bluuebunny/crossref_metadata_2025_split huggingface dataset:
Model-based methods in analysis of biomedical images
Authors: Cootes Tim 
Published: November 2023

Abstract
Biomedical images usually contain complex objects, which will vary in appearance significantly from one image to another. Attempting to measure or detect the presence of particular structures in such images can be a daunting task. The inherent variability will thwart naive schemes. However, by using models which can cope with the variability it is possible to successfully analyse complex images. Here we will consider a number of methods where the model represents the expected shape and local greylevel structure of a target object in an image. Model-based methods make use of a prior model of what is expected in the image, and typically attempt to find the best match of the model to the data in a new image. Having matched the model, one can then make measurements or test whether the target is actually present.
Assay of membrane transport in cells and membrane vesicles
Authors: Jarvis Simon M 
Published: November 2023

Abstract
Membrane transport is a vectorial process involving the translocation of ions or solutes from one compartment to another. With the exception of simple diffusion, specific proteins catalyse transport. The last decade has seen an explosion in studies applying molecular biology techniques to the identification, classification, localization, and regulation of the transport proteins (see other chapters in this book). An ultimate goal of many studies is to relate structure to function. Transport function is characterized by studying properties of substrate specificity, species of co-substrates and counter-substrates, inhibitors, activators, pH dependence, permeant concentration dependence, and temperature dependence. These functional characteristics rely upon measurements of the initial rates of transport.
Expression of foreign transport proteins in yeast
Authors: Sauer N , Stolz J 
Published: November 2023

Abstract
Over the last few years we have learned from sequencing projects for various organisms that up to 5% of all nuclear genes encode putative transport proteins, emphasizing the importance of these proteins for compartmentation and transmembrane fluxes. The number of cloned transporter genes is increasing exponentially, and the functional analysis of these transporters is an inalienable requirement for understanding their specific functions and their influence on the underlying physiological processes. For that reason, expression of transporter genes or cDNAs in heterologous expression systems has become increasingly important, and expression in yeast has, in particular, turned out to be extremely useful. Besides functional analysis, which includes determination of substrate specificities, ion and pH-dependences, KM values, electrochemical properties, and energy-dependences, expression in yeast has also been the basis for the biochemical characterization of recombinant transport proteins. Numerous well-characterized yeast promoters and yeast mutants represent excellent tools for the large-scale preparation of transport proteins and also for the identification of transporter genes by complementation analyses.
Two-dimensional crystallization of membrane proteins
Authors: Ringler Philippe , Heymann Bernard , Engel Andreas 
Published: November 2023

Abstract
While structure determination of soluble proteins by X-ray crystallography and nuclear magnetic resonance (NMR) imaging has progressed at a remarkable rate, it has been far less successful for membrane proteins, as manifested by the small number solved to atomic resolution. The fact that membrane proteins must be solubilized using detergents leads to large complexes unsuitable for study by NMR. Also, solubilized membrane proteins are often destabilized by the detergent and are thus unsuitable for 3-D (three-dimensional) crystallization. 3-D crystals that have allowed atomic resolution by X-ray crystallography have so far all been obtained from proteins known for their high structural stability. These include four bacterial porins (for a review, see ref. 1), the photosynthetic reaction centre (2), two cytochrome c oxidases (3, 4), two forms of the bovine mitochondrial cytochrome bc1 complex (5, 6), the purple bacterium light-harvesting complex II (7), bacteriorhodopsin (8), and a microbial K+ channel (9). In addition, a membrane inserting toxin (a-haemolysin) has recently been crystallized in 3-D (10).
Mapping genomes
Authors: Denny Paul , Brown stephen D M 
Published: November 2023

Abstract
It is an exciting time in mammalian genetics, as the power of molecular genetic and genomic tools allows a genetic approach to many different biological problems in both mouse and man. The rapid pace of development means, however, that this chapter will quickly become out of date, so we advise the reader to consult the literature for novel and improved methods. The methods described here outline our approach to the generation of genetic and physical maps. There are alternative approaches but there is insufficient space to pro vide a completely comprehensive set of protocols. We describe the use of genetic and genomic resources to construct maps de nova as well as the use of published genetic maps for the construction of higher resolution physical maps such as for positional cloning of an unknown gene.
Mutagenesis of the mouse germline
Authors: Justice Monica J 
Published: November 2023

Abstract
Concern about genetic risk from radiation and chemicals has, in the past, directed the research of germline mutagens in the mouse. Now, however, the Human Genome Project compels functional studies of the mammalian genome (1). This requires new mutations, changing the focus for mouse mutagenesis from studies of genetic risk to isolating new variants at a high frequency (2,3). The value of mutagenesis in the mouse is compounded by the highly developed comparative genetic linkage map between mouse and human, which allows mammalian gene functions to be defined in conserved regions and provides mouse models of human diseases.
Directed mutagenesis in embryonic stem cells
Authors: Plagge Antonius , Kelsey Gavin , Allen Nicholas D 
Published: November 2023

Abstract
Since the first description of homologous recombination in mouse embryonic stem (ES) cells (1,2), gene targeting has become a routine technology in mouse molecular and developmental genetics. At its simplest it allows the introduction of disabling mutations into a gene of interest in ES cells and passage of the disrupted allele through the mouse germ line in order to assess gene function from the phenotypic consequences. Mutations in some hundreds of genes have now been made by this route (see the transgenic/targeted mutation database and Mouse Knockout and Mutation Database (MKMD);. Over the past few years, approaches have become more sophisticated and the potential for more refined sequence manipulation is being realized (see Table J). Amongst these refinements are, for example, conditional mutations which can be induced at defined times in development, or within specific lineages, to assess aspects of function of a gene for which a constitutively null mutation is lethal. In addition, targeted alleles can be ‘cleaned up’ by removing selectable markers to avoid potentially confounding effects arising from the presence of heterologous sequences. Finally, it is feasible to introduce subtle mutations, even single nucleotide exchanges, into genes. Many of these extra possibilities have come about through the exploitation of site-specific recombinases (reviewed in ref. 3). The potential of such technology should be carefully considered before embarking on a simple targeting experiment. Although generation of a more elaborate construct may be more time-consuming, in comparison with the time involved in targeting ES cells and obtaining germline transmission it is insignificant and the additional time investment in designing a more versatile construct might have significant longer-term benefits. In this chapter, we provide an overview of targeting strategies and the factors to be taken into account in the design of a targeting experiment, from initial construct preparation to the generation and analysis of mice carrying the desired mutation.
Optimization
Authors: Ezan Eric , Grassi Jacques 
Published: November 2023

Abstract
The ultimate goal of immunoassay optimization is to satisfy previously determined criteria of accuracy and precision along with the achievement of adequate sensitivity and practicability. All of these four basic characteristics must be kept in mind at all three stages of assay development:
Choice of assay format and reagents,
Setting assay conditions (optimization),
Validation, including application to biological samples.
lmmunoaffinity chromatography of macromolecules
Authors: Hobbs Steve 
Published: November 2023

Abstract
The epitope specificity of an antibody combining site allows the antibody to bind to its corresponding ligand in the presence of other complex molecules such as whole cell lysates or animal sera. If the antibody is immobilized to a solid surface the complex can easily be separated from the mixture, and the reversible nature of the antibody-antigen interaction allows the ligand to be recovered by dissociation once the unbound molecules have been washed away. This process is called immunoaffinity chromatography, a technique now in widespread use (1, 2). It can be used either to purify an antigen, or to remove a specific antigen from a solution in which case recovery of biologically active molecules is not an issue (3). The advent of monoclonal antibody technology (4) has enabled the preparation of homogeneous populations of antibody molecules with identical structure, epitope specificity, and affinity. These features are ideally suited to solid phase antigen isolation because the supply of antibody is reproducible, and elution of antigen from the immobilized antibody will take place under a narrow range of conditions.
Detection of chemically modified DNA in lymphocytes of patients undergoing chemotherapy
Authors: Tilby Michael J 
Published: November 2023

Abstract
Antibodies that recognize DNA occur spontaneously in people and animals exhibiting certain types of ‘autoimmune’ diseases. In rodents not exhibiting autoimmune symptoms, DNA is a weak immunogen, possibly because B cells exhibiting specificity for DNA are deleted (1). However, it was discovered that alteration of the normal structure of DNA by irradiation with lN light greatly increased its immunogenicity and the antibodies, that were then produced, specifically recognized the lN-induced thymine dimers (2). In the 1950s it was also reported that the immunogenicity of DNA could be enhanced by forming an electrostatic complex with methylated bovine serum albumin (2). The mechanisms by which antibodies are made against DNA are not understood. They show the conventional features of oligoclonality, somatic mutation, gene re arrangements, and class switching, but there is no known way that DNA molecules can be processed and presented to T cells in the context of HIA class II molecules (1, 3). Since the 1960s a large number of antisera and monoclonal antibodies have been produced which recognize various DNA structures or modifications. These can be divided pragmatically into three broad groups:
Monoclonal antibody therapy in rheumatoid arthritis
Authors: Choy Ernest H S , Kingsley, Gabrielle H , Panayi Gabriel S 
Published: November 2023

Abstract
Rheumatoid arthritis (RA) is the commonest inflammatory and destructive arthropathy in the world with a prevalence of 1000 per 100000. It is characterized by a chronic symmetrical polyarthritis that leads to joint damage in most patients. The commonest affected joints are the small joints of the hands and feet but it can involve any synovial joint. Rheumatoid factor is present in over 70% of the patients. The long-term prognosis of RA is poor as most patients develop significant disability despite treatment (1). Hence it is a major socio economical burden. In the UK, the cost of RA was estimated at £1.3 billion/year in 1992 (2).
Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates
Authors: Van Der Geer Peter , Luo Kunxin , Sefton Bartholomew M , Hunter Tony 
Published: November 2023

Abstract
Phosphopeptide mapping enables the investigator to study the phosphorylation of a protein of interest in detail. The technique can be used to estimate the number of sites of phosphorylation in a protein, to determine the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for the phosphorylation of specific sites. Additionally, comparative phosphopeptide mapping is an invaluable tool for the determination of the identity, or lack of identity, of phosphoproteins obtainable only in trace amounts.
Assay of protein kinases and phosphatases using specific peptide substrates
Authors: Ruzzene Maria , Pinna Lorenzo A 
Published: November 2023

Abstract
The successful and widespread use of peptide substrates for the assay of protein kinases is grounded on the observation that the great majority of these enzymes are able to recognize phosphoacceptor sites defined by local structural features within their target proteins. Although this local sequence specificity is not the only tool ensuring the selectivity of protein kinases (indeed, in some cases, notably protein-tyrosine kinases, it may not even be a major one) many members of the protein kinase family phosphorylate short peptides reproducing their natural phosphoacceptor sites. The advantage of synthetic peptides, compared with protein substrates, for kinase assays and specificity studies include: they can be readily obtained in large amounts.
Interaction cloning of protein kinase partners
Authors: Stone Julie M , Walker John C 
Published: November 2023

Abstract
Historically, protein kinase partner proteins have been isolated by biochemical means, which requires purification of the interacting protein for antibody production or microsequencing before a cDNA clone can be obtained. However, two molecular biological approaches, the yeast two-hybrid system (Chapter 14) and interaction (or expression) cloning (1), directly yield a cDNA clone encoding the interacting protein. In this chapter we describe some of the recent advances in interaction cloning as a simple, rapid, and powerful alternative to biochemical approaches to identify protein kinase partners. The recombinant protein kinase of interest is enzymatically labelled with 32P by virtue of a five amino recognition site for cAMP-dependent protein kinase (PKA), to facilitate the identification of cDNA clones encoding interacting partners. Briefly, expression of polypeptides encoded by cDNA inserts from a bacteriophage X. expression library is induced, and the polypeptides adsorbed on to nitrocellulose filters. The library is then screened with the radiolabelled protein probe to identify cDNA clones expressing interacting protein kinase partners (see Figure 1).
Identifying protein kinase substrates by expression screening with solid-phase phosphorylation
Authors: Fukunaga Rikiro , Hunter Tony 
Published: November 2023

Abstract
The biological activities of protein kinases are evoked through phosphorylation of their substrate proteins. Phosphorylation of target proteins causes changes in their structure, stability, enzymatic activity, ability to interact with other molecules, or subcellular localization, leading to regulation of a wide variety of cellular processes. Indeed, the identification of physiological targets has been a high priority ever since the first protein kinase was purified. However, the conventional approach of purifying substrate proteins by biochemical techniques is laborious and time consuming, and is especially difficult in the case of scarce proteins. Among the approaches developed to identify protein kinase substrates in a systematic manner are various techniques for determining consensus phosphorylation site sequences using oriented peptide libraries (1) (see Chapter 16), interaction screening for protein substrates by the far-Western method (2), or the yeast two-hybrid system (3, 4) (see Chapter 14).
Analysis of protein kinase interactions using the two-hybrid method
Authors: Jiang Rong 
Published: November 2023

Abstract
The yeast two-hybrid system is a genetic approach that detects proteinprotein interactions (1, 2). This method relies on the functional reconstitution of transcriptional activators, such as Gal4, in the nucleus of yeast cells. The interaction of two-hybrid proteins, one bearing a DNA-binding domain and the other bearing a transactivation domain, results in transcriptional activation of a reporter gene. Certain features of the two-hybrid system make it particularly suitable to analyse protein kinase interactions. First, it is highly sensitive and can detect transient, as well as stable, interactions. This probably results from the fact that the two-hybrid system uses gene expression as a readout to amplify the signal generated by protein-protein interactions. The two-hybrid method has uncovered interactions not easily revealed with other methods. For example, the interaction between the Raf kinase and Ras is readily detectable by the two-hybrid method, but not by co-immunoprecipitation (3, 4). Secondly, as the two-hybrid system analyses interactions within the environment of a eukaryotic cell, many types of post-translational modifications critical for interaction are preserved. While yeast is the popular host for this method, a mammalian version of the two-hybrid system has also been developed (5, 6). Thirdly, the two-hybrid method detects bridged as well as direct interactions, which facilitates the dissection of protein interactions within kinase complexes. Such analyses have revealed important roles for scaffold proteins, bridging proteins, and adaptor proteins in kinase signalling. Finally, many kinases contain separable catalytic and regulatory domains, making protein kinases themselves amenable to the structure/function analysis offered by the two-hybrid system.
Simultaneous in situ detection of DNA and proteins
Authors: Ersfeld Klaus , Stone Elisa M 
Published: November 2023

Abstract
The development of fluorescent in situ hybridization (FISH) in combination with digital imaging technology has contributed greatly to our current view on how the nucleus of eukaryotic cells is organized. It has been shown that chromatin is not randomly distributed but is confined to defined sub-regions of the nucleus (1–4). There is mounting evidence that the non-random chromatin distribution is caused by anchoring of chromosomal sub-regions to elements of the nucleoskeleton, such as the nuclear lamina. The compartmentalization of genetic material and its interaction with specific proteins has important functional implications for various processes such as replication (5), transcription (6) or gene silencing (7–10) and chromosome partitioning (11-15) (Figure JA-G).
Evolutionary biology of the nuclear receptor superfamily
Authors: Escriva H , Robinson M , Laudet V 
Published: November 2023

Abstract
The study of the interplay between gene regulation, embryonic development, and the evolution of animal forms is receiving a growing interest. Numerous authors have studied the Hox gene complexes which control in all metazoan phyla. the establishment of antero-posterior polarity of organisms. By deciphering the phylogenetic relationships between individual genes of the Hox complex, as well a through isolation of homologues of Hox genes in a wide variety of organisms, from Hydrato various insects and vertebrates. the major role played by these genes in the evolutionary process is under intense scrutiny (reviewed in ref.1).
Analysis of steroid/nuclear receptor phosphorylation
Authors: Rowan H G , Weigel N L 
Published: November 2023

Abstract
As with other transcription factors. phosphorylation of nuclear receptors has been shown to be important in regulating receptor function. Studies in this area have utilized a variety of approaches and methodology to analyse receptor phosphorylation and the relation of phosphorylation to receptor function. This chapter will outline these different approaches and the current methodologies available lo identify phosphorylation sites. identify candidate kinases. and assay the functional significance of nuclear receptor phosphorylation. The advantages and disadvantages of each methodology are presented as well as a discussion of general considerations for examining receptor phosphorylation. This information should enable: the beginning investigator to choose the mos1 appropriate method to study receptor phosphorylation in different model systems.
Ligand- and cofactor-regulated transcription with chromatin templates
Authors: Kraus W L , Kadonaga J T 
Published: November 2023

Abstract
Nuclear hormone receptors function as DNA binding, ligand-, and cofactor-regulated transcription factors in the chromatin environment of the cell nucleus (1). Experiments aimed at understanding the detailed mechanisms of transcriptional activation by nuclear hormone receptors should use a bio-chemical approach and should include both the receptor and non-receptor (i.e. co-activators, co-repressors, chromatin, ligands) components of the nuclear hormone receptor signalling pathway. This chapter describes biochemical approaches for the analysis of lignnd- and cofactor-regulated transcription by nuclear hormone receptors. Methods are presented for the synthesis and purification of nuclear hormone receptors and co-activators (Protocols 1and 2). as5embly of chromatin templates i11 virro (Protocols 3and 4).and ligand- and co-activator-regulated transcription of the assembled chromatin template in vitro (Protocols 5—7).
Rho
Authors: Ridley Anne J 
Published: November 2023

Abstract
Rho was first identified in 1985 as a cDNA encoding a small GTP-binding protein related to Ras. Later, Rho was identified as the target of the Clostridium botulinum exoenzyme C3 transferase, which had been shown previously to induce changes to the morphology of mammalian tissue-culture cells. Subsequently, Rho and then a closely related protein, Rae, were shown to be major regulators of the actin cytoskeleton in all eukaryotic cells. A major surprise at that time was that Rae was also required for activation of the NADPH oxidase enzyme complex in phagocytic cells, a process seemingly independent of the actin cytoskeleton. Since then, additional members of the Rho family have been identified and many other cellular responses have been linked to them, including activation of MAPK cascades and regulation of transcription factors, secretion, endocytosis, cell polarity, and the cell cycle. Members of the Rho family have emerged as important coordinators of signal transduction processes activated by a wide variety of both extracellular and intrinsic cell cycle linked signals. Accordingly, they play crucial roles in the development and behaviour of multicellular organisms.
Rab
Authors: Collins Ruth N , Brennwald Patrick 
Published: November 2023

Abstract
A hallmark of eukaryotic cells is their possession of a set of membrane-enclosed organelles. The traffic of proteins between different organelles of the secretory and endocytic pathways is mediated by membrane-bound containers, termed vesicles. Figure 1 illustrates the basic stages of transport. Vesicles form by budding off from a donor compartment. The vesicle is transported to the acceptor compartment where it docks onto the membrane. This is followed by the final stage of fusion which releases the vesicle contents into the acceptor compartment. A variety of experimental strategies have begun to identify components of the transport machinery, which appear to be conserved in all eukaryotic cells. Every transport event is thought to require representatives from a set of conserved protein families. One of these families is that of the Rab proteins, a group of small, monomeric GTP-binding proteins related to Ras. The Rab GTPases will be the focus of this chapter.