field-swept, continuous-wave electron paramagnetic resonance spectra using Fourier transform-based deconvolution methods is presented. Basic steps that are involved in resolution enhancement of calculated spectra using an implementation based on complex discrete F...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2015.06.009
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abstract::RNA folds during transcription in the cell. Compared to most in vitro studies where the focus is generally on Mg(2+)-initiated refolding of fully synthesized transcripts, cotranscriptional RNA folding studies better replicate how RNA folds in a cellular environment. Unique aspects of cotranscriptional folding include ...
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abstract::The genome integrity of living organisms is constantly threatened by endogenous cellular metabolic processes and environmental agents. To quantify these low, physiologically relevant levels of DNA damage, a single-cell gel electrophoresis (comet) combined with strand-specific fluorescence in situ hybridization (FISH)-...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2017.03.023
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abstract::Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. ...
journal_title:Methods in enzymology
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abstract::Cryptococcus neoformans is a yeastlike fungus that causes a lethal meningoencephalitis in a broad spectrum of immunocompromised patients and has become the most common cause of meningitis due to AIDS-related infections in Africa. Key to the development of new agents to control and prevent this infection is the identif...
journal_title:Methods in enzymology
pub_type: 杂志文章
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abstract::Ultraviolet photodissociation time-of-flight (TOF) mass spectrometry (MS) is described as a method for determination of peptide ion primary structure. Monoisotopic selection and bond-specific activation, combined with the rapidity of TOF MS analysis, render this technique invaluable to the rapidly expanding field of p...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
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abstract::Tumor cells acquire distinct genetic characteristics as a means to survive and proliferate indefinitely. Changes in the genetic code can also translate in changes at the protein level, therefore creating a distinguishable signature unique for tumor cells, and absent in normal tissues. The presence of discernable moiet...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.10.003
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abstract::Microsatellite markers are quickly becoming the molecular marker of choice for studies of parentage and kinship in animals. In this chapter, we review methods and give protocols for screening potential microsatellite markers, as well as protocols for genotyping individuals with useful markers once they have been ident...
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abstract::A genetic interaction occurs when the combination of two mutations leads to an unexpected phenotype. Screens for synthetic genetic interactions have been used extensively to identify genes whose products are functionally related. In particular, synthetic lethal genetic interactions often identify genes that buffer one...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)70007-0
更新日期:2010-01-01 00:00:00
abstract::Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.027
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abstract::The binding of the chemokine [C-X-C motif] ligand 12 (CXCL12 or stromal cell-derived factor 1alpha [SDF-1alpha]) constitutively produced by bone marrow stromal cells and osteoblasts, to the CXC receptor (CXCR) 4, a transmembrane chemokine receptor expressed on hematopoietic stem and progenitor cells (HSPCs), has emerg...
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abstract::Studies on DNA replication in S. pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled. These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and geneti...
journal_title:Methods in enzymology
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abstract::Cell migration plays a key role both in physiological conditions, such as tissue repair or embryonic development, and in pathological processes, including tumor metastasis. Understanding the mechanisms that allow cancer cells to invade tissues during metastasis requires studying their ability to migrate. While spectac...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)00429-6
更新日期:2008-01-01 00:00:00
abstract::The lung is composed of two major anatomically distinct regions-the conducting airways and gas-exchanging airspaces. From a cell biology standpoint, the conducting airways can be further divided into two major compartments, the tracheobronchial and bronchiolar airways, while the alveolar regions of the lung make up th...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)19012-6
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abstract::The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions va...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385120-8.00020-6
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abstract::Mathematical computer-assisted simulations of episodic hormone, substrate, or metabolite release can be accomplished using explicit algebra and high-speed microprocessors. Such simulations are useful to describe the behavior of single or multiple pulse generators, as well as the expected output of these systems. Simul...
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abstract::Integrins are large modular cell-surface receptors that regulate almost every aspect of cellular function through bidirectional signals transmitted across the lipid bilayer. Regulation of integrin activity is accomplished by complex and still incompletely understood biochemical pathways that modify integrin ligand bin...
journal_title:Methods in enzymology
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abstract::One of the greatest advances in molecular genetics has been the application of selectable transposons in molecular biology. After 30 years of use in microbial genetics studies, transposons remain indispensable tools for the generation of null alleles tagged with selectable markers, genetic mapping, manipulation of chr...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)21002-4
更新日期:2007-01-01 00:00:00
abstract::Immunogenic cell death (ICD), induced by certain anticancer chemotherapeutics, leads to the emission of danger associated molecular patterns (DAMP) by cancer cells, which facilitates the attraction, activation and maturation of dendritic cells (DC) as well as the subsequent priming of effector T cells. In this context...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.07.021
更新日期:2020-01-01 00:00:00
abstract::Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397925-4.00017-1
更新日期:2014-01-01 00:00:00
abstract::Enzymatic processes have great potential for applications in industrial biocatalysis, biosensing, and biomedical engineering. However, poor stability and difficulty in the reuse of enzymes still create challenges for their use in catalysis and other applications. In recent years, enzyme-incorporated metal-organic fram...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2016.12.002
更新日期:2017-01-01 00:00:00
abstract::The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.09.028
更新日期:2016-01-01 00:00:00
abstract::Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family. It is expressed in the inner mitochondrial membrane and plays a role in the control of free radical production, oxidative damage, insulin secretion, and fatty-acid peroxide exportation. Although UCP2 expression occurs in several tissues, some of...
journal_title:Methods in enzymology
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更新日期:2009-01-01 00:00:00
abstract::The kinetics of biomolecular systems can be quantified by calculating the stochastic rate constants that govern the biomolecular state vs time trajectories (i.e., state trajectories) of individual biomolecules. To do so, the experimental signal vs time trajectories (i.e., signal trajectories) obtained from observing i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.08.021
更新日期:2016-01-01 00:00:00
abstract::EPR long-range distance measurements on spin-labeled macromolecules have recently become a popular tool in structural biology. The method can be used to obtain coarse-grained structures of biomolecules, to track conformational changes and dynamics, to dock macromolecular complexes, or to localize spin centers within m...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.06.006
更新日期:2015-01-01 00:00:00