Showing papers in "Methods in Enzymology in 2004"

Protein Structure Prediction Using Rosetta

Abstract: Publisher Summary This chapter elaborates protein structure prediction using Rosetta. Double-blind assessments of protein structure prediction methods have indicated that the Rosetta algorithm is perhaps the most successful current method for de novo protein structure prediction. In the Rosetta method, short fragments of known proteins are assembled by a Monte Carlo strategy to yield native-like protein conformations. Using only sequence information, successful Rosetta predictions yield models with typical accuracies of 3–6 A˚ Cα root mean square deviation (RMSD) from the experimentally determined structures for contiguous segments of 60 or more residues. For each structure prediction, many short simulations starting from different random seeds are carried out to generate an ensemble of decoy structures that have both favorable local interactions and protein-like global properties. This set is then clustered by structural similarity to identify the broadest free energy minima. The effectiveness of conformation modification operators for energy function optimization is also described in this chapter.

Computation and Analysis of Protein Circular Dichroism Spectra

Abstract: Publisher Summary This chapter presents computation and analysis of protein circular dichroism (CD) spectra. The origins of electronic CD in proteins, theoretical methods for computing protein CD, and empirical analysis of CD for estimating structural composition of proteins are described. The phenomenon of CD involves the absorption of light and it can be considered as a special type of absorption spectroscopy. The CD spectra of proteins are generally divided into three wavelength ranges, based on the energy of the electronic transitions that dominate in the given range. The basic approach used to compute the CD of complex systems, such as proteins and nucleic acids, is the divide and conquer approach. In addition to the isotropic atomic polarizabilities, the anisotropic polarizability of the first electrically allowed peptide transition was included in the dipole interaction model for protein CD calculations. Polarizability anisotropy data for simple amides were used for obtaining polarizability parameters. It is found that the CD spectrum of the typical β sheet has a negative band near 215-nm and a positive band near 198 nm.

Tumor hypoxia and malignant progression.

Abstract: Publisher Summary This chapter discusses tumor hypoxia and malignant progression. Hypoxic (or anoxic) areas arise as a result of an imbalance between the supply and the consumption of oxygen. Whereas in normal tissues or organs the O2 supply matches the metabolic requirements, in locally advanced solid tumors the O2 consumption rate of neoplastic as well as stromal cells may outweigh an insufficient oxygen supply and result in the development of tissue areas with very low O2 levels. Major pathogenetic mechanisms involved in the emergence of hypoxia in solid tumors are (a) severe structural and functional abnormalities of the tumor microvessels (b) a deterioration of the diffusion geometry, and (c) tumor-associated and/or therapy-induced anemia leading to a reduced O2 transport capacity of the blood. This chapter discusses current information from experimental and clinical studies, which illustrates the interaction between tissue hypoxia and the phenomenon of malignant progression. Evidence, characterization and pathogenesis of tumor hypoxia, and the role of hypoxia in malignant progressionare discussed.

Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy.

Abstract: Publisher Summary Chromatin-binding proteins play a crucial part in every aspect of chromatin structure and gene expression. An experimental approach to studying the binding of protein to chromatin in living cells is the use of photobleaching methods. In these experiments a fluorescently tagged protein of interest is introduced into cells and its apparent mobility is measured as an indicator of its dynamic properties. Because nuclear proteins move passively by rapid diffusion through the nuclear space, binding of a protein dramatically affects its overall mobility and therefore the measured mobility contains information about the in vivo binding properties of a protein. Qualitative analysis of photobleaching data gives an impression of whether a protein binds stably or transiently to chromatin in vivo. In addition to the standard qualitative analysis of photobleaching experiments, kinetic modeling methods can be applied for data analysis to permit extraction of quantitative information about simple biophysical properties of chromatin proteins. This method can be used to determine the residence time of a protein on chromatin, the size of the bound and free pools, and the number of kinetically distinct fractions of a protein.

Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles.

Abstract: Publisher Summary This chapter focuses on calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles. Modern sedimentation velocity methods using solutions of the Lamm equation assume a specific model for macromolecules under study, calculate their sedimentation behavior, and fit the model to the experimental data by least-squares techniques. The chapter focuses on the currently most general direct boundary model, which is that of a continuous distribution of noninteracting proteins. It provides a robust estimation of the populations of species with different sedimentation rates, at a high resolution, and along with a description of the diffusion of the ensemble via a weight-average frictional ratio. The numerical methods for the continuous sedimentation coefficient distribution analysis are also discussed. They are applied in the software SEDFIT for analysis of experimental analytical ultracentrifugation data. Several variations are described that can give molar mass distributions, or permit the use of different prior knowledge to constrain the model, such as, for proteins with conformational changes or known molar mass, large particles, sedimenting cosolutes, compressible solvents, and others. A brief description of the experimental procedures providing the best data basis for the c(s) analysis is given followed by several examples of practical applications.

NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase), functions and pharmacogenetics.

Abstract: Publisher Summary This chapter provides an overview of NAD(P) H:quinone oxidoreductase 1 (NQO1). NQO1 is a flavoprotein that is known to catalyze two electron reduction of a broad range of substrates. Because of the many deleterious effects of quinonoid compounds, including their capacity to arylate nucleophiles and generate aggressive oxygen species via redox cycling mechanisms, removal of a quinone from a biological system by NQO1 has been considered to be a detoxification reaction. Because NQO1 is expressed at high levels throughout many human solid tumors, compounds efficiently bioactivated by NQO1 have been designed for the therapy of tumors rich in NQO1. The chapter summarizes the role of NQO1 in chemoprotection and its contrasting role in bioactivation of antitumor quinines, and both the gene and protein structure of NQO1. The purpose of this chapter is to introduce the enzyme, to review the possible functions of NQO1, and to summarize current information on polymorphic forms of NQO1. The chapter also summarizes the relevance of the common NQO1 polymorphism for chemoprotection, susceptibility to disease and cancer chemotherapy.

Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes.

Abstract: Publisher Summary This chapter examines Nrf2 and other cap “n” collar (CNC) family factors—namely Nrf1 and Nrf3, which cross-talk with the Nrf2–Keap1 regulatory pathway. The emergence of the Nrf2–Keap1 regulatory system attracts a wide range of interests from a variety of fields in medical and biological sciences. The chapter reveals, through a consensus binding sequence comparison with antioxidant responsive element (ARE), that transcription factor Nrf2 (NF-E2–related factor 2) might be an important regulatory factor, which activates transcription of these cytoprotective genes. Cellular homeostasis against oxidative stress and xenobiotic insults is accomplished by the coordinate expression of antioxidant and drug detoxifying genes. These cytoprotective genes include the phase 2 detoxification enzyme genes, such as NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs); and antioxidant genes, such as heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS). Extensive analyses of the regulatory mechanisms for the phase 2 enzyme genes revealed that the inducible expression of these enzymes is attained at the transcriptional level through a cis-acting element called ARE or electrophile responsive elements (EpRE). Examination of Nrf3-deficient mice also supports the contention that Nrf3 does not contribute to the inducible expression of the nqo1 gene in the liver and intestine. These results, taken together, argue that the Nrf2–Keap1 regulatory system is the mainstream pathway for the regulation of antioxidant and phase 2 detoxifying enzyme gene expressions.

Poisson-Boltzmann methods for biomolecular electrostatics.

Abstract: Publisher Summary This chapter presents Poisson-Boltzmann (PB) methods for biomolecular electrostatics. The understanding of electrostatic properties is a basic aspect of the investigation of biomolecular processes. Structures of proteins and other biopolymers are being determined at an increasing rate through structural genomics and other effort. The nonlinear equations obtained from the full PB equation require more specialized techniques, such as Newton methods, to determine the solution to the discretized algebraic equation. The increasing size and number of biomolecular structures have necessitated the development of new parallel finite difference and finite element methods for solving the PB equation. These parallel techniques allow users to leverage supercomputing resources to determine the electrostatic properties of single-structure calculations on large biological systems consisting of millions of atoms. In addition, advances in improving the efficiency of PB force calculations have paved the way for a new era of biomolecular dynamics simulation methods using detailed implicit solvent models. It is anticipated that PB methods will continue to play an important role in computational biology as the study of biological systems grow from macromolecular to cellular scales.

Preparation of Magnetically Labeled Cells for Cell Tracking by Magnetic Resonance Imaging

Abstract: Publisher Summary This chapter describes the preparation of magnetically labeled cells for cell tracking by magnetic resonance imaging (MRI). Magnetic resonance (MR) tracking of magnetically labeled cells following transplantation or transfusion is a rapidly evolving new field. At one hand, MR cell tracking with its excellent spatial resolution can be used as a noninvasive tool to provide unique information on the dynamics of cell movement within and from tissues in animal disease models. As for MR contrast agents, gadolinium is the most effective paramagnetic contrast agent, owing to its seven unpaired electrons, but its relaxivity is far lower than the so-called superparamagnetic iron oxides. A significant improvement of labeling of nonphagocytic cells has been achieved by linking the particles to the human immunodeficiency virus (HIV) tat peptide. The distribution of magnetic microsphere-labeled porcine mesenchymal stem cells has been studied in a swine myocardial infarct model using MRI. Control cells are run side-by-side to determine the percent increased reactive oxygen species (ROS) production in the labeled cells. A transient increase in ROS production is also observed for the labeled cells.

Cooperativity principles in protein folding.

Abstract: Knowledge of the physical driving forces in proteins is essential for understanding their structures and functions. As polymers, proteins have remarkable thermodynamic and kinetic properties. A well-known observation is that the folding and unfolding of many small single-domain proteins, of which chymotrypsin inhibitor 2 is a prime example, appear to involve only two main states—N (native) and D (denatured). These proteins’ folding/unfolding transitions are often referred to as ‘‘cooperative’’ because of their phenomenological similarity to ‘‘all-or-none’’ processes. Traditionally, only N, D, and a small number of postulated intermediate states were invoked to account for experimental protein folding data. Under such an interpretative framework, two-state folding is described by the reaction N Ð D, and different properties are ascribed to N and D to account for different proteins. Although useful, this approach does not address the microscopic origins of experimentally observed two-state–like behavior. Traditional analyses simply assume that there are a small number of conformational states. But proteins are chain molecules. Physically, it is obvious that a polymer chain can adopt many conformations, ranging from the most open to maximally compact, and all intermediate compactness in between. Thus, whether and how the multitude of conformations available to a protein may be grouped into two or more ‘‘states’’—as traditionally assumed— should be ascertained through a fundamental understanding of the effective intrachain interactions involved. In the protein literature, however, folding energetics are often discussed in terms of the sum of contactlike energies of a fully folded native structure versus that of a random-coil–like state or a certain other prespecified unfolded conformational ensemble. Such analyses have yielded important insight. But they obscure the remarkable nature of protein cooperativities. This is because cooperativity has already been presumed in these discourses by their preclusion of many a priori possible conformations—notably compact nonnative conformations—from the energetic equation. To gain a consistent understanding

Engineering antibody affinity by yeast surface display.

Abstract: Publisher Summary This chapter elaborates the engineering antibody affinity by yeast surface display (YSD). YSD is a powerful tool for engineering the affinity, specificity, and stability of antibodies, as well as other proteins. The methods for displaying an antibody on yeast, creating mutant libraries and sorting libraries for improved clones are presented. YSD involves the expression of a protein of interest on the yeast cell wall, where it can interact with proteins and small molecules in solution. The protein is expressed as a fusion to the Aga2p mating agglutinin protein, which is in turn linked by two disulfide bonds to the Aga1p protein linked covalently to the cell wall. Labeling yeast that are displaying an antibody or antibody library with a fluorescent or biotinylated antigen allows quantification of binding affinity, and enables library sorting by fluorescence-activated cell sorting. A second fluorophore conjugated to an antibody is used to detect the epitope tag C-terminal to the scFv, which allows for the normalization of expression and eliminates nondisplaying yeast from quantification. It is found that a complete cycle of mutagenesis and screening, from wild-type clone to improved mutant clone requires conservatively approximately 3–6 weeks.

The "Prochaska" microtiter plate bioassay for inducers of NQO1.

Abstract: Publisher Summary This chapter provides details of ‘‘Prochaska’’ microtiter plate bioassay for inducers of NQO1. The microtiter plate bioassay for NQO1 was first developed in the laboratory by Hans Prochaska as a rapid and direct assay of quinone reductase (NQO1; QR; DT-diaphorase) activity in cultured cells, suitable for identifying, purifying, and determining the potency of inducers of this detoxication enzyme. The Prochaska microtiter plate bioassay has been exceptionally useful for identifying and isolating inducers of NQO1 from natural sources, and in guiding the synthesis of more potent analogs of isothiocyanates, dithiolethiones, and curcuminoids. It has also been used to survey screen, and compare the potency of various plant sources in both plant breeding and molecular genetics applications. An overview of Prochaska bioassay protocol is presented in this chapter, elaborating the technical improvements of the assay. Novel findings made with the Prochaska bioassay are also described in the chapter. The chapter elaborates the and limitations of the Prochaska bioassay. Test compound matrix, dosing, and bioassay variability are also analyzed in the chapter.

Analysis of Heterogeneous Interactions

Abstract: Publisher Summary This chapter presents the mathematical formalism to describe heterogeneous equilibria, with particular attention to the relationship between the macroscopic and microscopic equilibrium constants. Identification and characterization of critical macromolecular interactions within the cell are central research problems of the postgenomic era. Quantitative methods capable of accurately defining the stoichiometry, affinity, cooperativity, and thermodynamics are required for a fundamental mechanistic understanding and to effectively target molecular interactions for therapeutic intervention. Although many biologically significant interactions involve association of identical subunits, a much larger class of binding events involves interactions of dissimilar partners. Rigorous investigation of heterogeneous interactions presents particular challenges for experimental design, data analysis, and interpretation. Heterogeneous interactions involve at least two distinct macromolecular components. The features of several biophysical methods that are commonly used to characterize heterogeneous interactions are compared. Equilibrium and velocity analytical ultracentrifugation are particularly useful as baseline methods to define assembly models and extract equilibrium parameters. The focus in this chapter is on sedimentation equilibrium and this approach is illustrated with an analysis of a nonspecific protein–nucleic acid interaction.

DNA microarray time series analysis: automated statistical assessment of circadian rhythms in gene expression patterning.

Abstract: Publisher Summary This chapter describes the use of DNA microarrays in elucidation of network dynamics of circadian gene regulation Application of this technology to circadian biology is particularly apropos given the molecular basis of intracellular circadian regulation, a system of transcriptional–translational feedback control The COSOPT algorithm evolved as an extension of the CORRCOS algorithm and has been applied to search for diurnal/circadian gene expression profiling in mammalian and Drosophila microarray time series Simulated data sets were prepared to approximate gene chip profiles from the experimental examinations of expression time series The straightforward, direct approximation by nonlinear least squares optimization of a single-frequency cosine function to the data in question produced admirable performance under conditions in which pointwise uncertainties are not considered, but produced the highest false-positive rate of all the methods when pointwise uncertainties were considered It is found that COSOPT also reports information about mean expression level and can use this information as an exclusionary criterion

AtRGS1 function in Arabidopsis thaliana.

Abstract: Arabidopsis thaliana RGS1 is a novel "regulator of G-protein signaling" (AtRGS1) protein that consists of an N-terminal seven transmembrane domain characteristic of G-protein-coupled receptors and a C-terminal RGS box. AtRGS1 modulates plant cell proliferation. Atrgs1 mutants are insensitive to glucose and less sensitive to fructose and sucrose, suggesting that sugar signaling in Arabidopsis involves AtRGS1. In addition, sugar metabolism and phosphorylation by hexokinase (HXK) are not required for AtRGS1-mediated sugar signaling, suggesting that AtRGS1 functions in a HXK-independent glucose signaling pathway.

Atomic simulations of protein folding, using the replica exchange algorithm.

Abstract: Publisher Summary This chapter discusses the atomic simulations of protein folding, using the replica exchange (RE) algorithm. In REs method, several copies or replicas of a system are simulated in parallel, only occasionally exchanging temperatures through a Monte Carlo (MC) move that maintains detailed balance. This algorithm is ideal for a large cluster of poorly communicating processors because temperature exchanges can be relatively infrequent and require little data transfer. It was adapted for use with molecular dynamics and named the replica exchange molecular dynamics (REMD) method. The essence of the REMD method is to use the molecular dynamics to generate a suitable canonical ensemble in each replica rather than MC. REMD normally occurs in coordinate and momentum space instead of just coordinate space. The typical temperature fluctuations and their relation to conformational changes are demonstrated from an actual REMD simulation of a solvated peptide. The use of REMD to study the thermodynamics of helix-coil transitions in peptides that are experimentally characterized is also elaborated.

Analysis of Circular Dichroism Data

Abstract: Publisher Summary This chapter focuses on the analysis of circular dichroism (CD) data to determine thermodynamic parameters of folding, binding constants, and estimates of secondary structure. Proteins and polypeptides have CD bands in the far ultraviolet region that arise mainly from the amides of the protein backbone and are sensitive to their conformations. Proteins have CD bands in the near ultraviolet and visible regions, which arise from aromatic amino acids and prosthetic groups. CD can be used to determine the enthalpy, entropy and midpoints, and values of unfolding/refolding transitions of a protein if they are reversible as a function of temperature or denaturant. The change in CD as a function of ligand concentration has been used to study numerous systems. It is found that if two proteins bind to each other only when they are folded and the protein complex unfolds cooperatively and reversibly to give two unfolded monomers, it is easy to determine the binding constant by determining the thermodynamics of folding of the complex compared with the thermodynamics of folding of the monomers.

Mixed-model regression analysis and dealing with interindividual differences.

Abstract: Publisher Summary This chapter considers mixed-model regression analysis, which is a specific technique for analyzing longitudinal data that properly deals with within- and between-subjects variance. The term ‘‘mixed model’’ refers to the inclusion of both fixed effects, which are model components used to define systematic relationships such as overall changes over time and/ or experimentally induced group differences; and random effects, which account for variability among subjects around the systematic relationships captured by the fixed effects. To illustrate how the mixed-model regression approach can help analyze longitudinal data with large inter-individual differences, the psychomotor vigilance data is considered from an experiment involving 88 h of total sleep deprivation, during which subjects received either sustained low-dose caffeine or placebo. The traditional repeated-measures analysis of variance (ANOVA) is applied, and it is shown that that this method is not robust against systematic interindividual variability. The data are then reanalyzed using linear mixed-model regression analysis in order to properly take into account the interindividual differences. The study concludes with an application of nonlinear mixed-model regression analysis of the data at hand, to demonstrate the considerable potential of this relatively novel statistical approach.

Chemical Structures of Inducers of Nicotinamide Quinone Oxidoreductase 1 (NQO1)

Abstract: Publisher Summary This chapter describes the chemical structures of inducers of nicotinamide quinone oxidoreductase 1 (NQO1). Some studies propose the use of activity of NQO1 as a rapid screening procedure for the identification of protectors against carcinogenesis. It is suggested that the protective function of phenolic antioxidants against tumor development in animals could be ascribed to the induction of the phase 2 response. The structure-activity studies revealed that a wide array of compounds caused coordinate induction of phase 2 enzymes and protected against carcinogenesis. The development of a cultured cell microtiter plate bioassay for NQO1 by Hans Prochaska provided a quick and highly quantitative system for evaluation of the potencies of inducers and for screening pure compounds, as well as complex mixtures such as plant extracts, for their inducer activity. Identification of the chemical signals for monofunctional phase 2 gene induction is discussed in the chapter. The chapter presents an overview of monofunctional and bifunctional inducers. Nine chemical classes of inducers are described in the chapter. Implications of the chemical structures of inducers for their mechanism of action are also explained in the chapter.

Measuring changes in tumor oxygenation.

Abstract: Publisher Summary This chapter discusses the measurement of changes in tumor oxygenation. The oxygen pressure determinations could be of great clinical value and they are also vital to many laboratory investigations of new drugs and studies of tumor development. Blood oxygen level dependent (BOLD) contrast proton nuclear magnetic resonance (NMR) facilitates rapid interrogation of vascular oxygenation, and is particularly appropriate for examining dynamic responses to interventions. Both spectroscopic and imaging approaches have been applied to tissue oxygen pressure measurements depending on the available signal to noise. It appears that uptake and distribution efficiency vary with tumor type, but in general, maximum signal is detected from the tumor periphery corresponding with regions of greater perfusion. The various aspects of fluorocarbon relaxometry using echo planar imaging for dynamic oxygen mapping (FREDOM) are elaborated. The results suggested FREDOM as a valuable tool for assessing the dynamic time course of interventions to provide clear insight into the mode of action of therapeutic approaches and aid in the high-throughput screening of new drugs, such as vascular targeting and antiangiogenic agents.

Doxorubicin cardiotoxicity and the control of iron metabolism: quinone-dependent and independent mechanisms.

Abstract: Publisher Summary This chapter reviews an experimental evidence suggesting that Doxorubicin (DOX) and other anthracyclines can act at an intracellular level, perhaps by altering the function of iron regulatory proteins (IRP) that serve to maintain low molecular weight iron pool (often referred to as the labile iron pool, LIP) within physiologic concentrations. DOX is the leading compound of a broad family of extractive or pharmaceutically engineered anticancer anthracyclines. Since its introduction in several investigational and approved chemotherapy regimens, DOX has contributed to the improved life expectancy of countless patients affected by carcinomas, sarcomas, or lymphomas. The activity of DOX against tumors is nonetheless accompanied by acute and chronic toxicities to the heart. The acute toxicity develops immediately after initiation of DOX treatment and consists of arrhythmias or hypotensive episodes, which do not represent an indication to discontinue the anthracycline regimen. The unique reactivity of DOXol toward aconitase/IRP-1 and the precise mechanisms of null protein formation clearly call for validation in clinical settings or proper animal models of in vivo cardiotoxicity. Likewise, preliminary observations that DOX-sensitive or DOX-resistant tumor cells exhibit different ratios of aconitase to IRP-1 might anticipate a possibility to target iron trafficking in sensitive cells while not affecting iron homeostasis in the heart. Research efforts are now going in this direction.

Generation and characterization of methyl-lysine histone antibodies.

Abstract: Publisher Summary This chapter summarizes the development and characterization of rabbit polyclonal antibodies named histone that are directed against the methylated H3-K9 position. It provides protocols for peptide design, rabbit immunizations, and quality controls of methyl-lysine histone antibodies, followed by their in vivo characterization using indirect IF of inter-and metaphase chromatin in wild-type (wt) and mutant mouse cells that are deficient for the Suv39h histone methyltransferases (HMTases). Histone amino-termini (tails) protrude from the nucleosome core and are subject to a variety of post-translational modifications, including acetylation (on lysine residues), phosphorylation (on serine and threonine residues), methylation (on lysine and arginine residues), ubiquitination (on lysine residues), and ADP-ribosylation (on glutamic acid residues). In addition to their structural roles, histones play important functions in the control of gene expression by regulating access to the underlying nucleosomal template. It is without doubt that the development of high-quality, position-specific methyl-lysine histone antibodies can provide important tools for the further decoding of the epigenetic information, which is in part, indexed by distinct methylation states of selective lysine residues in the histone amino-termini. A comparative analysis indicates significant discrepancies in the specificity and avidity of the available methyl-lysine histone antibodies and highlights the need for extensive quality controls, such that experimental data can be correctly interpreted despite the exquisite complexity of histone lysine methylation.

Quinone chemistry and melanogenesis.

Abstract: Publisher Summary The divergence of the melanogenic pathway occurs after the initial oxidation step that yields dopaquinone. The current analytical approach to the classification of melanins depends on the assessment of the comparative levels of degradation products that are considered characteristic of indoles and benzothiazine residues. Several physical methods for the analysis of melanins are available, including solid-state nuclear magnetic resonance (NMR) spectroscopy using 13 C and 15 N as probes, and electron paramagnetic resonance (EPR) spectroscopy. The most reliable method of quantitative analysis of eumelanins and pheomelanins relies on chemical degradation and high-performance liquid chromatography (HPLC) separation of the degradation products. The basis of this method is the yield of (1) pyrrole-2, 3, 5-tricarboxylic acid (PTCA) from 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) (2.8%) and 5, 6-dihydroxyindole (DHI) (0.03%) after permanganate oxidation as an indication of eumelanin and (2) aminohydroxyphenylalanine (AHP) from benzothiazine derivatives by hydriodic acid hydrolysis as an indicator of pheomelanin content. These products are separated by HPLC and quantified by ultraviolet (UV) detection. Three important alternative modes of reaction of ortho-quinones in biological systems can be identified: (1) addition, (2) reduction, and (3) tautomerism. These pathways are illustrated in the chapter with selected examples.

Methods in optical oxygen sensing: Protocols and critical analyses

Abstract: Publisher Summary This chapter discusses the protocols for different methods in optical oxygen sensing. It focuses on basic principles of quenched-luminescence oxygen sensing and provides general strategies for the selection of the appropriate oxygen probe, detection principle, assay format, and experimental setups. Several typical applications and assay protocols have high practical utility and employ commercial instrumentation, materials, and accessory tools— namely, (1) noninvasive measurement of the oxygen concentration in a sealed vessel, (2) monitoring of oxygen uptake rates by living organisms and cells, and (3) microplate-based screening assays for cell viability and effector action on cells. These systems are then analyzed critically to outline their characteristics features, performance characteristics, advantages and limitations, and potential biolanalytical uses. Principles of quenched-luminescence oxygen sensing and protocol on contactless monitoring of oxygen concentration in a sealed vessel using the phase-fluorimetric oxygen sensor system are also discussed.

Histone methylation: recognizing the methyl mark.

Abstract: Publisher Summary Histone N-terminal tails are subject to a variety of covalent modifications that ultimately affect chromatin structure. One such modification is N-methylation, which occurs on Lys (K) and Arg (R) amino acids. The enzymes performing these modifications are the histone N-methyl transferases (HMTs) that catalyze the transfer of a methyl group from s-adenosylmethionine (SAM) to the ɛ-amino groups of lysine and/or arginine residues within histones. HMTs can be divided into two groups—namely, (1) histone lysine N-methyltransferases and (2) histone arginine N-methyltransferases. All of the enzymes known to methylate lysines within histone N-terminal tails contain a conserved methyltransferase domain termed an SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain. In vivo methylated lysines may be found in the mono-, di-, or trimethylated State. It is becoming clear that these states of methylation have differing effects with respect to chromatin structure and transcription. Perhaps the most powerful tools in the study of histone methylation are site-specific antibodies capable of discriminating between the different methylated forms. These can then be used in numerous analyses aimed at unraveling the complexities of histone methylation. Of course, analysis of the HMTs themselves is often desirable. The chapter also describes various methods and protocols that are useful in the study of the enzymatic activity associated with histone methyltransferases.

Modeling of oscillations in endocrine networks with feedback.

Abstract: Publisher Summary This chapter focuses on the mathematical approximation of endocrine oscillations in the framework of a modeling process structured in three formal phases. The mathematical methods presented are tailored to quantitatively interpret formal endocrine networks with (delayed) feedbacks. The goal is to illustrate different conditions, under which oscillations can emerge. The formal network itself consists of nodes and conduits, and is based on a qualitative analysis of available experimental data. In the presentation, the nodes are hormone concentrations in abstract pools, in which hormones are released or synthesized, under the control of other hormones. The conduits specify how the nodes interact within the network. The quantitative analysis of the formal network is based on approximation of the rate of change of a single system node. This essentially means that the dynamics of the hormone concentration is described with a single (delayed) ODE. A single half-life elimination model is used and the control of the synthesis is expressed as a combination of sigmoid Hill functions, depending on the related nodes. The derivation of the ODE is demonstrated, along with a brief analysis of the properties of its solution to facilitate the actual determination of all system parameters.

Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors.

Abstract: Publisher Summary This chapter describes methods for the use of chromatin immunoprecipitation (ChIP) in the genome-wide identification of binding sites in mammalian cells. This approach combines the adaptation of ChIP in the enrichment of mammalian transcription factor target genes with DNA microarray technology. This method enable researchers to overcome limitations in the study of eukaryotic transcriptional regulation by allowing the identification of direct, physiological targets of DNA-binding proteins in an unbiased, genome-wide manner. Genome-wide location analysis is based on the rapid purification of specific genomic fragments associated with a particular protein followed by detection of enriched sequences withDNA microarray technology. This method is a direct, high-throughput and general approach to locate protein-DNA interactions in mammalian cells. Since most transcription factors function by binding to DNA and regulating gene expression, this method allows for the rapid, specific identification of physiologically relevant target genes and provide key insights into the molecular mechanisms through which it functions.

High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays.

Abstract: Publisher Summary Sequencing of the human genome provides a wealth of information that allows rapid advances in the understanding of gene regulation. For example, 5′ exon prediction programs provide estimates of the number of promoters in the human genome. Similarly, scanning the human genome for Myc binding sites identifies a large set of promoters that contain Myc consensus sites. Follow-up analyses of the predicted E2F and Myc target promoters using chromatin immunoprecipitation (ChIP) assays indicate that many of the putative targets are in fact bound by E2F or Myc family members in living cells. However, there are two major concerns that arise when one relies solely on a bioinformatics approach. First, many consensus sites may reside within inactive chromatin and may not be available for interaction with a factor due to inaccessibility. A major problem in studying human transcription factors is the fact that no appropriate microarrays are widely available that can be used to study intergenic DNA; that is, all commercially available microarrays are allowed only for the detection of transcribed sequences. It can be hoped that the technologies and products arising from such projects allow much greater access of all investigators to the required reagents needed for a thorough analysis of the regulatory elements encoded in the human genome. Similarly, a binding site may reside within active (euchromatin) but be inaccessible due to specific nucleosomal positioning.

Structure and mechanism of NAD[P]H:quinone acceptor oxidoreductases (NQO).

Abstract: Publisher Summary This chapter discusses about the structure and mechanism of NAD[P]H:quinone acceptor oxidoreductases (NQO). NQO are flavoenzymes that catalyze the obligatory 2-electron reduction of quinones to hydroquinones. This reaction prevents the reduction of quinones by one-electron reductases that results in the formation of reactive oxygen species (ROS), generated by redox cycling of semiquinones in the presence of molecular oxygen. In addition to its possible role in the detoxification of dietary quinones, the enzyme has been shown to catalyze the reductive activation of quinolic chemotherapeutic compounds such as mitomycins, anthracyclines, and aziridinyl-benzoquinones. This chapter analyzes the primary structure of NQO. NQO type 1 is a 274-residue enzyme expressed under oxidative or electrophilic stress among a battery of phase II enzymes. An overview of structure-based mutagenesis is presented in the chapter. The chapter presents details of monomer structure, catalytic site, FAD binding site, electron donar binding, and substrate acceptor binding. Crystallography studies of complexes of hNQO1 with chemotherapeutic compounds are also discussed in the chapter. The chapter describes computer modeling of other complexes of NQO1.

Induction of Quinone Reductase as a Primary Screen for Natural Product Anticarcinogens

Abstract: Publisher Summary This chapter discusses induction of quinone reductase as a primary screen for natural product anticarcinogens. NAD(P)H:quinone reductase (NQO1), one of the phase II drug-metabolizing enzymes, plays an important role in the mechanism of cancer chemoprevention, presumably at the initiation stage of carcinogenesis. Because induction of quinone reductase can be detected readily by using murine hepatoma Hepa 1c1c7 cells, this cell line has been used for the discovery of novel natural product anticarcinogens. A large number of natural product quinone reductase inducers are known. This chapter describes phytochemical inducers that include flavonoids, steroids, isothiocyanates, and dithiolethiones. Details of using the quinone reductase induction assay as a monitor for drug discovery, as well as a strategy for the discovery of active compounds from plant sources are provided in the chapter. Role of phase II enzymes in cancer and quinone reductase induction and cancer chemoprevention are also described in the chapter. The chapter presents an overview of in vitro quinone reductase assay. The chapter elaborates screening of medicinal plants and describes quinone reductase inducers present in edible plants.