Library Science

A central challenge of the post-genomic era is to understand how the 30,000 to 40,000 unique genes in the human genome are selectively expressed or silenced to coordinate cellular growth and differentiation. The packaging of eukaryotic genomes in a complex of DNA, histones, and nonhistone proteins called chromatin provides a surprisingly sophisticated system that plays a critical role in controlling the flow of genetic information. This packaging system has evolved to index our genomes such that certain genes become readily accessible to the transcription machinery, while other genes are reversibly silenced. Moreover, chromatin-based mechanisms of gene regulation, often involving domains of covalent modifications of DNA and histones, can be inherited from one generation to the next. The heritability of chromatin states in the absence of DNA mutation has contributed greatly to the current excitement in the field of epigenetics.

The past 5 years have witnessed an explosion of new research on chromatin biology and biochemistry. Chromatin structure and function are now widely recognized as being critical to regulating gene expression, maintaining genomic stability, and ensuring faithful chromosome transmission. Moreover, links between chromatin metabolism and disease are beginning to emerge. The identification of altered DNA methylation and histone acetylase activity in human cancers, the use of histone deacetylase inhibitors in the treatment of leukemia, and the tumor suppressor activities of ATP-dependent chromatin remodeling enzymes are examples that likely represent just the tip of the iceberg.This 3-volume set of Methods in Enzymology provides nearly one hundred procedures covering the full range of tools—bioinformatics, structural biology, biophysics, biochemistry, genetics, and cell biology—employed in chromatin research. Volume 375 includes a histone database, methods for preparation of histones, histone variants, modified histones and defined chromatin segments, protocols for nucleosome reconstitution and analysis, and cytological methods for imaging chromatin functions in vivo. Volume 376 includes electron microscopy and biophysical protocols for visualizing chromatin and detecting chromatin interactions, enzymological assays for histone modifying enzymes, and immunochemical protocols for the in situ detection of histone modifications and chromatin proteins. Volume 377 includes genetic assays of histones and chromatin regulators, methods for the preparation and analysis of histone modifying and ATP-dependent chromatin remodeling enzymes, and assays for transcription and DNA repair on chromatin templates. We are exceedingly grateful to the very large number of colleagues representing the field’s leading laboratories, who have taken the time and effort to make their technical expertise available in this series.

As such, the field is attracting new investigators who enter with little first hand experience with the standard assays used to dissect chromatin structure and function. In addition, even seasoned veterans are overwhelmed by the rapid introduction of new chromatin technologies. Accordingly, we sought to bring together a useful ‘‘go-to’’ set of chromatin-based methods that would update and complement two previous publications in this series, Volume 170 (Nucleosomes) and Volume 304 (Chromatin). While many of the classic protocols in those volumes remain as timely now as when they were written, it is our hope the present series will fill in the gaps for the next several years.

Ed. Carl Wu, C. David Allis

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One of the most intriguing problems in biological energetics is that of cooperativity. From the discovery of cooperativity and allostery in hemoglobin 100 years ago (Bohr et al., 1904)1 to the characterization of cooperativity in a myriad of processes in modern times (i.e., transport, catalysis, signaling, assembly, folding), the molecular mechanisms by which energy is transferred from one part of a macromolecule to another continues to challenge us. Of course, the problem has many layers, as a molecule as ‘‘simple’’ and familiar as hemoglobin can simultaneously sense the chemical potential of each physiological ligand and adjust its interactions with the others accordingly. Ironically, the very allosteric intermediates that hold the structural and energetic secrets of cooperativity are the same whose populations are suppressed and, in many instances, largely obscured by the nature of cooperativity itself. Thus, innovative methodologies and techniques have been developed to address cooperative systems, many of which are presented in this volume Energetics of Biological Macromolecules Part D and its companion volume, Part E. The reader will observe remarkable similarities among the wide range of experimental strategies employed, attesting to fundamental issues inherent in all cooperative systems.

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One of the most intriguing problems in biological energetics is that of cooperativity. From the discovery of cooperativity and allostery in hemoglobin 100 years ago (Bohr et al., 1904)1 to the characterization of cooperativity in a myriad of processes in modern times (i.e., transport, catalysis, signaling, assembly, folding), the molecular mechanisms by which energy is transferred from one part of a macromolecule to another continues to challenge us. Of course, the problem has many layers, as a molecule as ‘‘simple’’ and familiar as hemoglobin can simultaneously sense the chemical potential of each physiological ligand and adjust its interactions with the others accordingly. Ironically, the very allosteric intermediates that hold the structural and energetic secrets of cooperativity are the same whose populations are suppressed and, in many instances, largely obscured by the nature of cooperativity itself. Thus, innovative methodologies and techniques have been developed to address cooperative systems, many of which are presented in this volume Energetics of Biological Macromolecules Part E and its companion volume, Part D. The reader will observe remarkable similarities among the wide range of experimental strategies employed, attesting to fundamental issues inherent in all cooperative systems.

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The speed of laboratory computers doubles every year or two. As a consequence, complex and time-consuming data analysis methods that were prohibitively slow a few years ago can now be routinely employed. Examples of such methods within this volume include wavelets, transfer functions, inverse convolutions, robust fitting, moment analysis, maximum-entropy, and singular value decomposition. There are also many new and exciting approaches for modeling and prediction of biologically relevant molecules such as proteins, lipid bilayers, and ion channels.There is also an interesting trend in the educational background of new biomedical researchers over the last few years. For example, three of the authors in this volume are Ph.D. mathematicians who have faculty appointments in the School of Medicine at the University of Virginia. The combination of faster computers and more quantitatively oriented biomedical researchers has yielded new and more precise methods for the analysis of biomedical data. These better analyses have enhanced the conclusions that can be drawn from biomedical data and they have changed the way that the experiments are designed and performed. This is our fifth ‘‘Numerical Computer Methods’’ volume for Methods in Enzymology. The aim of volumes 210, 240, 321, 383, and the present volume is to inform biomedical researchers about some of these recent applications of modern data analysis and simulation methods as applied to biomedical research.

Ed. Michael L. Johnson and Ludwig Brand

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Developments in genomics and proteomics rapidly generated focus on new -omics, particularly metabolomics and phenomics. Quinones, hydroquinones, semiquinones and their metabolites are naturally occurring compounds that serve as wonderful examples for this new paradigm of interdigitating ,-omics. In addition to a role as substrates and products in metabolism, quinone compounds are intermediates in many pathways of gene regulation, enzyme protein induction, feedback control, and waste product elimination. Quinones play a pivotal role in energy metabolism (Peter Mitchell’s proton-motive, Q cycle’), many other key processes, and even in chemotherapy where redox cycling drugs are utilized.

The present volume of Methods in Enzymology on quinones and quinone enzymes serves to bring together current methods and concepts on this topic. It focuses on the role in the so-called Phase II of drug metabolism (xenobiotics), but include aspects on Phase I (CYP, cytochromes P-450) and Phase III (transport systems) as well. This volume of Methods in Enzymology, Part B addresses mitochondrial ubiquinone and reductases, anticancer quinones, and the role of quinone reductases in chemoprevention and nutrition, as well as the role of quinones in age-related diseases, whereas (Part A) focused on quinones and quinone enzymes in terms of coenzyme Q (detection and quinone reductases), plasma membrane quinone reductases, and the role of quinones in cellular signaling and modulation of gene expression. Phase II Enzymes, Part C, will be focusing on glutathione, glutathione S-transferases, and other conjugation enzymes.

The enzyme, NAD(P)H:quinone oxidoreductase, is the subject of a major section in this volume. This enzyme, discovered in 1958 in Stockholm by Lars Ernster, and named DT-Diaphorase by him, has multiple roles, some of which were only recently discovered.

Human polymorphisms exist in these enzymes that relate to variations in cancer risk, and enzymes targeted by quinones are being investigated. Modern methods in assaying quinone reactions and, indeed, various quinones themselves, are also included in this volume.

Following its discovery in 1957, ubiquinone (coenzyme Q10) as a major naturally occurring quinone became a highlight of scientific interest and an established role in mitochondrial electron transport by Frederick Crane. Fundamental contributions were made by Karl Folkers on its supplemental use for health benefits in disease prevention and by Andre? s O.M. Stoppani, a pioneer of Argentinian biochemistry, in utilizing quinones for the treatment of Chagas disease.

Ed. Helmut Sies and Lester Packer

quinones-and-quinone-enzymes-part-b.pdf

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The speed of laboratory computers doubles every year or two. As a consequence, complex and time-consuming data analysis methods, that were prohibitively slow a few years ago, can now be routinely employed. Examples of such methods within this volume include wavelets, transfer functions, inverse convolutions, robust fitting, moment analysis, maximum-entropy, and singular value decomposition. There are also many new and exciting approaches for modeling and prediction of biologically relevant molecules such as proteins, lipid bilayers, and ion channels.

There is also an interesting trend in the educational background of new biomedical researchers over the last few years. For example, three of the authors in this volume are Ph.D. mathematicians who have faculty appointments in the School of Medicine at the University of Virginia. The combination of faster computers and more quantitatively oriented biomedical researchers has yielded new and more precise methods for the analysis of biomedical data. These better analyses have enhanced the conclusions that can be drawn from biomedical data and they have changed the way that the experiments are designed and performed. This is our fourth ‘‘Numerical Computer Methods’’ volume for Methods in Enzymology. The aim of volumes 210, 240, 321, and the present volume is to inform biomedical researchers about some of these recent applications of modern data analysis and simulation methods as applied to biomedical research.

Authors: Ludwig Brand and Michael L. Johnson

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As these volumes were being completed, American Paul C. Lauterbur and Briton Sir Peter Mansfield won the 2003 Nobel Prize for medicine for discoveries leading to the development of MRI.

The Washington Post story on October 6, 2003 announced the accolade, noting: “Magnetic resonance imaging, or MRI, has become a routine method for medical diagnosis and treatment. It is used to examine almost all organs without need for surgery, but is especially valuable for detailed examination of the brain and spinal cord.” Unfortunately, the article overlooked the growing usefulness of this technique in basic research.

Authors have been selected based on research contributions in the area about which they have written and based on their ability to describe their methodological contributions in a clear and reproducible way. They have been encouraged to make use of graphics and comparisons to other methods, and to provide tricks and approaches that make it possible to adapt methods to other systems. MRI, along with other imaging methods, has made it possible to glance inside the living system. For patients, this may obviate the need for surgery; for researchers, it becomes a noninvasive method that enables the model systems to continue “doing what they do” without being disturbed. The value and potential of these techniques is enormous, and that is why these once clinical methods are finding their way to the laboratory.

Ed. P. Michael Conn

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As these volumes were being completed, American Paul C. Lauterbur and Briton Sir Peter Mansfield won the 2003 Nobel Prize for medicine for discoveries leading to the development of MRI.

The Washington Post story on October 6, 2003 announced the accolade, noting: “Magnetic resonance imaging, or MRI, has become a routine method for medical diagnosis and treatment. It is used to examine almost all organs without need for surgery, but is especially valuable for detailed examination of the brain and spinal cord.” Unfortunately, the article overlooked the growing usefulness of this technique in basic research. MRI, along with other imaging methods, has made it possible to glance inside the living system. For patients, this may obviate the need for surgery; for researchers, it becomes a noninvasive method that enables the model systems to continue “doing what they do” without being disturbed. The value and potential of these techniques is enormous, and that is why these once clinical methods are finding their way to the laboratory.

Authors have been selected based on research contributions in the area about which they have written and based on their ability to describe their methodological contributions in a clear and reproducible way. They have been encouraged to make use of graphics and comparisons to other methods, and to provide tricks and approaches that make it possible to adapt methods to other systems. 

Authors: P. Michael Conn

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