bluuebunny/crossref_metadata_2025 huggingface dataset:
Achieving Global Justice in Health through Global Research Ethics:
Authors: Meslin∗ Eric M
Published: November 2023

International research raises challenging ethical issues for researchers, study participants, review committees, investigators, sponsors, institu- tions, and the countries in which the research is undertaken. This is so because in addition to the problems of informed consent, recruitment of participants, and protection of confidentiality, additional features such as geography, economic status, culture, and politics influence the conduct of research.1 But even more than this, we have come to appreciate that international research—in its simplest form, research undertaken by researchers from one country in another country—is inextricably linked to broader questions of international health and health care. Ruth Macklin recognizes this link and argues forcefully for the ‘‘interconnectedness of the research enterprise and global health.
Reid’s Rejection of Intentionalism
Authors: Ganson Todd Stuart
Published: November 2023

One of the central disputes, if not the central dispute, in contemporary philosophy of mind concerns the distinctive mark of the mental.According to intentionalism, a view usually traced back to Franz Brentano, intentionality is what sets mental states apart from everything else.Thomas Reid is sometimes heralded as the forerunner of the rival view that some mental states are non-intentional, not about anything.For he claims that sensation, unlike other mental states, ‘has no object’ (EIP 38, 196).
Practical Truth in Aristotle
Authors: Kenny Anthony
Published: November 2023

In 1965, having been newly appointed a philosophy tutor at Balliol, I was assigned to teach the Nicomachean Ethics to Jonathan Barnes, then in his final year of Literae Humaniores. One week I assigned as a topic ‘Practical Truth ‘, with special reference to chapter 6. 2 of the Ethics. The essay he handed in was one of the best I ever encountered in a dozen years of Greats tutoring: I was so impressed that I asked him if I could keep it. Its theme was that Aristotle had no concept of practical truth: that was a fiction foisted on him by commentators. Sadly, I can no longer find the essay among my papers, so I cannot refresh my memory of the arguments it contained. However, in Barnes ‘s recently published Locke lectures On Truth etc the index contains no entry for practical truth, and there is no reference in the Index Locorum to NE 6. 2. So I conclude that the John Locke lecturer is in agreement with the fourth-year undergraduate: there is no such thing as practical truth.
‘ As If We Were Investigating Snubness’: Aristotle On The Prospects For A Single Science Of Nature
Authors: Lennox James G
Published: November 2023

This paper began as a presentation to a Werkmeister conference in March 2003 honouring Russ Dancy. I thank Michael Ruse for the opportunity to take part in that wonderful event. I thank Myles Burnyeat, John Cooper, Mary Louise Gill, Deborah Modrak, and Steve Strange for helpful questions and comments on that occasion. I am also grateful for invitations to present these ideas from the Departments of Philosophy at the University of Texas, Austin, Ohio University, the University of Pisa, the University of Bologna’s Institute of Advanced Studies, which also provided support for work on them, and the Department of Philosophy at Concordia University. Most recently I presented some of these ideas to a seminar on Aristotle’s conception of natural science in my department, co-taught with Allan Gotthelf.
Prologue:
Authors: Scull Andrew
Published: November 2023

London, Friday, April 30, 1602. The last year of Elizabeth I’s reign. A 14-year-old girl, Mary Glover, the daughter of a well-to-do shopkeeper, left her house on Thames Street to run an errand for her mother. She soon arrived at her destination, the house of Elizabeth Jackson, a neighbor with whom she had quarreled in the past, where she sought to deliver her message. Seizing the opportunity, however, the old woman drew the teenager indoors, “locked the dore upon her,” and rained down imprecations and threats on the young girl’s head, cursing her for damaging her reputation in the community and for meddling with her own daughter’s apparel, and “wishing an evill death to light upon her.” For upwards of an hour, the old crone ranted and raved at Mary, till finally she relented and let her go, with the parting injunction that “My daughter shall have clothes when thou art dead and rotten.”
Reflexly Mad
Authors: Scull Andrew
Published: November 2023

The famous French philosopher Michel Foucault first rose to prominence by asserting that a Great Confinement of the mad was one of the defining features of the eighteenth century. Like many of his oracular pronouncements, the claim was factually mistaken, but on this occasion he was only off by a century or so. All across Western Europe and North America, the first half of the nineteenth century witnessed what David Rothman has called “the discovery of the asylum.”Differences in national cultures and political and social structures meant that the pathways to the incarceration of the insane in what was held to be a therapeutic isolation varied considerably, but the new commitment to locking up the lunatic, most often at state expense, could be seen in France and Germany just as easily as in England or the United States.
A Hysterical Circus
Authors: Scull Andrew
Published: November 2023

It was Jean-Martin Charcot (1825–93), the august Professor of Pathological Anatomy and later of Diseases of the Nervous System of the Paris Medical Faculty, the leading international neurologist of the nineteenth century, who made hysteria a spectacle and a circus. It was a scandalous circus that attracted the attention of tout Paris, one that regularly featured scantily clad women disporting themselves in unmistakably erotic cataleptic poses, or writhing and moaning in ways that mimicked orgasms on a public stage, before an understandably rapt audience—an audience soon drawn not just from the highest ranks of French society, but also from those attracted to Paris by news of these extraordinary Leçons du Mardi. The photographs of these occasions, captured in carefully staged arrangements before the supposedly objective lens of the camera and thus transmuted into indelible visual representations for a vastly greater virtual audience, have survived for later generations to inspect, and have become the iconic images of a disorder seen as at once sexual and feminine.
The Wounds of War
Authors: Scull Andrew
Published: November 2023

On August 14, 1914, the war to end all wars began. It was destined to be, in the words of the eminent German sociologist Max Weber, a "great and wonderful war."1 Civilization (whichever version of civilization one’s side was defending) would triumph, and the barbarians on the other side would be decisively defeated. And it would all be over by Christmas. Perhaps sooner: the Kaiser, seeing his troops off to the front in the first week of August, informed them: "You will be home before the leaves have fallen from the trees."
L’hystérie Morte?
Authors: Scull Andrew
Published: November 2023

Diseases disappear. Occasionally they vanish when public-health campaigns succeed in eliminating all existing outbreaks, and the pathogen responsible for the illness needs a human reservoir and vector to survive. Such was the case with smallpox, whose last remaining case was documented in 1977, and whose demise was announced by the World Health Organization in May 1980. Only the survival of the virus in biological warfare laboratories gives us reason to be concerned about the possible reappearance of an illness that once disfigured and killed on a massive scale. Perhaps polio may soon follow, for new cases of the disease now number in the low hundreds each year, compared with the ten million cases reported as recently as 1988. Occasionally, the discovery of a medical magic bullet ushers a dread disease off the stage. In the early twentieth century, between 15 and 20 per cent of male admissions to New York State̓s mental hospitals suffered from the ravages of general paresis. In its early stages, the disorder was visible only through subtle signs, overlooked by all but the trained eye—minor disturbances of gait and articulation, the unequal reaction of the pupils to light.
Introduction
Authors: Shafer-Landau Russ
Published: November 2023

Oxford Studies in Metaethics is designed to provide an annual selection of some of the best new work being done in this exciting field. I think that the articles collected here have done an excellent job in fulfilling the aims of this series. They also display, perhaps more than any previous volume in the series, the breadth of issues that nowadays falls within the boundaries of metaethics.

We begin with Andrew Sepielli’s “What To Do When You Don’t Know What To Do.” Sepielli considers a little-discussed problem in metaethics—how to behave when we are unclear about which normative considerations are most relevant to the situations we encounter. We often receive differing advice from competing normative theories, while unsure of which (if any) theory is correct.
Introduction. The boy companies
Authors: Chambers E K
Published: November 2023

The present chapter contains detailed chronicles-too often, I fear, lapsing into arid annals of performances at Court or in the provinces-of all the companies traceable in London during any year between 1558 and 1616. The household and other establishments to which the companies were attached are taken as the basis of classification. This principle is open to criticism. Certainly it has not always the advantage of presenting economic units. It is improbable that there was any continuity as regards membership between the bodies of actors successively appearing, of ten after long intervals, under the names of Sussex or Hunsdon or Derby. On the other hand, particular associations of actors can sometimes be discerned as holding together under a change of patrons.
Kan T: From Freedom To Morality
Authors: Rwin Terence I
Published: November 2023

We have seen that not all of Kant’s views on freedom depend on his acceptance of incompatibilism or on the aspects of noumenal causation that involve indeterminism. He is right to claim that his conception of freedom is not the simple compatibilist conception of Hobbes and Hume; he does not identify practical freedom with the causation of our actions by our thoughts and desires. In his view, freedom is independence of non-rational desire in our choice and motivation. The source of this independence is our capacity to act on practical reason apart from non-rational motives.
KAN T: Meta-Ethical Questions
Authors: Rwin Terence I
Published: November 2023

After examining the connexions between Kant’s moral philosophy and his metaphysics, in discussing his views on freedom and God, we may turn to his metaphysics of morals in a narrower sense; does he take moral principles to correspond to facts about reality independent of the moral beliefs of rational agents? We have already given some reasons, explicitly or implicitly, for attributing this realist view to Kant, but we need to examine these reasons more closely. The questions that arise here renew a theme that we have pursued through the chapters on Kant, about the relations between his views and traditional eudaemonism and naturalism. We have noticed some important differences, especially on the relation of the moral law to the good.
Schopenhauer
Authors: Rwin Terence I
Published: November 2023

Schopenhauer’s attitude to morality is most strongly influenced by Kant. He does not discuss any of Kant’s predecessors in any detail. He has more to say about Kant’s successors, but he believes they are misguided; he disagrees both with previous attempts to develop Kant’s general position and with Hegel’s attempt to treat problems of moral theory historically. He believes he knows which parts of Kant’s philosophy are genuine insights, and which parts are errors that need to be discarded. He attacks Kant’s rationalist conception of morality, and argues that morality is based on the feeling of compassion, not on rational principle. Here he seems close to the view of Hutcheson and (in some places) Hume.
Mill: a Revised Version of Utilitarianism
Authors: Rwin Terence I
Published: November 2023

If we approach Mill’s Utilitarianism in the light of his earlier essays, its strategy is easier to understand, and some relevant questions emerge. Mill may have begun work on it around the time of his essay on Whewell. In that essay, Mill tries a new defence of utilitarianism. In his view, Whewell’s criticisms of Paley and Bentham do not damage the basic utilitarian position. Though Mill dissociates himself from Paley, he defends Bentham, on the ground that Whewell’s criticisms attack Bentham’s strong points, but overlook Bentham’s real weaknesses (174). He offers an indirect utilitarian defence of Bentham’s appeal to utility as the test of rightness, and he supports Bentham’s view on the pleasures of animals against Whewell. He alludes to Bentham’s philistinism and disregard of character (173–4), but this criticism does not take Mill far from Bentham’s principles.
Sidgwick: Axioms of Morality
Authors: Rwin Terence I
Published: November 2023

One aspect of Sidgwick’s dialectical strategy is close to the method that Rawls calls Aristotelian. According to Rawls, its aim is to achieve ‘reflective equilibrium’; the principles we find by reflexion on common sense are those that make most sense of the common-sense judgments we start with, though our initial judgments may also be adjusted in the light of the theory. In some cases, we may achieve equilibrium either by adjusting our initial judgments to fit the theory or by the reverse process.
Green
Authors: Rwin Terence I
Published: November 2023

Green’s Prolegomena to Ethics is different from Bradley’s Ethical Studies in its structure. It begins with a long discussion (in Book i) of metaphysics, arguing for a Hegelian conclusion, and only then (in Book ii) turns to the will and (in Book iii) to moral theory. Hegelian metaphysics forms (as Green’s title for the whole book might suggest) the prolegomena to ethics. The rest of the work, however, appears, at first sight, to be less metaphysical than Ethical Studies. It appears to be intelligible without constant reference to the metaphysical argument, and it does not seem to be part of a cumulative metaphysical argument of the sort that Bradley develops.
Ross
Authors: Rwin Terence I
Published: November 2023

Ross is an objectivist in the metaphysics of morality, a cognitivist and descriptivist in semantics, and an intuitionist in epistemology. He develops his position by a close criticism of Moore. His comments often throw light on Moore and on the difficulties that his position raises. Like Moore, he discusses Sidgwick’s non-naturalism, intuitionism, and utilitarianism. But, in contrast to Moore, he revives the ‘dogmatic intuitionism’ (as Sidgwick calls it) of Price, Reid, and Whewell, and defends a non-utilitarian doctrine of the right. In contrast to Sidgwick and Moore, Ross and other 20th-century intuitionists believe that appeals to intuition support a pluralist conception of moral first principles, and so they reject Sidgwick’s defence of utilitarianism on an intuitional basis.
Existentialism
Authors: Rwin Terence I
Published: November 2023

In 1946, Sartre published a short essay, ‘The humanism of existentialism’ (literally, ‘Existentialism is a humanism’). He mentions three reasons for writing it: (1) ‘Existentialism’ was widely used, but little understood. (2) The philosophical position referred to with this term was widely believed to have distinctive implications for morality. (3) These implications were widely believed to be hostile to morality. In Sartre’s view, an accurate understanding of existentialism shows that the second popular belief is true, and the third is partly false. He admits that existentialism supports the rejection of some traditional conceptions of morality, both theistic and non-theistic; but he insists that existentialism is not hostile to morality as such, but in fact supports a distinctive outlook on morality.
Feeling Fantastic? Emotions and Appearances in Aristotle
Authors: Dow Jamie
Published: November 2023

This paper takes up the claim of some recent commentators that Aristotle, in the Rhetoric, supposed that emotional experience involved things merely appearing to the subject to be as they are represented, rather than the subject’s actually taking them to be so. Supposedly this represents a better interpretation of the Rhetoric (especially Aristotle’s use of phantasia (appearance) and cognates in connection with the emotions) and a better philosophical position for Aristotle. I shall argue that the claim has neither of these merits.
Charcoal Adsorption for Separating Bound and Free Radioligand in Radioligand Binding Assays
Authors: Strange Philip G
Published: November 2023

As discussed extensively in this book, there is a requirement for accurate, reproducible, rapid, and cheap methods for separating bound and free radioligand in radioligand receptor binding assays. In other chapters (Chapters 6, 7) methods have been described for the assay of membrane bound receptors based on collection of the bound radioligand by filtration or centrifugation of the membranes. When studying a soluble receptor (either a naturally soluble cytosolic receptor, such as a steroid hormone receptor, or a detergent solubilized receptor, formerly membrane bound) these methods are not applicable. A popular technique in such studies is the charcoal adsorption assay which relies on the ability of charcoal to selectively remove the free radioligand from the mixture of bound and free radioligand.
Analysis and Interpretation of Binding at Equilibrium
Authors: Wells James W
Published: November 2023

The first steps involved in receptor-ligand interactions are always second order binding reactions. They may, or may not, involve conformational changes of the receptor protein. The velocity of binding may be extremely rapid for neuroreceptors but can be much slower for hormone receptors. This diversity leads to a variety of experimental methods employing mostly fluorescent or radioactive ligands. Likewise, the analysis of kinetic experiments is performed on very different levels lepending on the type and quality of the data. Usually, and as a first app oximation, simple second order association reactions are assumed, which are sufficient for the analysis of most receptor-binding kinetics (1). Possible deviations from these schemes may lead to new insights into the binding mechanism and are, therefore, the focus of this chapter. It should be remembered that disproving a given mechanism is much simpler than proving another one.
Lymphokine-activated killer cells: regulation of activity
Authors: Ortaldo J R
Published: November 2023

Interleukin-2 (IL-2) has been reported to play a major role in many types of immune responses. This lymphokine induces proliferation of T lymphocytes and cells mediating NK activity, as well as production of lFN-γ; it also results in the induction oflymphokine-activated killer (LAK) cells with cytotoxic activity against fresh tumour cell preparations and NK-resistant cell lines. Cells mediating LAK activity induced by IL-2 are potent cytotoxic cells that may have an important role in the immunotherapy of solid tumours.
Tumour escape mechanisms
Authors: Guillou P J
Published: November 2023

The introduction of interleukin-2 administration, with or without adoptive cellular immunotherapy of one form or another, represents a novel approach to the immunotherapy of several different tumour types. It is disappointing, therefore, that the almost universal success of this treatment in experimental animal tumour systems2 has not been parallelled by the clinical experience of this approach. Indeed, even with those tumour types such as malignant melanoma and hypernephroma which do appear to respond to IL-2 therapy, response rates of 20-30 per cent would seem to be the best that can be expected, others such as colorectal cancer seeming to fare less well (response rates of 17 per cent or thereabouts), and yet others not responding at all. There has been no recorded correlation between the degree of lymphocytotixicity generated in response to IL-2 and clinical response. This and other data suggest that the therapeutic effects of IL-2 may be indirect. While there is doubt about such fundamental issues as the mode of action oflL-2 and the necessity or otherwise of adoptive reinfusion of in vitro activated LAK, TIL or A-LAK cells, the design of further modifications of current therapeutic protocols involving IL-2 can only be empirical. This review aims to outline some of the mechanisms which tumours may exploit to subvert the possible immunological mechanisms of tumour cell lysis induced by IL-2. The assumption is made, perhaps unjustifiably, that IL-2 activated lymphocytotoxicity plays at least some part in the therapeutic effect, or is an epiphenomenon of the final common pathway whereby the effects are brought about.
Open paper and poster sessions
Authors: Gallagher G , Guillou P
Published: November 2023

After interleukin- I, interleukin-2 (IL-2) is arguably the most important immunological mediator, given its ability to support the growth and differentiation of many cell types, in addition to its activity as a T cell growth factor. While the bias of the short paper and poster sessions was towards the role of IL-2 in cancer treatment, other important aspects of this molecule’s function were described.
Isolation and characterization of ribonucleoprotein complexes
Authors: Lamond Angus I , Sproat Brian S
Published: November 2023

Detailed analyses of a wide variety of RNA processing reactions in vitro have shown that the processing machinery usually either comprises ribonucleoprotein (RNP) complexes or is dependent upon the formation of RNP complexes during the reaction mechanism. Major experimental goals for the analysis of RNA processing mechanisms therefore include.
Analysis of pre-mRNA splicing in yeast
Authors: Newman Andrew
Published: November 2023

Many simple but powerful techniques have been developed to investigate gene expression in Saccharomyces cerevisiae, and a number of these have been employed in the analysis of RNA splicing. Test substrates can be transcribed from expression cassettes introduced into yeast by transformation or transplacement, and splicing can be monitored by RNA analysis or assay of a suitable reporter gene product. Gene disruption and inducible expression systems have been invaluable for investigating the roles of components of the splicing machinery in yeast. Many of the genes for yeast splicing factors have recently been isolated by molecular cloning, which is facilitated by the compact nature of the Saccharomyces genome and the fact that these genes are present as single copies. Splicing of yeast mRNA precursors can also be studied in vitro, since for Saccharomyces a simple method has been developed for making cell-free extracts capable of splicing synthetic pre-mRNA substrates. Such studies have shown that the splicing pathway in yeast is very similar to that of higher eukaryotes and that there is considerable conservation of structure and function between splicing factors in yeast and mammalian cells. However, yeast is an extremely valuable system for the detailed analysis of splicing since it allows some approaches which are not possible with mammalian cells, particularly the genetic selection and isolation of splicing mutants.
Role of heat-shock proteins in steroid receptor function
Authors: Pratt William B
Published: November 2023

Because of their intrinsic interest as hormone-regulated transcriptional enhancers, the steroid receptors have attracted the curiosity of people with diverse backgrounds in biology. In recent years, the tools of modern molecular biology and protein chemistry have been used to define both steroid receptor primary structure and supramolecular structure. The demonstration that steroid receptors form stable interactions with heat-shock proteins (hsp) has contributed significantly to the understanding of steroid hormone action, and the study of receptor-associated proteins has evolved as a subfield of nuclear receptor research. It should not escape the notice of the general scientific community that this structural knowledge has made the receptor proteins themselves into powerful tools for probing the mechanisms of basic biological processes, such as protein folding, protein transport, and regulation of protein function. This chapter addresses the way in which the study of hsp-mediated receptor folding is leading to the development of some concepts that may have wide application in other biological systems.
The Telomere
Authors: Kipling David
Published: November 2023

Telomeres are specialised structures at ends of linear chromosomes. They are involved in a wide range of fascinating genetic phenomena ranging from novel biochemistry to a potential role in human ageing and cancer. This is the first book of its kind to cover this exciting and rapidly expanding field of molecular biology and genetics. Its aim is to introduce the concepts and central questions involved in the study of telomeres, along with the body of experimental data.
Telomere proteins
Authors: Kipling David
Published: November 2023

Telomere function requires not only telomeric DNA sequences but also various interacting proteins such as telomerase (reviewed by Zakian 1989; Blackburn 1991; Henderson and Larson 1991). Protecting a telomere from fusion and recombination may not be a direct result of the DNA sequence but rather a result of the action of telomere binding proteins. In S. cerevisiae the RAP1 protein, in concert with a range of other factors, is speculated to form a heterochromatin-like domain at the telomere, making the double-stranded terminus inaccessible to nucleases or recombination enzymes as well as ensuring that natural chromosome ends do not activate cell cycle checkpoints which monitor DNA damage (see Chapter 2). Other telomere functions which may well reflect the activity of telomere binding proteins include physical interactions with the nuclear envelope and telomere-telomere interactions. Although almost nothing is known of the molecular nature of the interactions with the nuclear envelope (discussed in Chapter 2) the role of telomere binding proteins in the interactions between ciliate telomeres has been studied in some detail.
Lysosomes and endocytosis
Authors: Gjøen Tor , Berg Trond Olav , Berg Trond
Published: November 2023

Endocytosis is a multistep process whereby molecules are internalized into cells and subsequently transported to one of four destinations: lysosomes, the opposite side of the (epithelial) cell, storage granules, or back to the plasma membrane from which internalization took place. The various steps in the endocytic process are mediated by specialized organelles. Internalization may take place inside or outside coated pits and therefore involves two different areas of the plasma membrane (1). Clathrin-dependent internalization is a well-known process whereas the structure of the plasma membrane that mediates clathrin-independent internalization is not known. Invagination of coated pits leads to coated vesicles from which ‘primary endosomes’ are formed. Clathrin-independent endocytosis leads directly to formation of smooth-surfaced primary endosomes. Primary endosomes carry their cargo to early endosomes that consist of tubules and luminal parts. In some cells the early endosomes may gradually develop into multivesicular bodies. This ‘maturation’ involves formation of internal vesicles and loss of tubular extensions (2). The early endosomes serve as a sorting station. Some endocytic receptors are returned to the plasma membrane in ‘receptosomes’ whereas others follow a transcytotic route to the opposite side of the cells. Some receptors follow the ligand to the lysosomes. In other cells the transport from early to late endosomes may not primarily be due to a maturation process. Instead, specific carrier vesicles may bud off from early endosomes and these are subsequently moved along microtubules to late endosomes (3). The mechanism whereby receptor-ligand complexes are transported from endosomes to lysosomes is not known. Moreover, lysosomes may exist in different states of activity, and functions previously ascribed to lysosomes (e.g. proteolysis), may certainly also take place in endosomes (4). In some cells, such as macrophages, the dense lysosomes may primarily serve as storage granules while the main degradation takes place in a prelysosomal, late endosomal compartment. Transport to lysosomes may be mediated by vesicles budding off from the prelysosomal compartment. In other cells, late endosomes may fuse with lysosomes.
The membranes of the secretory and exocytic pathways
Authors: Graham John M
Published: November 2023

This chapter is concerned primarily with isolation of plasma membrane, Golgi membranes, and endoplasmic reticulum from rat liver. The first sections concentrate on the preparative isolation of these three major membrane systems using routine centrifugation techniques, and by immunoaffinity and density perturbation techniques. The later sections are concerned with the use of centrifugation to analyse the events associated with exocytic or secretory pathways in other animal cells and in yeast, which in addition to the endoplasmic reticulum, Golgi membranes, and plasma membrane, involve a number of compartments including the trans-Golgi network and a variety of vesicle populations which shuttle between membranes and membrane domains.
Isolation and purification of functionally intact mitochondria and chloroplasts from plant cells
Authors: Moore Anthony L , Whitehouse David G
Published: November 2023

The photosynthetic higher plant cell is compartmentalized into organelles such as chloroplasts, mitochondria, peroxisomes, and vacuoles and is unique amongst higher plant cells in containing two bioenergetic organelles. The success and the survival of the plant cell depends upon the co-ordination and integration of the differing metabolic activities of the different organelles. Clearly the metabolic activities and roles of the organelles will vary not only with the developmental state of that cell but also with the type of cell, e.g. mesophyll, epidermal, or bundle sheath. Photosynthetic higher plant cell systems offer the opportunity to isolate both mitochondria and chloroplasts from the same cell at a similar stage of development, a factor of some importance when attempting to present a complete picture of the bioenergetics of that cell (1, 2). There is a large diversity of plant cells each with differing characteristics and developmental patterns and a complete description of the life cycle of a plant cell will only emerge when the integrated pattern of both metabolism and development is known. An answer to this question necessitates the isolation and purification of functionally intact organelles.
RNA-protein interactions in ribosotnes
Authors: Draper David E
Published: November 2023

In recent years, the functional role of ribosomal RNAs in protein synthesis has received a great deal of attention, as domains of highly conserved structure have been identified with the decoding site, the peptidyl transferase active centre, binding sites for the elongation factors, and other functional centres of the ribosome (1). Now that a large database of ribosomal protein sequences is available, it is also apparent that many of the ribosomal proteins are as highly conserved as the rRNAs, including many of the proteins which bind directly and independently to the RNA (2-4). The basic ribosomal machinery is probably an RNA-protein complex which traces its ancestry to the beginnings of biotic evolution.
RNA-protein interactions in the splicing snRNPs
Authors: Nagai Kiyoshi , Mattaj Iain W
Published: November 2023

In 1977, several groups made the striking discovery that some protein-coding eukaryotic genes are discontinuous, being interrupted by non-coding intervening sequences now known as intrans (1-3). The entire length of these genes, including the intrans, is transcribed into pre-mRNA. The intrans are subsequently excised within the nucleus to produce continuous protein-coding mRNA sequences prior to transport to the cytoplasm. This process is called pre-mRNA splicing and its molecular mechanism has been subject to intensive research. The establishment of in vitro splicing-assay systems using either nuclear extracts of HeLa cells (a trans formed human epithelial cell line) or yeast cell extracts has allowed detailed investigation of the catalytic mechanism as well as biochemical characterization of cellular components involved in this process (4-8). Yeast genetics has also facilitated identification of protein and RNA-splicing factors and characterization of inter actions between them (6-9).
Using peptides to study RNA-protein recognition
Authors: Frankel Alan D
Published: November 2023

In biology, as in other sciences, the study of a particular system often is made simpler if the system is first reduced to small components. In structural biology, studies of large macromolecules can be hampered if, for example a molecule has too many flexible segments that prevent it from crystallizing or if it exceeds the practical size limit for nuclear magnetic resonance (NMR), currently -20-30 kDa. To overcome these problems, structural biologists often try to identify small, stable subdomains that retain the essential characteristics of the larger system but behave well under NMR conditions or crystallize. Some types of proteins, such as transcription factors, seem particularly well-suited to this approach because many are constructed from small structural and functional domains that can be studied as isolated modules (1). The structures of several DNA-binding domains were solved only after the domains were localized to short polypeptides, typically <100 amino acids, that bound to DNA with the same specificity as the intact proteins.
Footprinting and modification–interference analysis of binding sites on RNA
Authors: Merryman Chuck , F. Noller Harry
Published: November 2023

In its intracellular state RNA is usually complexed with proteins, other RNAs, and various small ligands. Chemical probing is a convenient and rapid approach that can be med to identify nucleotides that are involved in ligand binding. The two most common strategies employed in chemical probing are footprinting and modification-interference. In footprinting experiments, nucleotides that are protected from chemical attack in the presence of a bound ligand are identified. In modification-interference experiments, nucleotides whose modification prevents ligand binding are identified.
Purification Of Proteolytic Enzymes
Authors: Wilk Sherwin
Published: November 2023

Proteolytic enzymes are purified according to well established and extensively reviewed procedures for purification of proteins in general. However, there are aspects of the purification of some proteolytic enzymes which may require special attention. Many intracellular proteinases are under tight regulation to prevent uncontrolled cellular proteolysis. Tissue disruption may release potent inhibitors, which, if tightly bound, can pose serious problems in purification. In addition. unlike other proteins, proteinases are susceptible to autolysis. The first section of this chapter discusses issues that must be dealt with prior to initiating purification, and also discusses potential problems and pitfalls that are associated with the purification of this class of enzymes in particular. The second section of this chapter deals with the purification procedure in general. Particular attention is paid to affinity chromatography as it relates to proteolytic enzymes. The third section deals with optimizing the purification protocol and judging homogeneity.
Finding, Purification And Characterization Of Natural Protease Inhibitors
Authors: Nagase Hideaki , Salvesen Guy S
Published: November 2023

The activity of a proteolytic enzyme in a living organism is precisely regulated by synthesis and secretion of the enzyme, by zymogen activation and frequently by inhibition. Although some proteinase activities may be limited by very restricted substrate selectivity, or by specific cellular location {e.g. on the cell surface), most proteases are regulated by endogenous inhibitors. Thus, to understand the biological regulation of proteolysis, one must understand the role of endogenous inhibitors. In this chapter, we describe general approaches of how to discover, purify, and characterize endogenous protease inhibitors.
Using Proteinases For Edman Sequence Analysis And Peptide Mapping
Authors: Shannon John
Published: November 2023

When identifying or comparing proteins, it is usually necessary to analyze the component peptides, despite the availability of analytical techniques for intact proteins. One and two-dimensional electrophoresis, mass spectrometry, chromatography, N-and C-terminal sequencing and amino acid analysis all characterize proteins but if differences between proteins are small, the differences may be lost when analyzing a whole protein. However a difference of one amino acid between two peptides from a protein can be detected.
The DNA mobility shift assay
Authors: Dent C L , Smith, M D , Latchman D S
Published: November 2023

A knowledge of the DNA binding protein content of a cell or tissue type can be an important aid towards a more thorough understanding of the functional role of that tissue. Important processes, such as the development of an adult organism from the single-celled zygote and the maintenance of the developed structure and biochemical characteristics of tissues, are increasingly being demonstrated to be dependent on DNA binding transcription factors. The spectrum of transcription factors present determines what genes may be transcribed in a cell type, including genes that encode further transcription factors. The expression of a single gene may be determined by a number of different transcription factors, and the ratio of their concentrations may be vital, particularly if competing positive and negative factors are involved. Factors other than those that bind the DNA may be involved, such as protein cofactors, metal ions, and ligand molecules, and a further important factor is the chromatin structure of the DNA. The gene must be available for binding of factors and subsequent transcription.
Analysis of transcriptional control in RNA virus infection
Authors: Makino S
Published: November 2023

This chapter will illustrate several commonly used methods for the analysis of mRNAs and their transcriptional control by RNA viruses. I will first describe the conventional methods for characterizing mRNAs in virus-infected cells. Subsequently, methods utilizing genetically engineered virus RNA or cDNA with or without reporter genes will be described. These methods enable the analysis of cis- and trans-acting RNA sequences to be determined.
Analysis of RNA virus quasispecies
Authors: Ball Jonathan
Published: November 2023

A virus quasispecies can be thought of as a complex population of genetically related yet distinct variants (1). The term has been used to describe populations of a number of RNA viruses, e.g. human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). Although this definition of a quasispecies appears simple in theory, in practice it can be rather ambiguous (2). For example, the term has been applied to describe unrelated viruses present in different individuals, unrelated viruses arising from multiple infections within the same individual, and to genetically related but distinct variants co-circulating within the same individual and arising from a single infection. It is probably less confusing to use the term to describe the virus population co-existing within a single individual.
Reporter gene systems for the study of G protein-coupled receptor signal transduction in mammalian cells
Authors: Rees Stephen , Brown Susan , Stables Jenny
Published: November 2023

Traditional protocols for the measurement of G protein-coupled receptor (GPCR) signal transduction in mammalian cells have relied upon a number of biochemical techniques. These include the measurement of the rate of guanine nucleotide exchange at the activated G-protein alpha subunit (1), the measurement of the changes in the level of one of a plethora of intracellular second messenger metabolites, such as cAMP, calcium, or inositol phosphates (2), or the activation or inhibition of an ion channel (3). In recent years these assays have been supplemented by the development of reporter gene systems for the study of GPCR signal transduction (4-5). In this chapter we shall describe a number of such reporter gene systems, we shall outline the advantages of reporter genes over standard biochemical analyses, and we shall describe a number of protocols developed for the study of GPCR signal transduction using reporter gene systems.
Differential scanning microcalorimetry
Authors: Cooper Alan , Nutley, Margaret A , Wadood Abdul
Published: November 2023

Differential scanning calorimetry (DSC) is an experimental technique to measure the heat energy uptake that takes place in a sample during controlled increase (or decrease) in temperature. At the simplest level it may be used to determine thermal transition (‘melting’) temperatures for samples in solution, solid, or mixed phases (e.g. suspensions). But with more sensitive apparatus and more careful experimentation it may be used to determine absolute thermodynamic data for thermally-induced transitions of various kinds. Formerly this was more the realm of the dedicated specialist, but now with the ready availability of sensitive, stable, user-friendly DSC instruments, microcalorimetry has become part of the standard repertoire of methods available to the biophysical chemist for the study of macromolecular conformation and interactions in solution at reasonable concentrations. And, to the extent that thermal transitions might be affected by ligand binding, DSC can provide useful information about protein ligand binding.
Structure and energetics of anti-lysozytne antibodies
Authors: Braden Bradford C , Poljak Roberto J
Published: November 2023

The formation of specific, stable protein-protein complexes is essential to biological processes. Since such complexes are fundamental for biological function, an understanding of their structure can lead to important clues about structure-function relationships in general. Such an understanding requires detailed characterization of the three-dimensional structure of the proteins as well as the physicochemical basis of interatomic interactions at the points of intermolecular association, i.e. the interface. Advances in the techniques of protein structure determination, site-directed mutagenesis for probing amino-acid substitutions, and methods for investigating the kinetic and thermodynamic behaviour of protein-protein association have greatly enhanced our understanding of the underlying forces involved in protein-protein recognition and complex formation.
RNA polymerase II transcriptional machinery
Authors: Hampsey Michael
Published: November 2023

Transcription initiation by RNA polymerase II (RNAP II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements that include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements that either enhance or repress transcription in a gene-specific manner. The core promoter is the site of assembly of the transcription preinitiation complex (PIC), which includes RNAP II and the general transcription factors (GTFs) TATA-binding protein (TBP), TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors to affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. Biochemical characterization of the requirements for activation identified another class of transcription factors, designated coactivators or mediators, that are required for activation by many, but not all, activators. Accordingly, coactivators are neither gene-specific factors nor GTFs and include the SRBMED, TFHD, and SAGA complexes, as well as other factors.
Yeast nuclear transport
Authors: Cole Charles N
Published: November 2023

The presence of a nucleus is one of the defining characteristics of eukaryotic cells. Nuclei are bounded by a double-membraned nuclear envelope. This separation of the cytoplasm from the nucleus compartmentalizes the genetic material in an environment that is physically and biochemically distinct from the remainder of the cell. Therefore, cellular components that function within the nucleus but are produced in the cytoplasm must enter the nucleus, and RNA molecules required in the cytoplasm must exit the nucleus. The double-membraned nuclear envelope is perforated by nuclear pore complexes (NPCs), which are the only chaimels for nucleocytoplasmic transport. The importance of nudeocytoplasmic transport to eukaryotic organisms is underscored by the extremely high degree of conservation, from yeasts to mammals, in the nuclear transport machinery (for reviews see References 1-4). Rapid progress has been made in recent years in defining the proteins involved in nuclear transport and in elucidating their mechanisms of action. In addition to NPCs, nuclear transport requires specific signals in the macromolecules (cargo) to be transported, soluble receptor proteins that recognize these signals and move across the nuclear envelope with the cargo, Gsp1p /Ran, a small ras-like GTPase, and additional factors.
Mutagenesis
Authors: Pelegri Francisco
Published: November 2023

The ability to carry out genetic screens in the zebrafish is one of its most attractive features. The development of the genetic trickery of ploidy manipulation almost 20 years ago by Streisinger and co-workers underscored the genetic potential of the zebrafish and led to the identification of a number of zygotic patterning and behavioural mutations (1). More recently, the optimization of inbreeding strategies has allowed carrying out large-scale screens for zygotic mutations affecting development (2, 3). In this chapter we present the most practical methodologies used to perform mutagenesis and genetic screens in the zebrafish.
Determination of the subunit composition of native and cloned NMDA receptors
Authors: Stephenson F Anne , Hawkins Lynda M
Published: November 2023

N-Methyl-D-aspartate (NMDA) receptors are a subtype of the excitatory 1-glutamate neurotransmitter receptor family. They are fast-actingcation channels with a high permeability for Ca+ that are activated by the binding of both 1-glutamate and the co-agonist, glycine. Six genes encode homologous NMDA receptor subunits. They are the NR1 subunit gene which undergoes extensive alternative splicing to yield eight variants, NR1-1a, -1b to NR1-4a, -4b; the NR2 subunit genes NR2A-NR2D, and the NR3A subunit gene. Expression of different NMDA receptor subunit combinations in mammalian cells or in Xenopus oocytes has shown that functional NMDA receptors are formed only when the NR1 subunit is co-expressed with an NR2 subunit and that NR1/NR2 NMDA receptor subpopulations have different pharmacological and biophysical properties (reviewed in refs 1 and 2). Although correlations can be made between the properties of cloned NMDA receptors versus native receptors, expression studies do not yield definitive information with regard to the subunit composition and subunit ratios of native NMDA receptors. Since the different NMDA receptor subunits share a high degree of amino acid sequence homology, it is necessary to generate research tools which discriminate each NMDA receptor NR1 splice variant and NR2 subunit in order to determine in vivo subunit combinations.