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1804 results in Molecular biology, biochemistry, and structural biology

Page 8 of 91

  • 23 - Enzymes and Receptors

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 809-863

  • Summary

    DEFINITION AND CLASSIFICATION OF ENZYMES

    Catalytic reactions in biological processes are facilitated by two types of catalysts, enzymes and ribozymes. Whereas enzymes are proteins, ribozymes consist of ribonucleic acid (RNA). Most enzymes are much larger than the substrates they process, but this is not a requirement (for example, restriction enzymes that cleave DNA). The catalytic features of enzymes arise through a particular three-dimensional arrangement of functional groups in a small number of amino acids (see Section 23.4.2) in the active site of the enzyme. The geometrical arrangement of such groups enables productive interactions with the bound substrate and leads to formation of a transition state for which the energy barrier (activation energy) is significantly reduced as compared to the non-catalysed reaction (see Figure 23.1). Consequently, the reaction rate is increased by several orders of magnitude relative to the non-catalysed reaction. Importantly, enzymes do not alter the position of equilibrium of the reversible reactions they catalyse, rather they accelerate establishment of the position of equilibrium for the reaction.

    Many enzymes are the key players in metabolic or signalling pathways. In these coordinated pathways, enzymes are collectively responsible for maintaining the metabolic needs of cells under varying physiological conditions (Section 23.5). Through their individual catalytic activities, they control the rate of a particular metabolic or signalling pathway. A range of regulatory mechanisms operates to allow short-, medium- and long-term changes in activity (Section 23.5.1). Therefore, the over- or under-expression of an enzyme can lead to cell dysfunction, which may manifest itself as a particular disease. Hence, enzymes have been the most important target in the development of therapeutics, and typical drugs for the treatment of pathological conditions act as inhibitors of particular enzymes (Sections 23.5.3). Other clinically relevant applications include monitoring of enzyme levels in assessment of disease states. For example, damage to the heart muscle as a result of oxygen deprivation following a heart attack results in the release of cellular enzymes into extra-cellular fluids and eventually into the blood. Such release can be monitored to aid diagnosis of the organ damage and to make a prognosis for the patient's future recovery (Section 10.3).

Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
  • 8th edition
  • Edited by Andreas Hofmann, Samuel Clokie
  • Published online:
    06 August 2018
    Print publication:
    19 April 2018
  • Bringing this best-selling textbook right up to date, the new edition uniquely integrates the theories and methods that drive the fields of biology, biotechnology and medicine, comprehensively covering both the techniques students will encounter in lab classes and those that underpin current key advances and discoveries. The contents have been updated to include both traditional and cutting-edge techniques most commonly used in current life science research. Emphasis is placed on understanding the theory behind the techniques, as well as analysis of the resulting data. New chapters cover proteomics, genomics, metabolomics, and bioinformatics, as well as data analysis and visualisation. Using accessible language to describe concepts and methods, and with a wealth of new in-text worked examples to challenge students' understanding, this textbook provides an essential guide to the key techniques used in current bioscience research.

  • 16 - Fundamentals of Bioinformatics

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 559-598

  • Summary

    INTRODUCTION

    Over the last decade, we have witnessed a massive expansion in demand for and access to low-cost high-throughput sequencing of nucleic acids, which can be predominantly attributed to the advent and establishment of so-called next-generation sequencing technologies (NGS; see Section 20.2.2). The availability and progressively decreasing costs of such technologies has been accompanied by an ever-increasing number of nucleic acid and protein sequences being deposited in public repositories and, in turn, by the need to draw biologically meaningful information or interpretations from these data. As a consequence, the discipline of bioinformatics has become instrumental in many areas of biology and, in particular, molecular biology.

    Bioinformatics can be defined as a ‘fusion’ of biology and informatics, which includes applied mathematics, computer sciences, information technology and statistics. This multi-disciplinary field of research includes two major components, one aimed at developing computational tools and algorithms to facilitate storage, analysis and manipulation of sequence data, and one aimed at applying such tools to the discovery of new biological insights on the organism(s) under consideration. Researchers involved in the field of bioinformatics comprise both algorithmor software- developers and end-users. While the main interest of the first group lies in writing sequence analysis programs and tools (programming, often called ‘ coding’), the second wishes to apply these tools to answer questions of biological relevance. Within this latter group, experienced end-users often download and maintain programs on their private personal computers or servers, analyse a number of sequences (thousands to millions) simultaneously, have a working knowledge of programming languages and are therefore skilled in the use of command-linebased software. On the other hand, occasional users mainly deal with a limited number of sequences and thus prefer the use of ‘user-friendly’, web-server-based tools, which often offer a reduced set of options and a limited capacity when compared with the corresponding downloadable software packages. This chapter intends to provide an overview of the basic methods and bioinformatics resources available for the analysis of nucleic acids and protein sequences, and is primarily addressed to occasional users. For details on bioinformatic analyses of largescale sequence datasets, such as those generated by NGS technologies, the reader is referred to Chapter 20, while programmatic or script access to programs will require more advanced programming skills, for example using the Python language (see Chapter 18).

  • 15 - Mass Spectrometric Techniques

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 535-558

  • Summary

    INTRODUCTION

    Mass spectrometry (MS) is an extremely valuable analytical technique in which the molecules in a test sample are converted to gaseous ions that are subsequently separated in a mass analyser according to their mass-to-charge (m / z) ratio and then detected. The mass spectrum is a plot of the relative abundances of the ions at each m / z ratio. Note that it is the mass-to-charge ratio (m / z), and not the actual mass, that is measured. For example, if a biomolecule is ionised in positive ion mode, the instrument measures the m / z after the addition of one proton (i.e. 1.0072772984 Da for exact mass or 1.0078 Da for average mass). Similarly, for a biomolecule ionised in negative ion mode, an m / z after the loss of one proton is measured.

    The essential features of all mass spectrometers are:

  • • G eneration of ions in the gas phase

  • • S eparation of ions in a mass analyser

  • • D etection of each species of a particular m / z ratio.

    • Several techniques exist to generate ions and are discussed below. Of these, the development of electrospray ionisation (ESI; Section 15.2.4) and matrix-assisted laser desorption ionisation (MALDI; Section 15.2.5), has effectively expanded the detectable mass range, enabling the measurement of almost any biomolecule. Mass analysers separate ions by use of either a magnetic or an electric field (Section 15.3); detectors produce a measurable signal in the form of either a voltage or a current that can be transformed by a computer into data to be analysed (Section 15.4). The symbol M r is used to designate relative molecular mass. As a relative measure, M r has no units.

    The treatment of mass spectrometry in this chapter will be rather non-mathematical and non-technical. Mass spectrometry has a wide array of applications, including drug discovery and the sciences of proteomics (Chapter 21) and metabolomics (Chapter 22).

    This chapter will focus on the fundamental principles of mass spectrometry. The intention is to give an overview of the different types of instrumentation that are available and discussion of their applications, complementary techniques and the advantages/disadvantages of each system. Sample preparation and data analysis will also be covered. A further reading list is provided, covering more technical and mathematical aspects of mass spectrometry.

  • Foreword to the Eighth Edition

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp xiii-xiv

  • Summary

    When the first edition of our book was published in 1975, biochemistry was emerging as a new discipline that had the potential to unify the previously divergent -ologies that constituted the biological sciences. It placed emphasis on the understanding of the structure, function and expression of individual proteins and their related genes, and relied on the application of analytical techniques such as electrophoresis, chromatography and various forms of spectrometry. In the ensuing 40 years the completion of the Human Genome Project has confirmed the central role of DNA in all of the activities of individual cells and the emergence of molecular biology as the means of understanding complex biological processes. This in turn has led to the new disciplines of bioinformatics, chemoinformatics, proteomics and metabolomics. The succeeding six editions of our book have attempted to reflect this evolution of biochemistry as a unifying discipline. All editions have placed emphasis on the experimental techniques that undergraduates can expect to encounter during the course of their university studies and to this end we have been grateful for the excellent feedback we have received from the users of the book.

    The point has now been reached where we believed that it was appropriate for us to hand over the direction and academic balance of future editions to a new editorial team and to this end we are delighted that Andreas Hofmann and Samuel Clokie have agreed to take on the role. We wish them well and look forward to the continuing success of the book.

  • 12 - Centrifugation and Ultracentrifugation

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 424-453

  • Summary

    INTRODUCTION

    Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. The smaller the particles, the higher the g-forces (see next section) required for the separation. It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and, with higher g-force instruments or ‘ultra’-centrifuges (up to 60 000 revolutions per minute corresponding to ~ 200 000×g) isolated macromolecules such as proteins or nucleic acids. Such high-speed devices require a vacuum to avoid overheating of samples. The development of the first analytical ultracentrifuge – with a specially designed optical system for monitoring and recording the sedimentation process – by Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades. Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all invasive subcellular studies. While analytical ultracentrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies, preparative centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest.

    Most undergraduate students will be exposed to preparative centrifugation protocols during practical classes and might also experience a demonstration of analytical centrifugation techniques. This chapter is accordingly divided into a short introduction into the theoretical background of sedimentation, an overview of practical aspects of using centrifuges in the biochemical laboratory, an outline of preparative centrifugation and a description of the usefulness of ultracentrifugation techniques in the biochemical characterisation of macromolecules. To aid in the understanding of the basic principles of centrifugation, the general designs of various rotors and separation processes are diagrammatically represented. Often, the learning process of undergraduate students is hampered by the lack of a proper linkage between theoretical knowledge and practical applications. To overcome this problem, the description of preparative centrifugation techniques is accompanied by an explanatory flow chart and the detailed discussion of the subcellular fractionation protocol for a specific tissue preparation.

  • 9 - Radioisotope Techniques

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 313-345

  • Summary

    WHY USE A RADIOISOTOPE?

    When researchers contemplate using a radioactive compound there are several things they have to consider. First and foremost, they must ask the question: is a radioisotope necessary or is there another way to achieve the objectives? The reason for this is that use of radioisotopes is governed by very strict legislation. The rules are based on the premise that radioactivity is potentially unsafe (if handled incorrectly) and should therefore only be used if there are no alternatives. Then, once it is decided that there is no alternative, the safest way of carrying out the work needs to be planned. Essentially this means using the safest isotope and the smallest amount possible. But why do we use radioisotopes in the first place? First, it is possible to detect radioactivity with exquisite sensitivity. This means that, for example, the progress of an element through a metabolic pathway or in the body of a plant or animal can be followed relatively easily. Very small amounts of a radioactive molecule are needed, and detection methods are well established. Second, it is possible to follow what happens in time. Imagine a metabolic pathway such as carbon dioxide fixation (the Calvin cycle). All the metabolites in the cycle are present simultaneously; so a good way to establish the order of the metabolism is to add a radioactive molecule (in this example, 14 C-labelled carbon dioxide in the form of sodium bicarbonate) and see what happens to it by extracting the metabolites from the plant and identifying the radioactive ones. Third, it is possible to trace what happens to individual atoms in a pathway. This is done, for example, by creating compounds with a particular isotope in specific locations in the molecule. Fourth, we can identify a part or end of a molecule, and follow reactions very precisely. This has been very useful in molecular biology, where it is often necessary to label one end of a DNA molecule (e.g. for techniques such as gel mobility shift assay or DNA footprinting, methods for investigating sequence-specific DNA protein binding), or immunochemical diagnostics; see Section 7.2.9).

  • 9 - Salvation through Reductionism: The National Institute of Mental Health and the Return to Biological Psychiatry

  • Edited by Stephen T. Casper, Delia Gavrus
  • Book:
    The History of the Brain and Mind Sciences
    Published by:
    Boydell & Brewer
    Published online:
    26 May 2021
    Print publication:
    01 June 2017, pp 229-256

  • Summary

    It has become our mantra at NIMH that mental disorders can be addressed as disorders of brain circuits.

    —Thomas Insel, director, National Institute of Mental Health, 2010

    The guideline of the institute, to quote the law, is “to provide support of research on the etiology, diagnosis, treatment, prevention, and control of mental illness and the promotion of mental health.” This broad mandate contains no restrictions on approaches or disciplines.

    —Article encouraging submissions by anthropologists to the National Institute of Mental Health, 1963

    Since its founding in 1949, the National Institute of Mental Health (NIMH) has been a major force in shaping America's understanding of mental illness, ushering the shift back toward a biological perspective. This move was not simply the logical outcome of scientific progress, but rather an admission of failure to meet the nation's growing mental health problems and a hope for salvation through reductionism. To facilitate this shift, NIMH modeled, underwrote, and broadcast the new biological revolution in psychiatry. Fulfilling its mission to disseminate mental health information, starting in the 1950s NIMH disseminated press releases, radio spots, television programs, and most recently websites and podcasts to broadcast the message that mental illnesses are fundamentally physical ailments whose cure depends on scientific medicine. The messages NIMH conveyed about how mental illness should be approached had a dramatic disciplinary de facto effect; such messages helped steer psychiatry away from psychosocial and psychodynamic explanations and toward a neurological picture of the mind and mental disturbances.

    At the outset, it is prudent to bear in mind Daniel Breslau's cautionary words: “The dramatic reversal of the relationship between the declining psychodynamic orientation and the ascendant biological and nosological approach is inexplicable with reference only to the rhetoric of science.” Although this claim is undoubtedly true, it is the argument of this chapter that rhetorical strategies nevertheless did significantly help advance the cause of biological psychiatry. Ostensibly just reporting the latest research, NIMH's messages increasingly suggested that brain science will solve the problem of mental illness.

  • 1 - “We Are Veritable Animals”: The Nineteenth-Century Paris Menagerie as a Site for the Science of Intelligence

  • Edited by Stephen T. Casper, Delia Gavrus
  • Book:
    The History of the Brain and Mind Sciences
    Published by:
    Boydell & Brewer
    Published online:
    26 May 2021
    Print publication:
    01 June 2017, pp 25-47

  • Summary

    Introduction

    In his paper “The Mental Hospital and the Zoological Garden” (1965), the psychiatrist and historian Henry E. Ellenberger drew a provocative parallel between the histories of these two institutions. He pointed out a chronological synchronicity in the emergence of the appearance of a recognizable modern version of the asylum and the zoo: both were products of the era of the French Revolution and its aftermath. There was even a certain overlap in the personnel who oversaw these developments. Philippe Pinel (1745–1826), best known as an alienist famed for striking off the chains of the inmates at Bicêtre, was a member of the commission that in 1792 drew up a blueprint for a new Menagerie in the Jardin des Plantes in Paris.

    Ellenberger, moreover, discerned a certain structural homology between the asylum and the zoo. One shared feature was a category that Ellenberger designates “Authorities.” In the case of the asylum, these Authorities were the alienists who provided medical direction of the establishment. In the Paris Menagerie, the zoologist possessed a comparable status. Corresponding to the asylum patients were the animals housed in the Menagerie. Both may be seen as subject to—perhaps even constituted by—the gaze of the presiding Authority. The asylum and menagerie also shared a third, often overlooked yet significant, category: the lay staff who were responsible for the daily running of these institutions. The care and control of asylum patients was largely delegated to attendants drawn from a variety of backgrounds but with little or any in the way of specialist training. Still less is known about the keepers who performed a similar indispensable role in the menagerie, seeing to the daily needs of the animals.

    Ellenberger also parenthetically notes that in respect of the role of the public there is, however, a divergence. While the public was largely excluded from the nineteenth-century asylum—in contrast to the voyeurism of earlier epochs—part of the rationale for the reformed post-revolutionary Menagerie was that it should be open to the citizenry. Such a populist ethos was in explicit contrast to the elitism of the old royal menagerie at Versailles.

The History of the Brain and Mind Sciences
  • Technique, Technology, Therapy
  • Edited by Stephen T. Casper, Delia Gavrus
  • Published by:
    Boydell & Brewer
    Published online:
    26 May 2021
    Print publication:
    01 June 2017
  • How did epidemics, zoos, German exiles, methamphetamine, disgruntled technicians, modern bureaucracy, museums, and whipping cream shape the emergence of modern neuroscience?

  • 5 - Epilepsy and the Laboratory Technician: Technique in Histology and Fiction

  • Edited by Stephen T. Casper, Delia Gavrus
  • Book:
    The History of the Brain and Mind Sciences
    Published by:
    Boydell & Brewer
    Published online:
    26 May 2021
    Print publication:
    01 June 2017, pp 136-163

  • Summary

    That's a popular misconception about science, Nett. But, Science, on the contrary, is very humanly warm and frail. It makes very human mistakes and has to change its views often so that, like the rest of humanity, it can go blundering steadily forward.

    —Edward Dockrill, mid-1930s

    In the interwar period, Gerry Armstrong, a technician working at a prestigious Canadian hospital, spoke passionately against the invisibility of laboratory workers. Dependent on the patronage and research funds of elite doctors and scientists, the technicians who mastered staining techniques received no formal acknowledgment for their scientific endeavors. “I’m paid to produce results,” Armstrong reflected bitterly. “Whoever pays me has the social and economic right to take what they are paying for.” This invisible labor formed the “stepping stones for somebody else's rise to fame,” Armstrong lamented, while technicians were too worried to demand public recognition for fear of losing their jobs and “falling into the gutter.” Mustering all the courage and frustration he felt, Armstrong did confront his superiors on several occasions, receiving either blank incomprehension or hollow placations.

    Gerry Armstrong did not actually exist. He was a character conjured up by Edward Dockrill, a real-life laboratory technician who wrote a manuscript titled “The Means Are Nothing,” a complex, nearly four-hundred-page novel that never saw the light of print. Set mostly in the fictional bilingual town of Tasville—a lightly disguised Montreal—the novel is a bildungsroman that slowly unspools the young hero's life as he arrives to North America from England with the explicit goal of proving his worth. Armstrong is dazzled at first by the hospital and its chief, Dr. Meadowes, and he throws himself passionately into his work in the pathology laboratory. Soon, however, disillusion sets in. After many nights of exhausting labor to perfect histological techniques, he watches helplessly as one of the senior doctors appropriates one of his own breakthroughs, publishing it to great acclaim and giving Armstrong no credit, despite having promised to share the laurels of scientific discovery. The story's climax stages an explosive encounter between Armstrong and Meadowes, the technician accusing the chief and his acolytes of “becom[ing] famous with work you never did, paid for by money you never owned.”

Page 8 of 91