bluuebunny/crossref_metadata_2025 huggingface dataset:
Parasitism and hybrid zones
Authors: Moulia Catherine , Joly Pierre
Published: November 2023

Hybridization between animal taxa was underestimated for a long time and its contribution to the dynamics and evolution of biodiversity has been understated. The first studies and analyses explained hybrid zones as transient phenomena and barriers preventing genetic exchanges between two taxa. Then, further studies describing long-lasting hybridizations with substantial genetic exchanges revealed that, depending on the case, the fitness of the hybrids could be inferior, equal, or superior to the fitness of the parents. The issue became how to determine the selective factors favouring or disfavouring hybrids, and among them the internal (genetic) or external (environmental) factors. Of the biotic factors, parasitism represents a major evolutionary constraint which may influence hybridization favourably or unfavourably.
Introduction: Words for Music: In Search of the Irish Omphalos
Authors: White Harry
Published: November 2023

What is the stone that marks the centre of the Irish world? Some remembrance would surely be graven upon it. Some formula, or incantation, that if repeated often enough would return all things to one thing, and all expression to one mode of expression. A bardic impulse to recall and convey might be discerned in this motif at the Irish epicentre. If we could adequately imagine such a thing, such a phrase rich in its fine-spun capacity to generate the future tense of all experience within its own remembrance, the ‘blunt and falling music’ of a pump would be synonymous with its description, and not only in English. The music of language and the language of music would be one and the same thing. Perhaps, as some historians of language suggest, they once were.2 No one, in any case, could dispute the growth of music in western culture as a provisional support for language.
W. B. Yeats and the Music of Poetry
Authors: White Harry
Published: November 2023

Two years before his death, W. B. Yeats broadcast a series of programmes on the BBC in which his poems were recited and sung. In his prefatory remarks to the first of these broadcasts, Yeats was careful to distinguish clearly for his audience what he meant by ‘singing’: Both the folk singers and the speaker of verse must keep within the range of the speaking voice and the range of the speaking voice is small. Perhaps the folk singer may go a little beyond it here and there but if he goes far he becomes a bird or a musical instrument, and his proper place is the concert platform. Why not fill up the space between poem and poem with musical notes and so enable the mind to free itself from one group of ideas, while preparing for another group, and yet keep it receptive and dreaming? Furthermore to rest and vary the attention I have suggested that certain parts of poems should be sung. There is, however, one point on which I have been emphatic.
Basic principles of ultrasonography
Authors: Marhofer Peter
Published: November 2023

In order to achieve ultrasound images sufficient for regional anaesthetic peripheral nerve block techniques, some initial considerations about basic principles of ultrasonography are mandatory. The following sections outline essential knowledge about this important topic.
Future perspectives
Authors: Marhofer Peter
Published: November 2023

Currently, learning tools for, education about, and certification of the use of ultrasound-guided regional anaesthesia are under discussion. The consensus is that new learning tools are required to improve teaching in this fast-growing field of anaesthesia. Most of the traditional textbooks already include chapters concerning ultrasound-guided nerve blockade, and new textbooks such as this that focus solely on ultrasound-guided nerve blocks are available. We sincerely hope that our practical and scientific experience, obtained over the past 15 years of clinical experience with ultrasound-guided regional anaesthesia, will further the worldwide enthusiasm in this field.
Representation versus action
Authors: Berthoz Alain , Petit Jean-Luc , Macann Christopher
Published: November 2023

Today we are witnessing a breakdown of our scientific discourse on mind and brain into a multitude of disciplines, a plurality of models and levels of approach or explanations. No wonder the plural has been adopted to describe this situation: ‘the cognitive sciences’ or ‘the sciences of cognition’. The same holds at the level of the leading ideas for the cognitive sciences. We are going to claim that the brain is essentially an organ for action, whereas others hold that, on the contrary, the brain is an organ of representation. Many have concluded that the basic division is between those who hold the view of representation for action, and those who hold the contrary view, of action for representation.
A kinaesthetic theory of constitution
Authors: Berthoz Alain , Petit Jean-Luc , Macann Christopher
Published: November 2023

Having dealt with Husserl’s kinaesthetic theory in the preceding chapter, we are now going to consider specific aspects of this theory, which should help us to reflect upon the foundations of a physiology of action. Physiology and modern psychology owe much to certain of Merleau-Ponty’s writings. By going back to his Husserlian sources we will perhaps be able to develop new hypotheses. Reconsidered from a physiological standpoint, Husserl’s theory does indeed appear to have anticipated many of the recent discoveries on the functioning of the brain, from which one might be led to think that it can yield still more new ideas.
A Fossilized Jelly Baby
Authors: Brasier Martin
Published: November 2023

A group of us was standing by a hole in the ground, chatting innocently about trilobites. Suddenly, we were startled by a whoop of joy: ‘Wow! Come and look at this!’ Palaeontologist Vibhuti Rai had been hammering away at the honey-coloured mudstones when out leapt a fossil in a billion—it looked like a shrimp, with all its soft parts preserved. We crowded round to take a closer look. Coated in rust-red iron oxide, it was replete with antennae, gills, legs, and a tail. It even had little eyes on stalks. My own eyes grew on stalks in response. For a brief moment, we viewed Vibhuti with admiration. Then with something close to envy. And then our mood turned ugly. We began bashing away at the cliff like demented dwarves, each fighting for space along the ledge.
Exploitation of Patchy Resources
Authors: Goulson Dave
Published: November 2023

Within the foraging range of a bee, there will probably be many different patches of flowers, varying in size and in the plant species of which they are composed. Patches of a particular plant species may vary in the rewards that they provide, according to local microclimate, soil quality or genetic differences. Each individual bee has to choose which patch(es) to exploit. One might naively predict that, all else being equal, large patches should be favoured over small ones, since this would minimize travelling time between flowers. However, if all bees adopted this strategy, then large patches would become overrun with bees, and flowers in small patches would contain much more nectar because they would never be visited. Whatever size of patch a bee chooses, it must then decide how long to stay.
The Literary World: Context and Circulation
Authors: Shawcross Teresa
Published: November 2023

Upon the death in 1396 of the Grand Master of the Hospitallers, Juan Fernández de Heredia, some pains were taken by his former pupil, now king, Juan I of Aragon, to acquire from his effects ‘three volumes of large books containing the histories of Greece’. Bound in these may have been the Aragonese version of the Chronicle of Morea, the Libro de los fechos. If so, then its acquisition by the royal library of Aragon marked the end of a complex process of transmission. That process can be reconstructed in some detail, for the circumstances which brought Heredia into contact with the world of Greece, its histories, and the men able to translate them are well-documented, as are the techniques employed by those who worked under his patronage.
Structure
Authors: Shawcross Teresa
Published: November 2023

In studying the structure of the Chronicle of Morea, a distinction should be made between ‘story’ and ‘narrative’: the former is simply the raw material or content, while the latter is the artistic construction into which that content is moulded with the help of devices such as temporal deformation. A comparison of the temporal order of the succession of events in the story and the pseudo-temporal order of their arrangement in the narrative reveals that the overall shape of the Greek and French versions of the Chronicle conforms to the pressures of chronological progression. Thus, the two versions begin with the First Crusade and, having covered—in sequence—the Fourth Crusade, the capture of Constantinople and creation of the Latin Empire, the conquest of the Peloponnese by Guillaume I de Champlitte, and the reigns of Geoffroy I de Villehardouin and his sons Geoffroy II and Guillaume II, break off in the time of Princess Isabeau, the fourth Villehardouin ruler. Even so, a number of deviations, or ‘anachronies’, may be discerned within that broad schema. These anachronies are of particular interest. In a situation where reception is achieved through the written word, if for any reason there is ignorance or confusion regarding the context out of which emerges the material being read at a particular moment, that context can be rediscovered with relative ease by glancing back or forward over the material selectively. By contrast, when oral discourse is involved, this possibility obviously does not present itself, for an utterance is by its very essence ephemeral, confined to the moment when it is pronounced. So as to compensate for the difficulties posed by its medium, a non-written narrative has recourse to distinctive devices. As we shall see, specific types of anachrony in the Greek and French versions of the Chronicle of Morea reflect narrative methods characteristic of orality.
Tense-Switching
Authors: Shawcross Teresa
Published: November 2023

A comparison of the Greek and French versions of the Chronicle of Morea reveals considerable discrepancy between them with regard to the usage oftense.1 This is apparent if one examines any attempt, brief or sustained, made in the versions to relate events that have taken place at an earlier date.
Losing France and Becoming England
Authors: Watkins John
Published: November 2023

On January 7, 1558, the English Middle Ages ended. On that day, the Duke of Guise defeated the English at Calais and reclaimed the city for the King of France. Mary I famously proclaimed that the loss was her deathblow and told her subjects that if they opened her corpse, they would find ‘Calais’ inscribed on the heart of her corpse. English monarchs had claimed territory in France since the Norman Conquest, and they had claimed the French Crown since 1328. Although English monarchs styled themselves Kings and Queens of France until 1800, the title became a romantic anachronism. Within a half-century after Mary’s death, English expansion.
Post-hole and pit: interpreting timber structures
Authors: Harding D W
Published: November 2023

In lowland environments in Britain for the best part of a century the post-hole has been the fundamental unit of construction in Iron Age building. Students of archaeology were taught how to recognize them and section them, while members of the public were bemused by the fascination that these ephemeral traces of buildings evidently had for professional archaeologists. Equal only in basic signiWcance was the pit, almost ubiquitous in prehistoric settlement, as if the burrowing instinct was primeval to humans and beasts. The idea that pits might have served as dwellings, as we have seen, remained in contention well into the 20th century, eVectively until Bersu’s excavation at Little Wood- bury, carried out on the eve of the Second World War, aVorded a more realistic perception of Iron Age houses.
Galleries, cells, and corbelling: interpreting stone structures
Authors: Harding D W
Published: November 2023

Iron Age buildings in lowland Britain, as we have seen, may be constructed exclusively of timber. In upland regions where stone was readily available this was commonly used, especially for the foundations of external walls, but otherwise the design and layout of many of these buildings remain essentially the same as the timber round-houses of lowland Britain. In fact the external stone walls were evidently in many cases little more than foundations to lift the timber superstructure oV the ground, thereby avoiding rotting of timbers. Whether there was an external timber-framed wall mounted on this foundation, or whether the roof-rafters were bedded directly on the low external wall, as shown in Fox’s (1955) reconstructions of Hut 1 and the central house in the Round Pound at Kestor, Devon, remains unclear; the basic principle of construction remains the use of an internal ring of uprights to support the roof. In one instance, from Trevelgue Head at Newquay in Cornwall (Fig. 20), the external foundations of the round-house consisted of alternate orthostats and thin slabs of horizontal dry-stone coursing, which seem almost to simulate the pattern of upright timbers with wattle panels that was doubtless the basis of timber-wall construction throughout lowland Britain.
Origins and antecedents
Authors: Harding D W
Published: November 2023

For the past seventy years, since Bersu’s excavation at Little Woodbury, the large, timber round-house has been an icon for the southern British Iron Age. For a good many years brochs have fulWlled a similar role in the northern British Iron Age, though for earlier generations of antiquaries they were familiarly known as ‘Pictish towers’, a label that has now rightly fallen into abeyance. Though there is every reason to suppose cultural continuity from the earlier Iron Age, Picts only enter the historical record after ad 297, and the occupation of broch sites that corresponds to the later Iron Age is represented by a variety of more ephemeral buildings, rather than the monumental towers themselves. Furthermore, the geographical distribution of brochs is mainly in the northern and western Highlands and Islands, rather than in eastern Scotland where symbol-stones and other monuments that supposedly signify the Pictish ‘heartlands’ are concentrated. In both instances, however, these distinctive classes of building, as we have seen in Chapters 3 and 4, are just two in a much wider range of variants, and whilst they may represent the pinnacle of architectural achievement in their respective regions, neither are necessarily typical of the much greater diversity of buildings that are found in later prehistory in Britain.
Houses Wt for gods and heroes
Authors: Harding D W
Published: November 2023

To all intents and purposes, this structure is a typical round-house, of the general type known from many Iron Age sites in southern Britain. Architecturally, nothing distin- guishes it from a domestic structure, and it can best be considered as a house for the deity, of a type that would have been familiar to inhabitants of the region in a secular context. What served to diVerentiate it from a primarily domestic building, however, is the central pit . . . and the votive deposits. in the courtyard around the structure.
Houses, homesteads, villages, towns
Authors: Harding D W
Published: November 2023

From an archaeological perspective, a house consists of a principal structure that was the living accommodation of the family or household unit. Viewed purely from an architectural point of view, a house might comprise just one building of distinctive plan that might be accorded its place within a struc- tural typology. But this would not be a particularly meaningful analysis in terms of social or cultural archaeology.
The inhalants
Authors: Robson Philip
Published: November 2023

It is evident from cave drawings in Australia and Mexico, and from the writings of Greek and Persian scholars, that the inhalation of naturally occuring mind-altering gases or vapours is a practice which extends back into prehistory. Mystics, including the Delphic Oracle, used such emissions along with other mind-altering miasmas such as fumes from perfumes and burning spices to induce visions and enhance contact with the spirit world.
‘If He Were Living, and an Englishman’: Translation Theory in the Age of Dryden
Authors: Hopkins David
Published: November 2023

The writings of John Dryden constitute the most substantial and influential body of thinking about translation in the late seventeenth and early eighteenth centuries, and soon established a framework of principles and assumptions which would be constantly invoked in discussions of the subject. Dryden’s first essay on translation, the Preface to Ovid’s Epistles (1680), was immediately cited by other practitioners,1 and quotations from Dryden dominate the section ‘Concerning Translations’ in Sir Thomas Pope Blount’s collection of commonplaces about poetry.2 But to describe Dryden’s writings on the subject as ‘translation theory’ might suggest an original, comprehensive, static, and consistent body of doctrine, rather than – as is the case – a constantly-evolving set of programmatic statements and reflections, often developing from the work of predecessors, com- posed over the course of two decades as a working translator, and deriving their authority as much from the poet’s practice as from their cogency in the abstract. Dryden’s major statements on translation all occur in prefaces or dedications attached to particular works or collections: Ovid’s Epistles (1680), Sylvae (1685), The Satires of Juvenal and Persius (1692, dated 1693), Examen Poeticum (1693), Virgil’s Aeneis (1697), and Fables Ancient and Modern (1700).
Dryden and the Tenth Satire of Juvenal
Authors: Hopkins David
Published: November 2023

Recent commentary on Dryden’s translations of Juvenal can be broadly divided into four main categories. One group of scholars has devoted itself to assembling the range of materials which the poet had before him while composing his versions. As is now well established, Dryden did not simply work from plain texts of his originals, but drew on a remarkably wide variety of commentaries and earlier translations, continually incorporating, adapting, and combining interpretative glosses, phrasing, diction, rhymes, and end-words from these sources in his own rendering. Scholarship over the last fifty years has gone a long way towards establishing the particular editions and translations of Juvenal with which Dryden worked, and on whose phrasing and insights he drew when composing his own versions.
Translation, Metempsychosis, and the Flux of Nature: Dryden’s ‘Of the Pythagorean Philosophy’
Authors: Hopkins David
Published: November 2023

To turn from Dryden’s earliest discussions of translation to his last is to experience a shift from criticism focused primarily on questions of genre, method, and technique to writing which contemplates, in language of extraordinary metaphorical resonance and suggestive- ness, the most fundamental processes of inheritance, infiuence, transmission, and transformation – in nature and history as well as in art. In his earliest and most famous essay on translation, the Preface to Ovid’s Epistles (1680), Dryden, as we saw in Chapter 4, had proposed his celebrated tripartite division of translation into ‘Metaphase’, ‘Paraphrase’, and ‘Imitation’, in which ‘Paraphrase’ (‘or translation with Latitude, where the Author is kept in view by the Translator, so as never to be lost, but his words are not so strictly follow’d as his sense, and that too is admitted to be amplyfied but not alter’d’) had been offered as a via media between the crabbed obscurity of ‘Metaphrase’ and the unacceptable licence of ‘Imitation’.
Pope’s Trojan Geography
Authors: Hopkins David
Published: November 2023

The first readers of Alexander Pope’s translation of Homer’s Iliad, published in instalments between 1715 and 1720, would have noticed a number of striking features which differentiated Pope’s version from all that had preceded it. In addition to a substantial Preface, an ‘Essay on the Life, Writings and Learning of Homer’, and extensive notes on each Book commenting both on the meaning and beauties of Homer’s text and on the challenges presented by the task of rendering it into English, Pope offered the reader detailed help in determining both the general physical layout of Homer’s Trojan plain, and of the precise location within that terrain of each of the battles which comprise the bulk of the poem’s action. In the course of his notes on the Catalogue of the Ships in Book 2, he included a fold-out map [Fig. 1] in conventional format, specially engraved by the celebrated cartog- rapher John Senex, of ‘Graecia Homerica’ (comprising mainland Greece, the Aegean islands, and the west coast of Asia Minor), showing the locations from which each contingent in the Greek and Trojan armies had come.
Colonization, Closure, or Creative Dialogue? The Case of Pope’s Iliad
Authors: Hopkins David
Published: November 2023

The second half of the twentieth century witnessed a significant revival of academic interest in the literary form that has been a central concern of the present volume: English verse translation from classical poetry which aspires to the status of English poetry in its own right. An important agent in this process was the journal Arion, founded at the University of Texas in 1962 and edited by a group of American and expatriate British classicists, including William Arrowsmith, D. S. Carne-Ross, C. J. Herington, and J. P. Sullivan. Arion was the first classical journal with an avowedly literary-critical, as opposed to historical and philological, agenda. It would, its editors hoped, perform within classics a similar role to that of Scrutiny or Essays in Criticism in English studies, and would apply to the study of classical literature the critical methods and assumptions familiar at that date in university English departments.
The processing of audio-visual speech: empirical and neural bases
Authors: Campbell Ruth
Published: November 2023

In this selective review, I outline a number of ways in which seeing the talker affects auditory perception of speech, including, but not confined to, the McGurk effect. To date, studies suggest that all linguistic levels are susceptible to visual influence, and that two main modes of processing can be described: a complementary mode, whereby vision provides information more efficiently than hearing for some under-specified parts of the speech stream, and a correlated mode, whereby vision partially duplicates information about dynamic articulatory patterning.
Listening to speech in the presence of other sounds
Authors: Darwin C J
Published: November 2023

Although most research on the perception of speech has been conducted with speech presented without any competing sounds, we almost always listen to speech against a background of other sounds which we are adept at ignoring. Nevertheless, such additional irrelevant sounds can cause severe problems for speech-recognition algorithms and for the hard of hearing, as well as pose a challenge to theories of speech perception. A variety of different problems are created by the presence of additional sound sources: detection of features that are partially masked, allocation of detected features to the appropriate sound sources, and the recognition of sounds on the basis of partial information.
Fronto-temporal brain systems supporting spoken language comprehension
Authors: Tyler Lorraine K , Marslen-Wilson William
Published: November 2023

The research described here combines psycholinguistically well-motivated questions about different aspects of human language comprehension with behavioural and neuroimaging studies of normal performance, incorporating both subtractive analysis techniques and functional connectivity methods, and applying these tasks and techniques to the analysis of the functional and neural properties of brain-damaged patients with selective linguistic deficits in the relevant domains. The results of these investigations point to a set of partially dissociable sub-systems supporting three major aspects of spoken language comprehension, involving regular inflectional morphology, sentence-level syntactic analysis and sentence-level semantic interpretation.
The fractionation of spoken language understanding by measuring electrical and magnetic brain signals
Authors: Hagoort Peter
Published: November 2023

This paper focuses on what electrical and magnetic recordings of human brain activity reveal about spoken language understanding. Based on the high temporal resolution of these recordings, a fine-grained temporal profile of different aspects of spoken language comprehension can be obtained. Crucial aspects of speech comprehension are lexical access, selection and semantic integration. Results show that for words spoken in context, there is no ‘magic moment’ when lexical selection ends and semantic integration begins.
Speech perception at the interface of neurobiology and linguistics
Authors: Poeppel David , Idsardi, William J , Wassenhove Virginie van
Published: November 2023

Speech perception consists of a set of computations that take continuously varying acoustic waveforms as input and generate discrete representations that make contact with the lexical representations stored in long-term memory as output. Because the perceptual objects that are recognized by the speech perception enter into subsequent linguistic computation, the format that is used for lexical representation and processing fundamentally constrains the speech perceptual processes. Consequently, theories of speech perception must, at some level, be tightly linked to theories of lexical representation.
Tool use, communicative gesture and cerebral asymmetries in the modern human brain
Authors: Frey Scott H
Published: November 2023

The Palaeolithic record indicates that our ancestors began modifying rocks for pounding at least 2.5 Myr ago (Ambrose 2001). Subsequently, rocks were attached to sticks to create compound tools that were capable of generating higher impact forces. These hammers, as well as many other manipulable artefacts, have been refined into their various modern forms through a process of cumulative cultural evolution (Basalla 1988; Tomasello 1999).
The role of cultural practices in the emergence of modern human intelligence
Authors: Hutchins Edwin
Published: November 2023

Many accounts of how humans became the creatures they are rely on speculations about changes in the neural architecture of the human brain. For example, Clark (2001, ch. 8) says, ‘The idea is that some relatively small neural (or neural/bodily) difference was the spark that lit a kind of intellectual forest fire. The brain is, let us assume, wholly responsible (courtesy, perhaps of some quite small tweak of the engineering) for the fulfillment of some precondition of cultural and technological evolution’.
Neuroscience, evolution and the sapient paradox: the factuality of value and of the sacred
Authors: Renfrew Colin
Published: November 2023

The human mind over the past 10 000 years, in most areas of the world, has developed symbolic concepts of such evident factuality that it is difficult for us now to imagine life without them. These include notions of value (including money), of number and measure, of individual people of high status and power, and of material things that embody the sacred and the forbidden. These are often considered givens of the human condition. Yet these features are not ‘givens’ at all—they are emergent features of the past 10 000 years.
Detection, generation, and use of cytotoxic T lymphocyte (CTL) clones
Authors: Oldstone Michael B A
Published: November 2023

Cytotoxic T lymphocytes (CTL) recognize an infected target cell in a virusspecific major histocompatibility antigen (MHC) restricted manner (Figure 1 ). Target cells are selected on the basis of containing the correct MHC glycoproteins, permissibility for the infecting virus, ease of manipulation and carrying in culture, stability of the uninfected cell in releasing low amounts of internalized radiolabelled 5 Chromium, and ability of the cell to be lysed by CTL during which maximal amounts of 51Cr are released. Established MHC typed murine lines most often used because they fit the above criteria are listed in Table 1.
Methods for studying mouse natural killer cells
Authors: Welsh Raymond M
Published: November 2023

Natural killer (NK) cells are bone marrow-derived, thymus-independent, large granular lymphocytes (LGL) which, without specificity or antigenic memory, lyse on contact a variety of target cells (1, 2). The prototypic NK cells lack T cell receptor or CD3 mRNA or proteins (3). NK cells lyse a variety of tumour cells and certain normal cells such as thymocytes. Infection of target cells with any of a variety of viruses contributes to enhanced NK cell-mediated lysis of those targets. The cytotoxic capacity of NK cells can be greatly augmented by interferon (α, β, or γ) and by interleukin-2 (IL-2), resulting in enhanced kinetics of lysis of highly sensitive targets and a greatly expanded target range encompassing most cultured cell lines. Interferon and IL-2 also contribute to the proliferation of NK cells. Virus infection, as a result of stimulating very high levels of IFN (α and β), causes marked activation and proliferation of NK cells in vivo. Studies in the mouse have characteristically shown a peak in NK cell activity and number early in infection, followed by a peak in cytotoxic T cells later in infection (2). In keeping with the general orientation of this book, most of the following discussion will focus on NK cells in the mouse, but many of the techniques and general principles discussed can also be applied to studies in man and other animals.
Culture of adult mammalian peripheral neurons
Authors: Lindsay Ronald M , Evison Caroline J , Winter Janet
Published: November 2023

The vast heterogeneity of both the neuronal and glial elements which constitute the nervous system has proved to be a major barrier to studies to elucidate the precise physiological function and underlying biochemistry of individual subclasses of neurons and glia. As in many other systems, tissue culture has been widely used in attempts to simplify such studies. In the nervous system, tissue culture has been particularly useful in studying factors that regulate the development of the peripheral nervous system (PNS). Explant and dissociated cultures of peripheral neurons, for example, have been invaluable in the discovery and explanation of the key role played by growth and survival factors (e.g. nerve growth factor — NGF (1, 2), brain-derived neurotrophic factor — BDNF (3, 4), and ciliary neurotrophic factor-CNTF (5)), substrate adhesion factors (e.g. the extracellular matrix molecules such as laminin, collagen, and fibronectin), and cell-cell adhesion molecules (N-CAM, NG-CAM) in the development of the PNS (6).
Photolabile calcium buffers to selectively activate calciumdependent processes
Authors: Gurney Alison M
Published: November 2023

Optical probes provide a particularly powerful means for manipulating the concentration of free calcium inside or outside neurons. Photosensitive calcium buffers are molecules that either release or sequester calcium upon irradiation with appropriate wavelengths. The experimental usefulness of these probes lies in the speed of the photochemical reactions, as well as the ease with which the intensity, duration, and area of activating light can be varied. Thus the light could be directed at a whole population of cells, or be focused into a spot small enough to irradiate a single cell or part of a cell. The approach therefore allows the possibility of studying how a neuron responds to changes in the calcium concentration imposed throughout the cell, or localized to one part of the cell. The probes currently available include nitr-5 (1) and DM-nitrophen (2), which evolved from two separate series of molecules that release calcium upon photolysis. Both of these agents can be obtained from Calbiochem Corp., PO Box 12087, San Diego, California 92112-4180, USA, or Novabiochem (UK) Ltd, 3 Heathcoat Building, Highfields Science Park, University Boulevard, Nottingham NG7 2QJ. Agents that mop up calcium after photolysis have also been described (3, 4), but are not yet available commercially. All of these probes are calcium chelators that incorporate a photolabile group, which upon photolysis results in molecules with altered affinities for calcium. This chapter discusses practical aspects in the use of the currently available probes, nitr-5 and DM-nitrophen, for studying calcium-dependent phenomena in neurons.
Mutagenesis facilitated by the removal or introduction of unique restriction sites
Authors: Carter Paul
Published: November 2023

Over the last decade, a variety of efficient and reliable methods have been developed for the construction of site-directed mutations in DNA using synthetic oligonucleotides (reviewed in refs 1-3). This has revolutionized the study of gene regulation and protein -structure and function. All of these mutagenesis methods fall into one of three broad strategies. One approach uses an oligonucleotide complimentary to part of a single-stranded (ss) DNA template but containing an internal mismatch(es) to direct the desired mutation (see Section 1.1, and Chapters 2 to 5 and 9). A second strategy is to replace the region of interest with a synthetic mutant fragment generated by annealing complimentary oligonudeotides (‘cassette mutagenesis’ (ref. 4 and Chapter 10)), or by hybridization and ligation of a number of oligonucleotides (reviewed in ref. 5). The third and most recent strategy, has been to harness the power of the polymerase chain reaction (PCR) (6) to generate a mutant fragment starting from a double-stranded (ds) DNA template using mismatched oligonucleotides (refs 7 and 8, and Chapter 11). In this chapter an efficient method of site-directed mutagenesis using mismatched oligonucleotides (first strategy above) is discussed.
Cassette mutagenesis
Authors: Richards John H
Published: November 2023

Cassette mutagenesis provides a powerful and versatile approach for the generation of mutations and allows a wide range of studies of the relationship between base sequence and function in nucleic acids or between structure and function in proteins. The central aspect of this approach is the replacement of a region of DNA with new sequences that can be of any length, any sequence or any mixture of sequences. Thus, in studying the basis for protein function one can introduce not only changes of a few amino acids (an important aspect of primer-extension in vitro mutagenesis) but can also create chimeric proteins in which entire domains of a protein have been replaced with a region of totally different amino acid sequence.
Expression cloning: transient expression and rescue from mammalian cells
Authors: Aruffo Alejandro
Published: November 2023

Cloning strategies based on transient expression in mammalian cells have been used effectively to isolate CDNAs encoding secreted, cell surface, and intracellular proteins. The first application of transient expression in mam malian cells in cloning was the isolation of CDNAs encoding the lymphokine GM-CSF (granulocyte macrophage colony stimulating factor) (1, 2). This was followed by the use of transient expression in mammalian cells in conjunction with antibody panning (3) to isolate CDNAs encoding cell surface proteins (4, 5). Transient expression in mammalian cells has also been used to isolate cell surface receptors based on the ability of the transfected cells to bind to either fluorescein-conjugated (6) or radiolabelled (7) ligands or ligand immobilized on a solid phase (8, 9) or ligand expressed on a cell surface (10). Recently, transient expression in mammalian cells has also been used to isolate CDNAs encoding intracellular proteins (11, 12).
Biochemical methods for studying supramolecular complexes involving cell adhesion molecules, integral membrane proteins, and the cytoskeleton
Authors: Nelson W James , Wilson, Rachel , Mays Robert W
Published: November 2023

During development of complex tissue patterns, cells undergo dramatic structural and functional reorganization (1). The inductive process(es) leading to cellular differentiation often involves specific cell-cell and cell-substratum interactions (2, 3). These interactions result in new programmes of gene expression and the spatial redistribution of plasma membrane and cytoplasmic proteins in line with the specialized structural and functional requirements of the fully differentiated cell. Understanding the mechanisms involved in the regulation of protein distributions during cellular differentiation is one of the most interesting and experimentally challenging areas of cell and developmental biology.
Looking for protein-protein interactions
Authors: Midgley Carol A , Lane David P
Published: November 2023

The specific interactions of protein molecules to form homo- and heterooligomers underlie many key events in biology, from the assembly of viruses to the formation of multisubunit protein machines of great complexity. Traditionally the study of such interactions has demanded the availability of large amounts of purified components, and the members of a complex have been identified through their extensive copurification. These techniques are restricted to the analysis of tight associations between relatively abundant proteins, and cannot easily be used to study the biological regulation of a protein complex in different cellular environments.
Pre-mRNA splicing factors in the yeast Saccharomyces cerevisiae
Authors: Hodges Peter e. , plumpton, mary , Beggs jean D
Published: November 2023

The budding yeast Saccharomyces cerevisiae provides a model system to study pre-mRNA splicing using a combination of classical genetics, molecular biology, and biochemistry {see 1-4 for other recent reviews). Attention must be paid to those aspects of yeast splicing that are not general to all eukaryotes. Initial discoveries left some doubt about the universality of the yeast mechanism. For instance, the yeast splicing machinery has more stringent requirements for branchpoint and splice site sequences (4), yeast Ul and U2 snRNAs arc unusually large (5-7), and initial genetic screens for yeast splicing mutants failed to identify genes encoding any of the snRNP proteins known from mammals (see below). However, the universal nature of spliceosome function is now accepted; many splicing factor homologues are recognized as conserved between organisms, and the mechanisms of spliceo-some assembly, deduced from mammalian and yeast splicing systems, are identical. This review summarizes the discoveries found in yeast splicing, compares splicing in yeast and in other eukaryotes, and enumerates the advantages of working with yeast as a model system.
Drosophila Embryos
Authors: Forbes Zandy , Ingham Philip
Published: November 2023

Adult fruitflies of the species Drosophila melanogaster are easily maintained in the laboratory. At 25 °C, the generation time of Drosophila is 9 days and embryogenesis is completed in 22 h. Traditionally, large numbers of flies are grown in half-pint milk bottles with a solid nutrient medium in the base; alternative containers include plastic fruit juice bottles or urine sample containers. Stocks of flies can be maintained at 18 °C in smaller tubes with the same medium. For 25 bottles or 120 tubes containing nutrient medium, boil 8 g agar in 1 litre tap water for 3 min, add 80 g corn (maize) meal, 18 g dried yeast, and 10 g soya flour (premixed with some of the water), plus 80 g malt extract and 40 g molasses. Boil for 20 min, cool to 50-80 °C, add 6.6 ml acid mix (a mix of 500 ml propionic acid and 32 ml phosphoric acid), and pour.
Zebrafish embryos
Authors: Warga Rachel m.
Published: November 2023

Zebrafish (Brachydanio rerio) embryos have gained considerable popularity in recent years because they offer several advantages for developmental studies. They are cheap and easy to breed, can be obtained all year round and the embryos are transparent. The latter are easy to manipulate and develop quite rapidly: from egg to hatching takes roughly 3 days.
Xenopus
Authors: Altaba Ariel Ruiz I
Published: November 2023

Amphibian embryos have a prominent place among the species used to study vertebrate embryonic development. There is a long and distinguished history of experimental manipulations that were carried out in salamanders and newts. Perhaps the best known example is the 1924 organizer transplant experiment of Hilde Mangold (1), then a student in Hans Spemann’s lab. This experiment was not an isolated event, rather it was the culmination of a long search for the basis of the formation of embryonic tissues and their patterning in several laboratories, including that of Spemann. To understand the roots of present day molecular embryology, the reader is invited to read the fascinating book by Viktor Hamburger (2) written 50 years after the appearance of Spemann’s magistral work (3).
Induction Assays in Xenopus
Authors: Taba Ariel Ruiz I Al
Published: November 2023

The study of inductive interactions requires the identification of tissues that produce the inductive signal and tissues that are competent to respond (1). The identification of the signal as well as the study of the molecular mechanisms underlying induction requires the development of an assay system with which to test different preparations and factors. Amphibian embryos have been used to identify and to define a series of embryonic inductions. Two of these, the induction of mesoderm and the induction of neural tissue, are crucial in the formation of the vertebrate body plan and were first described in amphibian embryos (2-4). In the first case, vegetal cells signal overlying animal cap cells at the equator of the blastula-stage embryo to change fate from ectodermal to mesodermal. In the second case, mesodermal cells signal to adjacent animal cap cells to change fate from epidermal to neural during gastrulation.
Embryonic Stem Cells and Gene Targeting
Authors: Robertson Elizabeth J , Martin Gail R
Published: November 2023

Embryonic stem cells (ES cells), in conjunction with protocols for targeted gene disruption, have made it possible to introduce mutations into specific genetic loci in mammalian embryos. ES cells are permanent tissue culture lines of pluripotential stem cells derived from the inner cell mass of the normal mouse embryo (1,2). They can be readily manipulated in culture and genetically altered cell clones then used to generate chimaeric animals following their microinjection into preimplantation stage carrier embryos. As ES cells reproducibly populate both the somatic cell lineages and the germ line, they afford a means of introducing mutations into the mouse genome. The discovery that mammalian cells can undergo a process of homologous recombination (site-specific recombination between exogenously introduced cloned DNA and the chromosomal cognate gene), has facilitated the generation of mice carrying a range of targeted genetic alterations, from large deletions to single nucleotide changes (3).
Purification of over-produced proteins from E. coli cells
Authors: Grisshammer Reinhard , Nagai Kiyoshi
Published: November 2023

Many biologically interesting proteins are present naturally in small quantities and are not amenable for structural and biochemical characterizations. The molecular mechanisms of gene expression in Escherichia coli became well understood in the late 1970s, allowing the over-production of foreign proteins from their cloned genes. A large proportion of new protein structures reported in 1990 and 1991 were derived from heterologously over-produced proteins (1) and this unequivocally demonstrates that the technique of overproduction has now become an essential tool for the study of protein structure and function. We mainly focus in this chapter on the purification of overproduced proteins from E. coli cells. It is a well-known fact that overproduced proteins are not always soluble and active in E. coli and often aggregate as insoluble inclusion bodies. As re-folding of proteins is difficult, it is desirable to produce proteins in a soluble and active form. We first describe how inclusion body formation can sometimes be avoided. We then detail purification methods for both soluble and insoluble proteins. Each protein has its own ‘personality’ and the methods described in this chapter should be regarded as a collection of experiences which may direct readers to the successful purification of their own proteins.
Library construction in Pl phage vectors
Authors: Sternberg N
Published: November 2023

The Pl cloning system was developed as an alternative to YAC and cosmid technology for the cloning of high molecular weight DNA fragments. It permits the recovery of inserts in E. coli that are up to 95 kb in size (more than twice the size of inserts that can be recovered in cosmids) with an efficiency that is intermediate between ;... cosmid and YAC cloning; greater than 105 clones can be generated using the Pl system with 1-2 µg of vector arms and 2-4 µg of Sau3Al digested, size selected genomic DNA insert (1). While the size of cloned insert recovered with the Pl system are significantly smaller than arc those recovered with YACs, the former offen, several advantages: libraries representative of multiple copies of the genome arc more easily produced, larger quantities of a particular cloned DNA are more easily isolated, the cloning process may be more faithful (2, 3), and physical dissection of cloned inserts may be more easily accomplished. Indeed, the ability to analyse cloned DNA before and after attempting amplification with the Pilytic replicon permits one to assess whether amplification of the insert by a high copy number replicon can indeed occur, and if so, whether DNA integrity is maintained.
Genetic mapping with microsatellites
Authors: Naom Isam S , Mathew, Christopher G , Town Margaret-Mary
Published: November 2023

Simple tandemly repeated sequences, or microsatcllites, are ubiquitous in the genomes of a wide range of organisms, and the number of repeats within many of them is highly variable in the population of a particular species. The advent of the polymerase chain reaction (1) provided the means for rapid analysis of the repeat number, and several groups showed that these sequences were likely to provide a rich source of very informative markers for genetic mapping (2-4).
Transposon and T-DNA mutagenesis
Authors: Aarts Mark G M , Koncz Csaba , Pereira Andy
Published: November 2023

Insertional mutagenesis with a known mobile DNA insert can generate mutations that are marked by a molecular tag. When the insertion disrupts a gene, causing a mutant phenotype, the tagged mutant gene can easily be cloned using the DNA insert as a molecular probe. DNA sequences of the cloned mutant gene can then be used to isolate the corresponding wild-type allele. This procedure, known as gene tagging, facilitates the analysis of both mutant and gene.
Analysis of cell death by flow cytometry
Authors: Darzynkiewicz Zbigniew , Li Xun , Gong Jianping , Hara Shinsuke , Traganos Frank
Published: November 2023

Flow cytometry allows one rapidly and accurately to measure individual cells in large cell populations. Because several cell attributes can be measured simultaneously and the data are recorded in a mode that allows one to relate all the measured attributes to a particular cell (‘list mode’), their multivariate analysis provides information on the relationship between these attributes. In contrast to biochemical analysis in bulk, cellular heterogeneity can be estimated and cell populations with distinct characteristics can be discriminated. If needed, cells with particular features, can be electronically sorted. These advantages have all contributed to the fact that flow cytometry has become a methodology of choice in a variety of functional and structural assays of the cell, including cell viability.