bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structure And Conduct Of Theatres
Authors: Chambers E K
Published: November 2023

It remains to give some account of the iconographical material available. Of four representations of the interiors of play-houses, the only one of early date (c. 1596) is (a) Arend van Buchell’s copy of a drawing by Johannes de Witt of the Swan, published in 1888 by Gaedertz and in more accurate facsimile by Wheatley (vide supra). The other three are Caroline. (b) A small engraving in a compartment of the title-page of W. Alabaster, Roxana (1632), may be taken as representing a type of academic stage, as the play was at Trinity, Cambridge, c. 1592. (c) A very similar engraving in the title-page of N. Richards, Messallina (1640), if it represents a specific stage at all, is less likely to represent the second Fortune, as suggested by Skemp in his edition of the play, or the Red Bull, as suggested by Albright, 45, than Salisbury Court, where it is clear from Murray, i. 279, that most of the career of the Revels company, by whom it was produced, was spent.
Staging At Court
Authors: Chambers E K
Published: November 2023

WE come now to the problems, reserved from treatment in the foregoing chapter, of scenic background. What sort of setting did the types of theatre described afford for the plots, of ten complicated, and the range of incident, so extra-ordinarily’ wide, which we find in Elizabethan drama ?. No subject in literary history has been more often or more minutely discussed, during the quarter of a century since the Swan drawing was discovered, and much valuable spade-work has been done, not merely in the collecting and marshalling of external evidence, but also in the interpretation of this in the light of an analysis of the action of plays and of the stage-directions by which these are accompanied.1 Some points have emerged clearly enough ; and if on others there is still room for controversy, this may be partly due to the fact that external and internal evidence, when put together, have proved inadequate, and partly also to certain defects of method into which some of the researchers have fallen. To start from the assumption of a ‘typical Shakespearian stage ‘ is not perhaps the best way of approaching an investigation which covers the practices of thirty or forty playing companies, in a score of theatres, over a period of not much less than a century. It is true that, in view of the constant shifting of companies and their plays from one theatre to another, some ‘standardization of effects ‘, in Mr. Archer’s phrase, may at any one date be taken for granted.2 But analogous effects can be produced by very different arrangements, and even apart from the obvious probability that the structural divergences between public and private theatres led to corresponding divergences in the systems of setting adopted, it is hardly safe to neglect the possibility of a considerable evolution in the capacities of stage management between 1558 and 16421 or even between 1576 and 1616.
Staging In The Theatres: Sixteenth Century
Authors: Chambers E K
Published: November 2023

IN dealing with the groups of plays brought under review in the last chapter, the main problem considered has been that of their adaptability to the conditions of a Court stage. In the present chapter the point of view must be shifted to that of the common theatres. Obviously no hard and fast line is to be drawn. There had been regular public performances in London since the beginning of Elizabeth’s reign or earlier, and there is no reason to suppose that the adult companies at least did not draw upon the same repertory both for popular and for private representation. But there is not much profit in attempting to investigate the methods of staging in the inns, of which we know nothing more than that quasi-permanent structures of carpenter’s work came in time to supplement the doors, windows, and galleries which surrounded the yards ; and so far as the published plays go, it is fairly apparent that, up to the date of the suppression of Paul’s, the Court, or at any rate the private, interest was the dominating one. A turning-point may be discerned in 1576, at the establishment, on the one hand of the Theatre and the Curtain, and on the other of Farrant’s house in the Blackfriars. It is not likely that the Blackfriars did more than reproduce the conditions of a courtly hall. But the investment of capital in the Theatre and the Curtain was an incident in the history of the companies, the economic importance of which has already been emphasized in an earlier discussion.1 It was followed by the formation of strong theatrical organizations in the Queen’s men, the Admiral’s, Strange’s, the Chamberlain’s.
Moving Like a Stream: Protagoras’ Heracliteanism in Plato’s Theaetetus
Authors: Eck Job Van
Published: November 2023

In Plato’s Theaetetus Socrates equates Theaetetus’ account of knowledge as sense-perception (151 E) with Protagoras’ Man—Measure doctrine (152 A—C), which in its turn is being related to what Socrates calls the secret truth of this doctrine (152 c 8—E 1), a strong type of Heracliteanism which is worked out in 153 D—160 —. Briefly paraphrased in terms close to the text, this Secret Doctrine reads as follows.
Kan T: Some O Bjections And R Eplies
Authors: Rwin Terence I
Published: November 2023

From the summary account of the supreme principle of morality that we have considered, we can see some points of comparison between Kant’s view and the main views that he opposes. He claims to have presented the true account of morality that embodies the autonomy of the will, whereas the heteronomy of the will is the source of all the spurious principles that he rejects. To evaluate this claim properly, we need to examine Kant’s account of freedom and autonomy. But even before we discuss his attempt to connect morality with freedom and the rational will, we can see part of his case for preferring his account of morality over others. If we can see the strong and weak points in this case, we can see how much it matters whether he succeeds in his efforts to connect morality with freedom.
Hege L: History and Theory
Authors: Rwin Terence I
Published: November 2023

Hegel believes that the study of history is central in morality and moral philosophy, just as it is central in the history of thought and of philosophy as a whole. The history of thought is an account of how we understand and make explicit what we had inarticulately grasped all along. We need to reflect on the stages of the history of thought. If we impatiently try to grasp the product without the process that produced it, we will miss the whole point; for we cannot grasp the product separately from the process. We might suppose that once we have corrected our various false and one-sided views, or the outlooks of earlier stages in the history of thought, we can forget about them, and stick to our corrected version.
Kierkegaard
Authors: Rwin Terence I
Published: November 2023

In Either-Or Kierkegaard tries to capture an essential feature of the moral outlook. By this he means not the outlook of someone who accepts and follows the correct moral system or principles or virtues, but the outlook of someone who thinks it is worth looking for the correct moral system (etc.). Such people may not have grasped what is morally right, but they are the people who take questions about what is morally right or wrong, good or evil, to be practically important. Kierkegaard calls this outlook the ‘ethical’ outlook. He contrasts it with a non-moral attitude to one’s life that he calls the ‘aesthetic’ outlook. If we understand and accept the ethical outlook, we may not have found what is morally right, but at least we think it important for the conduct of our lives to find out what is right. From the aesthetic point of view, questions about moral rightness are purely theoretical questions that we do not regard as having any practical bearing on our lives.
Nietzsche
Authors: Rwin Terence I
Published: November 2023

Nietzsche believes that previous theorists have been asking the wrong question about morality. They have looked for the rational foundation of morality, on the assumption that morality could safely be taken as ‘given’, and beyond question. They have therefore overlooked some questions about morality that throw an unfamiliar and subversive light on the issues that they choose to discuss. Many readers of Nietzsche might agree with this summary of his approach to morality. But it is more difficult to find any more precise account of his subversive questions, or of what he takes them to show. Questions of interpretation are difficult to settle, partly because of the non-argumentative character of the texts, and partly because they depend on views about aspects of Nietzsche’s philosophy outside moral philosophy. The questions include these:
Bradley
Authors: Rwin Terence I
Published: November 2023

Green and Bradley are the British idealists who most deserve the attention of a moral philosopher. From the mid-19th century to the early part of the 20th, Hegel was a significant philosophical influence in Britain, especially in Oxford and in Scotland. But idealists other than Green and Bradley are either inferior or derivative or less interested in moral philosophy.
Lewis
Authors: Rwin Terence I
Published: November 2023

Lewis is a systematic philosopher. He defends his views in ethics by appeal to the principles that underlie his views in logic, epistemology, and metaphysics. He is an empiricist both about meaning and knowledge. He believes that judgments about objective states of affairs in the world are ‘non-terminating judgments’. They must eventually be explained by reference to ‘terminating judgments’ that are to be understood as predictions of possible experience. The relevant experience consists in apprehensions of what is given in experience. Apprehensions of the given are purely ‘expressive’; they do not constitute knowledge, because they are not subject to any possible error (AKV 183–4). Statements about them can be false only if one lies about them. They cannot be false because one honestly mis-describes them.
Rawls: The Just, The Fair, and The Right
Authors: Rwin Terence I
Published: November 2023

In the previous three chapters, we have pursued some questions in the moral philosophy of the present or the recent past, in order to discuss some of the developments in 20th-century meta-ethics (in Chs. 92–3) and their relevance to Aristotelian naturalism (in Ch. 94). In this chapter and the next, we take a step back chronologically, to Rawls’s Theory of Justice, published in 1971. Though this book is by no means the most recent contribution to normative moral theory, it is none the less a suitable focus for some further discussion of the questions that have arisen in the previous few chapters.
Rawls: The Right and The Good
Authors: Rwin Terence I
Published: November 2023

We have seen why Rawls’s defence of his description of the Original Position is open to objection. The appeal to judgments about fairness, regulated by the formal constraints of the concept of right, does not seem adequate for his purposes. We may be able to find a successful defence, however, if we consider what the various conditions of the Original Position seek to achieve, and we do not restrict ourselves to judgments about fairness in defending these conditions.
Malaysia
Authors: Wright Michael , Hamzah Ednin , Phungrassami Temsak , Bausa-Claudio Agnes
Published: November 2023

Malaysia (population 25. 35 million) covers an area of around 330 000 square kilometres. Its territory encompasses a peninsula which borders Thailand and the northern one-third of the island of Borneo, bordering Indonesia, Brunei, and the South China Sea (Fig. 2.1).
Thailand
Authors: Wright Michael , Hamzah Ednin , Phungrassami Temsak , Bausa-Claudio Agnes
Published: November 2023

The Kingdom of Thailand (population 65.18 million) is a country in Southeast Asia that encompasses 514 000 square kilometres and borders with Myanmar, Laos, Cambodia, and Malaysia (Fig. 3.1).
Pointless Suffering? How to Make the Problem of Evil Sufficiently Serious
Authors: McCann Hugh J
Published: November 2023

Most theodicies assume evil is completely alien to God’s purposes as Creator, and hence that in an ideally perfect world suffering would be minimized. There is however an alternative view: that part of God’s purpose in creating the world is the defeat of evil. On this assumption, we should not expect suffering to be minimized. Rather, it should be present in creation in a quantity and variety sufficient to enable its complete defeat. In particular, we should expect that there will be many cases of seemingly pointless suffering. Were it not so, victory over evil could only be incomplete, because an important dimension of it, and of the moral challenge it presents, would be lacking.
Relativism and Self-Refutation in the Theaetetus
Authors: Erginel Mehmet M
Published: November 2023

Relativism has always been a position of great interest to philosophers, and this interest has generally been accompanied by a desire to refute the position. Various kinds of relativism are available, but there has been a particularly strong interest in the view that every judgement is true for the person whose judgement it is—the view known as ‘relativism about truth’. Protagoras’ famous claim that ‘Man is the measure of all things’ has often been taken to represent this sort of relativism. Plato responds to Protagoras with what remains as the primary criticism of relativism about truth, that it is self-refuting.
Law and Value in the Stoics
Authors: Annas Julia
Published: November 2023

Katja ffogt’s book discusses some aspects of Stoic thought, concentrating on the community of all human beings, and law and reason in Stoic thought about the cosmos. A major aim of the book is ‘to relocate early Stoic political philosophy—from the margins of what we perceive as early Stoic philosophy to a position closer to its center’ (7).

After an introduction sketching the plan of the book, the first chapter discusses the ‘disturbing theses’ which give such ammunition to opponents of the Stoics in the ancient world. Many are connected to claims about Zeno’s Republic, in which it seems there would be ‘community of women’ and no temples, courthouses, or gymnasia.
In Vivo Labelling
Authors: Griffiths Robin
Published: November 2023

Following their development, radioisotope tracer techniques have become widely used in many applications; particularly in the tracing of dynamic processes such as flow, transport or chemical reactions (1-4). With the recent development of external detection capabilities such as positron emission computed tomography (5), tracer techniques became an integral part of the new methods for measuring many physiological or biochemical parameters in man (6, 7).
Radioisotopes and Immunoassay
Authors: Gearing Adrian F Bristow. Andrew J H , Thorpe Robin
Published: November 2023

Immunmissay is the quantitative measurement of a substance of interest (‘analyte’) using antibodies which bind specifically to that analyle. For the re t of this chapter, these two main components of the assay will be referred to as ligand and antibody respectively. In order to perform such an assay, it is necessary to measure the binding of the ligand to the antibody at very low concentrations and this has most commonly been achieved with the use of radiosotopes. The first practical application of the principle was the technique of radioimmunoassay, invented by Yalow and Berson in the late 1950s (1), for which the Nobel prize was subsequently awarded. This technique, and subsequently developed related techniques such as immunoradiometric assays and enzyme-linked immunosorbent assays, have revolutionized the biological sciences. Using these methods the minute quantities found in biological materials of a whole range of analytes such as protein hormones, steroids, viral antigens, cytokincs, and clotting factors can be accurately measured without relying on difficult and sometimes imprecise measurements based on their biological activities. Indeed, it is hard to overestimate the impact that the development of immunoassays has had on the life sciences.
Federal Regulations on Use of Radionuclides in the United States of America
Authors: Nelson Neal S
Published: November 2023

In 1967, the World Health Organization reported that regulation of devices and materials emitting ionizing radiation was divided among fifty state governments, several city governments, six Federal agencies, a Federal Council and a Federal Commission (1). In a 1978 review, five Federal agencies and twenty-five states were reported to be regulating some aspccb of radiation protection and control (2). Unfortunately the situation in the 1980s is not much improved.
Anti-receptor Antibodies as Ligands
Authors: Strosberg A D
Published: November 2023

In this chapter we will discuss three approaches to obtaining anti-receptor antibodies: through direct immunization, from patients’ sera, and via anti idiotypes. In each case we provide some of the specific procedures that have been used by various laboratories and discuss typical results.
Dopamine receptors: isolation and Molecular Characterization
Authors: Strange Philip G , Williamson richard A
Published: November 2023

The actions of the catecholamine neurotransmitter dopamine can be accounted for by the binding of dopamine to D1 and D2 dopamine receptors. The properties of the two receptor classes have been discussed in detail elsewhere (1,2) and they appear to be distinct proteins with different pharmacological properties linked to different biochemical and physiological events. The isolation and purification of the receptor proteins is essential in understanding how the two receptor systems function. These methods will further enable the composition of the two proteins to be determined, will enable studies on receptor reconstitution to be performed, and will assist in the cloning of the genes for the receptors. Even when the genes have been cloned and the primary structure of the receptors has been obtained, studies on the isolated proteins will be required in order to locate key regions of the receptor, e.g. the ligand-binding site. This underlines the need for methods of purification.
Measurement and control of intracellular calcium
Authors: Gurney Alison M
Published: November 2023

Calcium is an important intracellular messenger in transducing responses to activation of many receptor types. Its concentration is tightly regulated in cells, with most of it bound up in intracellular stores and only a small fraction freely available in the ionized form. In order to understand how Ca 2+ concentration is regulated in cells, as well as its role in transducing receptor signals, it is clearly important to know how the concentration of the free ion changes in response to various stimuli, and how cells respond when intracellular Ca 2+ is changed.
Phosphate-labelling studies of receptor tyrosine kinases
Authors: Mahadev Louis C , Bell john C
Published: November 2023

The extensive literature on receptor tyrosine kinases and the intense research effort it currently receives reflects its central role in the regulation of mitosis, aberrations of which result in uncontrolled proliferation of cells and the formation of tumours. This literature is comprehensively summarized and reviewed in several recent articles and is not to be considered here (1 3). This chapter deals with the practical aspects of demonstrating that a particular protein or protein complex is a receptor tyrosine kinase (RTK) which is defined as one in which the binding of a specific ligand results in the direct activation of tyrosine kinase activity intrinsic to the receptor. It is obvious immediately that there are two criteria that must be satisfied. First, the putative receptor must be shown to have the appropriate ligand-binding characteristics. Approaches to demonstrat ing respectable ligand binding (summarized in Section 2.1) are considered in detail in a companion book in this series (25) and are not described here, although a brief outline is given in Appendix 1.
A reconciled view of the non-MHC requiring cytotoxicity phenomenon
Authors: Triebel F , Hercend T
Published: November 2023

Non-MHC requiring cell-mediated cytotox1c1ty is now recognized as an important effector function generated during cellular immune responses. Many studies over the years have contributed to delineate both the different cell subpopulations and the membrane molecules involved in these cytotoxic reactions. Among human peripheral blood lymphocytes (PBL), two types of effector cells are now well defined: the natural killer (NK) cells and the activated cytotoxic T lymphocytes (CTL), wither TCR α/ β+or γ/ δ+, which mediate some non-MHC requiring killing.
Specificities involved in recognition of tumour target cells by IL-2 activated NK cells and T cells; ‘one receptor’ versus ‘multiple choice’ models
Authors: Kärre K
Published: November 2023

The specificity of cytotoxic activities associated with in vivo or in vitro administered IL-2 is usually discussed in terms of LAK activity. Without further specification, the concept of LAK cells (or LAK activity) may be misleading. Studies in recent years indicate that IL-2 administration not only can activate different effector cells, but that some of these can also use alternative recognition strategies. The discussion in this paper will therefore take into account different principles for cell mediated recognition that have emerged in the literature in recent years. This will include a multiple choice model for NK cells as well as the paradigm of peptide presentation by MHC molecules to CTL. The basis for the discussion of different effector cells and the effect of low dose versus high dose IL-2 will be introduced by a brief overview of the historical development in interpretations of lymphokine activated killing of tumour cells.
Involvement of cytokines in combined modality preclinical approaches to cancer treatment
Authors: Wiltrout R H , Hornung R L , Futami H , Sayers T J , Young H A
Published: November 2023

About 15000 to 20 000 new cases of renal cell cancer are diagnosed each year in the United States. Renal cell cancer is relatively resistant to most chemotherapeutic drugs, which makes it a candidate for alternative treatment approaches such as immunotherapy in combination with chemotherapy. The rationale for believing that such an approach may prove useful is based on the fact that primary renal cancers are often fairly vascularized and responses to biological response modifiers (BRM) such as interferon-0( (IFN-0() and interleukin-2 (IL-2) have been documented.1−4 These observations suggest that preclinical animal models for renal cancer could be useful for testing the antitumour activity of BRMs, either alone, or in combination with chemotherapeutic drugs or other BRMs. The model chosen for preclinical study in our laboratory is the Renea adenocarcinoma of BALB/c mice. The Renea tumour arose spontaneously and its growth and progression in syngeneic mice has been previously characterized. The tumour spontaneously metastasizes from an intrarenal (IR) implant to the regional lymph nodes, lungs, liver, and spleen, as well as some other organs.5,6 The immunogenicity of Renea has been determined to be relatively low.
Chromatin structure and position effects
Authors: Kipling David
Published: November 2023

In Saccharomyces cerevisiae telomeric DNA sequences can influence the expression of genes located close to them, and may also influence the timing of replication of adjacent genomic regions. Genetic analysis has revealed that this position effect on gene expression is closely related to the transcriptional repression that occurs at the silent mating-type loci. Because the latter has been characterized in great detail it will be described first, thus enabling telomere position effects to be reviewed in context.
Ribosomes and polysomes
Authors: Bommer Ulrich A , Burkhardt Nils , Junemann Ralf , Spahn Christian M T , Triana-Alonso, Francisco J , Nierhaus Knud H
Published: November 2023

Isolation of ribosomes and assay systems for testing the translational apparatus have been part of the experimental routine of many laboratories for more than two decades, and excellent collections of the methods have been published previously, including two books in this series. However, a number of methods have been gradually and continuously improved and optimized. Major developments have included the improvement of ribosome isolation procedures, the establishment of highly efficient assay systems, and the application of heteropolymeric mRNA. The protocols in this chapter are concerned with the isolation of polysomes, ribosomes, and ribosomal subunits from prokaryotic and eukaryotic sources, as well as test systems for both total protein synthesis and single ribosomal functions, taking into account the recent developments.
RNA-protein interactions in Rnase P
Authors: Gopalan Venkat , Talbot, Simon J , Altman Sidney
Published: November 2023

The endoribonuclease RNase Pis a ubiquitous and essential ribonucleoprotein. It cleaves the 5’ terminal leader sequences of precursor tRNAs (ptRNAs) to generate mature tRNAs (Figure 1). Much of our current understanding of this enzyme is derived from studies of RNase P from Escherichia coli and Bacillus subtilis (1, 2). In E. coli, RNase P consists of an RNA subunit (Ml RNA, 377 nucleotides (nt)) and a protein subunit (CS protein, 119 amino acid residues). Ml RNA and CS protein are encoded by the rnpB and rnpA genes, respectively. Apart from ptRNAs, the precursors to 4.5S RNA (p4.5S RNA) (Figure 1) and lOSa RNA (H. Inokuchi, personal communication) are also processed by RNase P from E. coli in vitro and in vivo. The observation that Ml RNA can catalyse the hydrolysis of ptRNAs in vitro even in the absence of CS protein demonstrated that RNase P owed its catalytic potential to its RNA subunit and that the protein subunit is a co-factor (3). How ever, both Ml RNA and CS protein are essential for the activity of RNase P in vivo. This latter observation emphasizes the importance of auxiliary role(s) played by the protein subunit in vivo (4, 5).
Study of RNA-protein recognition by in vitro selection
Authors: Bartel David P , Szostak Jack W
Published: November 2023

A major focus of biochemistry and genetics has been to deduce the determinants of molecular recognition and catalysis. In vitro selection of nucleic acids is a method that represents a synthesis of these two disciplines - a method that retains both the control of in vitro biochemical manipulation and the power of genetic selection. This new tool has been used successfully in many laboratories for applications ranging from defining DNA-binding sites of transcription factors to isolating new ribozymes. The basic features and applications of in vitro selection of nucleic acids have been reviewed extensively (1-6). After a brief overview of these features and applications, this chapter focuses on practical considerations for designing, implementing, and interpreting in vitro selection schemes for the characterization of RNA-protein interactions.
Yeast artificial chromosomes cloning using PFGE
Authors: Ougen P , Cohen D
Published: November 2023

The major goal of the human genome project includes the isolation of the entire human genome in overlapping clones and the development of physical maps of the cloned DNA. Cloning into yeast artificial chromosomes (YAC) represents the method of choice for genome mapping analysis in the megabase range.
PFGE analysis of yeast artificial chromosomes
Authors: Ragoussis J
Published: November 2023

There are two basic pulsed field gel electrophoresis (PFGE)-related techniques for mapping yeast artificial chromosomes (YACs). One is to digest the DNA with restriction endonucleases, detect specific fragments with defined probes, and construct a restriction map. The other technique is to generate YAC variants—(so called fragmentation products)—by homologous recombination with vectors containing a repeated (e.g. Alu), or specific, sequence. Using the recombination potential of yeast allows the construction of YAC derivatives for mapping of sequences within a YAC.
Analysis of the genomes of protozoan parasites using PFGE
Authors: Kemp David J , Cappai Roberto
Published: November 2023

Pulsed field gel electrophoresis (PFGE) has played a major role in developing our current knowledge of the genomes of a number of protozoan parasites as the genomes are small and the chromosomes usually fall within a usable size range. The studies on malaria and trypanosomatids described here demonstrate the applicability of the techniques to other protozoa.
Desalting inositol polyphosphates by dialysis
Authors: van der Kaay Jeroen , van Haastert Peter J M
Published: November 2023

In the study of the large spectrum of inositol phosphates and the enzymes involved in their metabolism, anion-exchange chromatography is commonly used for the separation, identification and quantitation of inositol phosphates. Desalting inositol phosphates after chromatography is often desired. The most commonly used method for desalting involves chromatography using a small column packed with mixed ion-exchange resin from which the inositol phosphates are washed with a volatile eluent (e.g. ammonium formate, HCI or Freon/octylamine) which is subsequently removed by vacuum concentration. Other methods used for desalting are gel-filtration or HPLC using a volatile eluent. The large volumes which have to be dried down make this procedure time-consuming.
Applications of chemically synthesized RNA
Authors: Naryshkin Nikolai A , Smith terence v. , Gait Michael J , Earnshaw David J , Farrow Mark A , Fogg Jan H , Grenfell Richard L
Published: November 2023

Many structural and biochemical projects on RNA and RNA:protein interactions have benefited from recent significant improvements in methods of chemical and enzymatic synthesis of oligoribonucleotides(1-6). These methods can also now be used to introduce site-specifically into RNA a range of nucleotide analogues containing modified functional groups or attachment sites for cross-linking (7-10). lt is not unusual in a new project involving RNA for all the three oligoribonucleotide synthesis techniques described in this book to be used together, namely chemical synthesis (this chapter), in vitro transcription (Chapter 2), and ligation (Chapter 3).
The electrophoretic mobility shift assay for RNA binding proteins
Authors: Black Douglas L , Chan Raymond , Min Hosung , Wang Jiwu , Bell Leslie
Published: November 2023

This chapter will discuss the identification and characterization of RNA binding proteins using the electrophoretic mobility shift assay (EMSA). The basis of EMSA is the change in the electrophoretic mobility of a nucleic acid molecule upon binding to a protein or other molecule. Initially a labelled RNA, containing the binding sequence of interest, is incubated with a sample containing RNA binding proteins and then analysed on an electrophorctic gel. The unbound RNA will have a characteristic electrophoretic mobility on a non-denaturing or native gel. If the RNA is bound by one or more proteins or other molecules, the movement of the RNA through the gel is retarded. This gives rise to a characteristic shift in the position of the RNA hand on the gel. The mobility shift method is a common technique for identifying DNA binding proteins (1).
Assays for virus infection
Authors: Gray J J
Published: November 2023

The laboratory diagnosis of virus infections utilizes a wide range of techniques, including virus isolation (Chapter 2), the detection of virus antigens and specific antiviral antibodies and the amplification and detection of virus genomes. This chapter will outline the principal methods used for the diagnosis of virus infections, and describe in detail those which are used most commonly. This section will outline methods used to identify and quantify viruses grown in cell culture, since detailed methods for the isolation of viruses are described in Chapter 2. Many of the cytopathic effects (CPE) seen in virus-infected cell cultures are characteristic of specific viruses or virus groups (Table 1), and the ability to recognize a specific CPE is the important first step in virus identification. Not all viruses growing in cell culture produce a CPE, and other methods have been devised for their identification (Section 2.7). The object of these assays is to measure the number of infectious particles in a suspension of viruses. This is done either by plaguing viruses on monolayer cell cultures in order to reveal and enumerate plaques which originate from a single infectious virus particle (pfu) (Protocol I), or by determining the highest dilution of the virus suspension which produces a CPE in 50% of the cell cultures inoculated (TCID50) (Protocol 2). The TCID50 value is calculated by use of the Reed-Muench formula or the Karber method (1). Calculation of the TCID50 is more commonly used to determine the dose of virus required in a neutralization test, although plaque assays give a more precise measurement of virus infectivity.
Virus vaccines
Authors: Jennings R , Potter C W
Published: November 2023

Any list of significant developments in the history of medicine would include the introduction of vaccines against infectious disease. History records epidemics and pandemics in earlier centuries which ravaged society and influenced or precipitated major events. The eighteenth century saw the dawning of the age of immunization, the nineteenth the wider introduction of vaccines against a variety of human and animal infections, through the work of many microbiologists. The present time has seen the eradication of smallpox, the predicted eradication of poliomyelitis and measles, and the control of many other infectious diseases. Vaccine development has been targeted to all virus infections which have a significant impact on human and animal health in terms of morbidity and mortality. The vaccine era is considered to have begun with the use of vaccinia virus to immunize against smallpox in the eighteenth century, and continued and expanded with the development of inactivated vaccines and live attenuated vaccines. Novel, modern vaccination strategies of the late-twentieth century are based on genetic engineering and the application of molecular biology, including the development of recombinant virus vaccines, replication defective and DNA vaccines, together with the use of natural or fabricated accessory molecules and formulations for enhancing and directing the immune response. Table 1 lists many of those virus vaccines presently commercially available for the immunization of man and animals, their form or type, and also indicates some virus vaccines currently under development and which may be generally available in the future.
Development of RNA virus vectors for gene delivery
Authors: Usmani B A , Fassati A , Dickson G
Published: November 2023

Experimental gene transfer and clinical gene therapy approaches rely on the efficient delivery of genes to desired target cells. The first approved human gene transfer study began almost a decade ago using an RNA virus vector derived from a murine oncoretrovirus to deliver a bacterial marker gene to patients’ cells (1). This was rapidly followed by protocols to treat severe combined immunodeficiency disease (SCID) and advanced melanoma. There are now hundreds of human gene therapy trials ongoing to treat many different classes of illness including inherited genetic disorders, cancer, virus infections, and cardiovascular disease, the majority of which use modified retrovirus vectors as gene delivery vehicles.
G proteins and their identification
Authors: Mullaney Ian
Published: November 2023

The demonstration that G proteins mediate many intracellular signalling processes has led to the development of specific techniques that can be used to identify which of these polypeptides is involved upon receptor activation by ligand. This chapter deals with the methodology involved to produce specific immunological tools, and the use of these reagents to probe functionally the specificity of receptor-G protein interaction.
Adenylyl cyclases and cyclic AMP
Authors: Ho Maurice K C , Wong Yung H
Published: November 2023

Adenylyl cyclase (AC; adenylate cyclase or ATP pyrophosphate lyase; E.C. 4.6.1.1) catalyses the formation of cyclic AMP (cAMP), using ATP as substrate. The theoretical topology of all ACs is similar, with both amino- and carboxyl-termini on the cytosolic side, twelve transmembrane domains divided into two groups, and linked with an extended cytosolic loop (1). The catalytic activity and the regulatory elements of AC appear to be concentrated on the cytosolic loop (C1 domain) and the carboxyl-terminal tail (C domain). To date, nine subtypes of mammalian adenylyl cyclases (A Cs) have been successfully cloned (1-15), and a partial cDNA fragment corresponding to a novel AC subtype (type X) has also been identified (16). All ACs are subjected to extensive regulatory signals, including Ca2+, G proteins, protein phosphorylation, and small molecules such as forskolin and adenosine analogues (also known as P-site analogues), as reviewed in refs. 17-19, and summarized in Table 1.
Phosphoinositide 3-kinases
Authors: Anderson K E , Stephens, L R , Hawkins P T
Published: November 2023

Phosphoinositide 3OH-kinases (PI3Ks) are enzymes which can phosphorylate one or more membrane inositol lipids in the 3-position of the inositol ring; in vitro they can make phosphoinositide(3)phosphate (PtdIns(3)P), phospho- inositide(3,4)bisphosphate (PtdIns(3,4)P2), and phosphoinositide(3,4,5)triphos- phate (PtdIns(3,4,5)P3) from phosphoinositide (Ptdlns), phosphoinositide(4) phosphate (PtdIns(4)P), and phosphoinositide(4,5)bisphosphate (Ptdlns(4,5) P2), respectively. The substrate specificity of PI3Ks in the intact cell is a matter of current debate, but most evidence suggests that the preferred substrate of the sub-family of PI3Ks that are agonist sensitive (type 1 PI3Ks) is Ptdlns (4,5)P2, and also possibly PtdIns(4)P. Hence the rapid rise in the levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 seen on stimulation is widely regarded as the major signal generated by this pathway.
Signal transduction by sphingosine kinase
Authors: Heringdorf Dagmar Meyer Zu , Koppen Chris J Van , Jakobs Karl H
Published: November 2023

Several new findings in the last few years have drastically challenged our view of the role of sphingolipids in cellular function. It is now evident that not only the complex sphingolipids of the plasma membrane, but also small molecules of sphingolipid metabolism, play an essential role in cellular regulation. Analogous to the breakdown products of glycerophospholipids, certain small sphingolipids are messenger molecules which transduce information both between cells and within cells. Of these, ceramide and sphingosine-1- phosphate (SPP) have gained major attention (1), although others may also have a signalling function. Ceramide is cleaved from sphingomyelin by sphingomyelinase(s) (see Figure 1), and apparently plays an important role in apoptosis. SPP was originally considered as an intermediate product in the breakdown of sphingosine, but now it is recognized that it controls a variety of cellular functions.
Molecular electro-optics
Authors: Porschke Dietmar
Published: November 2023

Molecular electro-optical techniques provide a high potential for the acquisition of a unique set of data on macromolecules in solution, including hydrodynamic, spectroscopic, and electrostatic parameters. The information obtained by molecular electro-optics is very useful for assigning macromolecular structures. A major advantage is the particular high sensitivity. Molecular electro-optics has been used for the characterization of many different types of macromolecules, including large numbers of proteins, and is also very useful for the analysis of protein-ligand interactions.
High-flux X-ray and neutron solution scattering
Authors: Perkins Stephen J
Published: November 2023

Solution scattering is a diffraction technique that is used to study the overall structure of biological macromolecules in random orientations (1-4). A sample is irradiated with a collimated, monochromatic beam of X-rays or neutrons, as the result of which a circularly-symmetric diffraction pattern is observed on a two-dimensional area detector at low scattering vectors Q, The intensities I(Q) are measured as a function of Q, where Q = 4 7T sin 0 / A (20 = scattering angle; A = wavelength). While X-rays are diffracted by electrons, and neutrons are diffracted by nuclei, the physical principles are the same in both types of scattering experiments. Classical solution scattering views structures at a low structural resolution of about 2-4 nm from data obtained in a Qrange of about 0.05-3 nm-1 (Figure 1). Analyses of I(Q) by Guinier plots lead to the overall molecular weight and the radius of gyration Re (and in certain cases those of the cross-section and the thickness). The Fourier transform of I(Q) gives the distance distribution function P(r), from which the maximum dimension of the macromolecule and its shape can be deduced. In recent years, a powerful method of constrained solution scattering has been developed (4), in which automated curve fit procedures based on known crystal structures are able to extract structural information to a precision of 0.5-1.0 nm, corresponding to medium resolution structures.
Protein-ligand interactions studied by Raman and resonance Raman spectroscopy
Authors: Withnall Robert
Published: November 2023

The occurrence of the phenomenon of inelastic light scattering (now often called ‘Raman light scattering’) was first demonstrated by C. V. Raman (1), who was awarded a Nobel prize in Physics in 1930 for his discovery.

The Raman light scattering process involves two photons, one of which is incident on the sample whilst the other is scattered from the sample. The scattered photons travel in all directions over the full solid angle of 41r steradians although, in the Raman experiment, it is common to detect either those which are back-scattered (back-scattering configuration) or those which are scattered at an angle of90° to the direction of propagation of the incident light (90° scattering configuration).
Nitroxide spin labels as paramagnetic probes
Authors: Middleton David A
Published: November 2023

This chapter describes how spin label electron paramagnetic resonance (EPR) spectroscopy can be used to examine aspects of molecular structure and dynamics, and changes therein, associated with the binding of ligands to receptors at their sites of action. The scope of the chapter is restricted to studies of proteins that function within cell membranes, where this approach is particularly relevant, although some of the experimental methods can be adapted to accommodate studies on soluble proteins.
Chromosomal DNA replication in yeast: enzymes and mechanisms
Authors: Macneill Stuart A , Burgers Peter M J
Published: November 2023

Chromosomal DNA replication in eukaryotic cells is a remarkable process requiring the complex interplay of a variety of distinct essential factors in a spatially and temporally coordinated manner. Each replication fork supports a large multiprotein complex containing as many as 30 to 40 proteins, the vast majority of which are essential for the successful duplication of the genome. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the molecular basis of the replication process has been studied in great detail. Indeed much of our current understanding of replication in higher eukaryotes is founded on work performed on these two distantly related ascomycetes. Each has a genome size of approximately 15 Mb and a doubling time in the range 110-240 min depending on strain characteristics and the precise growth conditions employed. S phase occupies approximately 10-15% of the cell cycle, or 15-20 min at optimal growth rates.
Mechanisms of nuclear division
Authors: Masuda Hirohisa , Hiraoka Yasushi
Published: November 2023

Mitotic nuclear division is the process that ensures proper segregation of duplicated copies of the genome at cell division. The orderly segregation of genetic information depends on its reorganization into condensed chromosomes, and on the assembly and functioning of the mitotic spindle. The mitotic spindle is the machinery that segregates sister chromatids into the two daughter nuclei at mitosis. These events are precisely regulated to produce genetically identical daughter cells.

In contrast, meiotic nuclear division is the process that generates genetic diversity in the offspring. Recombination of genetic information from cells with opposite mating types occurs at meiotic prophase. Meiosis encompasses two successive nuclear divisions: the first division involves cohesion of sister chromatids and disjunction of homologous chromosomes, while the second division disjoins sister chromatids in a process similar to mitotic di vision.

In this chapter we first describe mechanisms of mitotic nuclear division with an emphasis on the mechanistic aspect of the mitotic spindle and chromosomes; next, we describe mechanisms of meiotic nuclear division with a particular emphasis on chromosome organization specific to meiosis.
Radioisotope Use
Authors: Prime David , Frith Barry
Published: November 2023

The subject of this chapter is the safe use of radioisotopes in biological tracer work. The emphasis of the treatment will be on the practical aspects but a certain amount of a more fundamental nature will be included where this is necessary to clarify explanations and terminology. Since biological tracers are used in a wide variety of ways, it is only intended to discuss this topic in general terms, paying particular attention to the most commonly used radioisotopes.
The Scintillation Counter
Authors: Dodson Charles L
Published: November 2023

A radioactive atom or molecule emits small particles or photons or both. Monitoring radioactivity depends upon the detection of such emissions, counting either the number of particles or the number of photons. No method has been discovered to monitor radioactively emitted particles directly. Rather, we monitor the emitted photons or the effects produced by the particles interacting with selected matter. An instrument to measure radioactivity requires a sample, means of detection and means of recording. In experiments conducted early in the nineteenth century, radioactivity was monitored by dark-adapted human eyes counting scintillations produced by solid detectors and lead sulfide, and recorded by human hand. This chapter describes machines that produce the same end result by automation.