Abstract:
:There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that block Rho GTPase function indirectly, either by targeting enzymes involved in post-translational processing or downstream protein kinase effectors. We have reported the identification and characterization of the EHT 1864 small molecule as an inhibitor of Rac family small GTPases, placing Rac1 in an inert and inactive state and then impairing Rac1-mediated functions in vivo. Our work suggests that EHT 1864 selectively inhibits Rac1 downstream signaling and cellular transformation by a novel mechanism involving guanine nucleotide displacement. This chapter provides the details for some of the biochemical and biological methods used to characterize the mode of action of EHT 1864 on Rac1 and its impact on Rac1-dependent cellular functions.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Onesto C,Shutes A,Picard V,Schweighoffer F,Der CJdoi
10.1016/S0076-6879(07)00409-0subject
Has Abstractpub_date
2008-01-01 00:00:00pages
111-29eissn
0076-6879issn
1557-7988pii
S0076-6879(07)00409-0journal_volume
439pub_type
杂志文章abstract::This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VI...
journal_title:Methods in enzymology
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abstract::Two factors, climate change brought on by rising atmospheric CO2 levels and the accelerating shift toward renewable energy sources, have together worked to heighten interest in understanding how biological catalysts so effectively bring about the reduction of CO2 to formate, with potential applications for both biorem...
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abstract::The Microsealed Drug Delivery (MDD) system was developed to achieve several objectives: to maximize the bioavailability of a therapeutic agent in a target tissue; to minimize the adverse side effects of a therapeutic agent or its metabolites; to optimize the onset, rate, and duration of drug delivery; to maintain the...
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abstract::Recent studies using sulfotransferase-deficient mice have revealed various physiological functions of sulfated glycans. Studies using gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 showed that these sulfotransferases play critical roles in lymphocyte homing....
journal_title:Methods in enzymology
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abstract::Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were d...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2016.11.009
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abstract::Rarely is any solution simply solute and water. In vivo, solutes, such as proteins and nucleic acids, swim in a sea of water, salts, ions, small molecules, and lipids, not to mention other macromolecules. In vitro, virtually all solutions contain a mixture of aqueous solvents, or "cosolvents" [i.e., solvent(s) in addi...
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abstract::Modeling enzymatic reactions is a demanding task due to the complexity of the system, the many degrees of freedom involved and the complex, chemical, and conformational transitions associated with the reaction. Consequently, enzymatic reactions are not determined by precisely one reaction pathway. Hence, it is benefic...
journal_title:Methods in enzymology
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abstract::Survival of macrophages, which serve as the first-line defense against invading pathogens by invoking the overproduction of highly toxic peroxynitrite (ONOO-), depends on their ability to maintain the intracellular GSH level and to induce the expression of heme oxygenase-1 (HO-1). The ONOO- is produced by macrophages ...
journal_title:Methods in enzymology
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doi:10.1016/S0076-6879(05)96035-7
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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
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journal_title:Methods in enzymology
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更新日期:1989-01-01 00:00:00
abstract::Scanning fluorescence correlation spectroscopy (sFCS) is the generic term for a group of fluorescence correlation techniques where the measurement volume is moved across the sample in a defined way. The introduction of scanning is motivated by its ability to alleviate or remove several distinct problems often encounte...
journal_title:Methods in enzymology
pub_type: 杂志文章
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abstract::Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluore...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2016.03.018
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abstract::Covalent attachment of lipids appears to be an important mechanism by which many proteins interact with membranes. As we learn more about how lipids and adjacent amino acids participate in addressing proteins to specific membranes within the cell, it should be possible to design more elegant and precise membrane targe...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(95)50089-8
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abstract::Considering that HIV-1 accumulates and replicates actively within lymphoid tissues, any strategy that will decrease viral stores in these tissues might be beneficial to the infected host. Follicular dendritic cells (FDC), B lymphocytes, antigen-presenting cells like macrophages, and activated CD4(+) T cells are abunda...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)91019-7
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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::Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Droso...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2015.08.028
更新日期:2016-01-01 00:00:00
abstract::Fertilization by intracytoplasmic sperm injection (ICSI) is a powerful technique that can be used to understand better the biology of fertilization, in addition to a form of assisted reproduction both in humans and in endangered species. Mouse is often the model organism of choice to study mammalian fertilization. The...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)76014-6
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abstract::MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...
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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
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doi:10.1016/bs.mie.2015.09.028
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abstract::When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general s...
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journal_title:Methods in enzymology
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更新日期:2010-01-01 00:00:00
abstract::Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycos...
journal_title:Methods in enzymology
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doi:10.1016/B978-0-12-385928-0.00010-9
更新日期:2011-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05022-8
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abstract::There is currently no known genetic disease linked to prolactin (PRL) or its receptor (PRLR) in humans. Recently, we identified three missense variants of the PRLR in patients presenting with breast tumors. Two of them (named PRLR(I146L) and PRLR(I76V)) had been reported earlier, but failed to draw much attention beca...
journal_title:Methods in enzymology
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更新日期:2010-01-01 00:00:00
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journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)24007-8
更新日期:2007-01-01 00:00:00
abstract::Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metallopro...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2016.09.044
更新日期:2017-01-01 00:00:00
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journal_title:Methods in enzymology
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更新日期:1986-01-01 00:00:00
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::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 s...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)22019-6
更新日期:2007-01-01 00:00:00