bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inositol phosphatases: constructive destruction of phosphoinositides and inositol phosphates
Authors: Woscholski Rudiger , Parker Peter J 
Published: November 2023

Abstract
Phosphoinositide metabolism and signalling rely on the integrated action of several enzyme activities that act upon the inositol moiety. Phosphorylation and phosphate removal, performed by various kinases and phosphatases respectively, as well as the release of the inositol headgroup by phospholipases, are the crucial players that control the flux of inositol lipid metabolism (Fig. 1). While the pathway of the soluble inositol polyphosphates and their recycling to phosphoinositides is well established (1), phosphoinositide metabolism itself is still relatively unexplored. This has been made evident by the recent discovery of novel phosphoinositides such as phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), which defined a completely new set of pathways and controlling enzymes (2–4). In particular, the influence of certain lipid kinases, such as PtdIns and PtdIns(3)P 3and 5-kinases, on the level of PtdIns(3,5)P2has recently been explored. This particular lipid might be as important for cellular signalling as those already established for the phosphoinositide 3-kinase products—PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3—(see Chapter 2) (5–7) and diacylglycerol (DAG) derived from phospholipase C (PLC) (Chapter 1).
Antisense oligonucleotides: tools for elucidating gene function
Authors: Wolf John J , Michael Flanagan W 
Published: November 2023

Abstract
The goal of antisense technology is to design potent, sequence-specific inhibitors that can be used to elucidate the molecular basis of diseases, validate drug targets, and treat human diseases. Currently, one oligonucleotide (ON) has been approved by the Food and Drug Administration for the treatment of cytomegalovirus-induced retinitis in AIDS patients, and several additional antisense ONs are in various phases of clinical testing for the treatment of inflammatory diseases and cancer (1).
Confocal scanning microscopy in microarray detection
Authors: Schermer Mack J 
Published: November 2023

Abstract
All microarrays require fluorescence scanning to extract their experimental results. The confocal laser scanner delivers the highest image and data quality, a significant performance advantage. This chapter will describe the scanning process from an instrument point of view. It is divided into three parts. Sections 1 and 2 will describe the relevant characteristics of microarrays and all types of microarray scanners. Sections 3– 7 will describe the design options and critical characteristics of confocal scanners. Finally, Section 8 will describe one commercial confocal implementation, the ScanArray®. This section will describe the aspects of microarrays that are most relevant from the point of view of a scanning instrument. Microarrays consist of small samples of DNA or other biological matter arranged on a flat surface. The DNA or other material is tagged with a fluorescent probe so that a fluorescence measurement will reveal the concentration of the sample, with a typical fluorescence dynamic range of between about 400:1 and 4000:1 limited by background fluorescence. The flat substrate is generally made of chemically treated glass, and is often in the form of a 25 mm × 75 mm microscope slide. Microarrays are described at length in several other chapters; this chapter will confine itself to the aspects of microarrays related to fluorescence detection.
The cytoskeleton in the transduction of signal and regulation of cellular function
Authors: Carraway Coralie A Carothers 
Published: November 2023

Abstract
Cells are constantly bombarded with signals from their environments, and their responses to these cues determine all aspects of their function. Each type of cell responds to these environmental stimuli in a sensitive selective, and temporally ordered manner. Each signal is perceived and integrated into a carefully regulated cellular response which reflects a hierarchy imposed by each cell. Responses to stimuli can range from relatively simple ones, such as activation of a housekeeping metabolic pathway, to global reorganizations which encompass multiple metabolic pathways and elicit massive morpho logical alterations. In many cases there is at least minimal involvement of portions of the intracellular cytoskeletal network. In some instances, such as mitogenesis, complete disassembly and reassembly of all the cytoskeletal net works is required. In these cases morphology becomes destiny, and the pathways which link cell activation to cytoskeletal changes are key to the transmission of signals for the pleiotypic effects necessary for appropriate cellular response.
Signalling complexes: association of signalling proteins with the cytoskeleton
Authors: Carraway Coralie A Carothers , Carraway Kermjt I 
Published: November 2023

Abstract
A plethora of studies in the areas of membranes, cytoskeleton and. more recently. signal transduction have contributed to both concepts and technologies for the study of interactions of signalling proteins with the cytoskeleton (for overviews. see refs 1 and 2). These studies have implicated association of signalling proteins with the membrane and with the cytoskeleton in targeting these proteins to appropriate cellular loc,11ions for their participation in signal transduction events. Appropriate cellular localization is requisite for activation processes (3) and can occur reversibly. to turn off signal. The approaches used historically are diverse, ranging from tissue and cell localization by microscopy, to protein sequence analyses for the identification of domains for structure-function determinations to genetic perturbations. The recent explosion in genomics/proteomics and informatics is beginning to contribute in a major way to design and interpretation of cxpcrimenb based on sequence similarities, structural and functional domains. Further, these areas arc expected to play signilicant roles in crossfertilization of concepts among major areas of technology and of hiological investigation.
The study of mRNA-cytoskeleton interactions and mRNA sorting in mammalian cells
Authors: Bermano Giovanna , Hesketh John E 
Published: November 2023

Abstract
It is vital for a wide range of cell functions that newly synthesized proteins are delivered to the correct place in the cell at the appropriate time. There is abundant evidence for targeting motifs in proteins themselves which play a role in protein delivery (e.g. nuclear import signals), and there is increasing evidence that, for a least some proteins, the localization and targeting of their mRNAs plays a significant role in determining the site of protein synthesis within cells (1-3). Thus, in Drosophila embryos, the localization of certain mRNAs functions to ensure correct development by determining the site of synthesis, and in this way, the distribution of proteins critical in development (4). mRNA localization has also been demonstrated to occur in mammalian somatic cells (2), and again its role appears to be to determine or restrict protein distribution. This is likely to be important where there are closely similar isoforms and protein targeting alone could perhaps not segregate isoforms (e.g. actin, creatine kinase, metallothionein) (5-7), where the mRNA and protein are unstable (e.g. c-myc) (8) or where inappropriate deposition of the protein might cause disruption of cell organization (e.g. myelin basic protein, vimentin) (9,10).
Cytochemical detection of cytoskeletal and nucleoskeletal changes during apoptosis
Authors: Van Engeland Manon , Schutte Bert , Hopman Anton H N , Ramaekers Frans C S , Reutelingsperger Chris.P M 
Published: November 2023

Abstract
Apoptosis is an evolutionarily conserved form of physiological cell death by which redundant and damaged cells are eliminated during embryonic development and tissue homeostasis. In diseased tissue the process may be disturbed in such a way that accumulation of cells or increased cell loss may occur. Rapid elimination of the dying cell, without evoking an inflammatory response, is accomplished by disintegration of the cell into apoptotic bodies, which will then be phagocytosed (1). During this process, in which the plasma membrane remains intact, cleavage of cytoskeletal and nucleoskeletal proteins occurs by caspases, a family of cysteine proteases that cleave key proteins at conserved aspartic amino acid residues. The cleavage of cell-cell adhesion molecules leads to loss of contact with neighbouring cells (2), proteolysis of nuclear proteins facilitates nuclear disassembly (3), and the reorganization of actin filaments plays a role in the formation of apoptotic bodies (4).
Methods for detecting proteolysis during apoptosis in intact cells
Authors: Blajeski April L , Kaufmann Scott H 
Published: November 2023

Abstract
Over the past five years it has become clear that protease activation is the single most important event that characterizes the process of apoptosis. In the present chapter, we briefly review the evidence that proteases2014particularly caspases-play an important role in apoptosis. We then discuss various methods that have been applied to the study of caspases and their precursors, including immunoblotting, activity assays, and affinity labelling. We also describe various polypeptides whose cleavage can be used as a marker for caspase activation in situ. Finally, we outline an approach for determining whether a particular cleavage is mediated by caspases, focusing on the potential pitfalls in relying solely upon in vitro cleavage assays or inhibitor studies.
Comparison of protein sequences and practical database searching
Authors: Yona Golan , Brenner* Steven E 
Published: November 2023

Abstract
During the last three decades a considerable effort has been made to develop algorithms that compare sequences of biological macromolecules (proteins, DNA). The purpose of such algorithms is to detect evolutionary, and thus structural and functional, relations among sequences. Successful sequence comparison would allow us to infer the biological properties of new sequences from data accumulated on related genes. For example, a similarity between a translated nucleotide sequence and a known protein sequence suggests a homologous coding region in the corresponding nucleotide sequence. Significant sequence similarity among proteins may imply that the proteins share the same second ary and tertiary structures, and have close biological functions. The prediction of unknown protein structures is often based on the study of known structures of homologous proteins.
Principles of enzyme assay and kinetic studies
Authors: Tipton Keith F 
Published: November 2023

Abstract
The activity of an enzyme may be measured by determining the rate of product formation or substrate used during the enzyme-catalysed reaction. For many enzymes there are several alternative assay procedures available and the choice between them may be made on the grounds of convenience, cost, the availability of appropriate equipment and reagents and the level of sensitivity required. It would not be possible in this account to give detailed descriptions of all the assay mixtures and procedures that have been devised for individual enzymes. The examples that will be presented here are intended to illustrate general features of specific types of assay. Convenient recipes for the assay of individual enzymes can be found in a variety of sources, such as Methods in Enzymology (1), Methods in Enzymatic Analysis (2) and The Enzyme Handbook (3), as well as in the original literature.
Radiometric assays
Authors: Hughes Kelvin T 
Published: November 2023

Abstract
The accurate measurement of enzymatic activity in biological samples is important in many fields of cell biology, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological studies as well as in drug discovery and development. For an enzyme assay to be ‘fit for purpose’, it should be specific, sensitive, quantitative and, ideally, simple and rapid to perform. It should also allow the assay of enzymatic activity in both crude and purified enzyme preparations.
Cell culture
Authors: Touhey Samantha , Heenan Mary 
Published: November 2023

Abstract
Until animal cell culture emerged as a valuable research tool in the 1940s and 1950s, experimental approaches to the study of cellular physiology were limited to either studies in the whole animal or short-term studies with isolated cells or slices from various tissues. The development of cell culture led to a new experimental approach to cellular physiology in which isolated, functionally differentiated cells could be maintained in culture under conditions that allowed direct manipulations of the environment and measurement of the resulting changes in the function of a single cell type. Today many aspects of research and development involve the use of animal cells as in vitro model systems, substrates for virus replication, and in the production of diagnostic and therapeutic products.
In vitro reconstitution of early to late endosome transport: biogenesis and subsequent fusion of transport intermediates
Authors: Gu Feng , Gruenberg Jean 
Published: November 2023

Abstract
Vesicles formed via invaginations of the plasma membrane are responsible for the internalization and turnover of cell surface proteins and lipids, and for the uptake of extracellular ligands and solutes. Although evidence is accumulating for the existence of more than one internalization pathway, most receptors are internalized via the well characterized clathrin-dependent pathway. The bulk of internalized molecules, including solutes, ligands, and membrane constituents, are then delivered to common early endosomes, at least in most animal cell types. From there, many receptors and lipids, as well as a significant proportion of internalized solutes, are rapidly recycled back to the extracellular medium, via recycling endosomes. In contrast, molecules destined to be degraded, including all down-regulated receptors, are selectively incorporated into transport intermediates destined for late endosomes. Protein sorting thus occurs within these common early endosomes, hence they have been referred to as sorting early endosomes (1). In this chapter, we will describe assays which have been used to study the sequential formation and consumption by fusion of transport intermediates between early and late endosomes.
Functional analysis of ion channels: Planar patch clamp and impedance spectroscopy of tethered lipid membranes
Authors: Mayer Michael , Terrettaz Samuel , Giovangrandi Laurent , Stora Thierry , Vogel Horst 
Published: November 2023

Abstract
Ion channels play essential roles in cellular processes such as maintenance of the membrane potential, signal transduction, and osmoregulation and are therefore directly or indirectly targeted by many clinically used drugs (1). Despite the importance of ion channels, the molecular mechanisms of their function are largely unresolved. A nearly unlimited number of mutant proteins provided by combinatorial genetics (2, 3) and huge libraries of potentially active compounds produced by combinatorial chemistry (4) offer enormous possibilities for unraveling molecular details of channel function and for finding new medicines. Electrical recordings of ion channel activity provide the most detailed insight into the function of these important proteins (1, 5, 6); however, presently available techniques are not ideally suited for efficient screening of the existing large numbers of compounds.
Northern analysis, RT-PCR, RNase protection, cDNA array systems, and in situ hybridization to detect cytokine messenger RNA
Authors: Scotton Chris , Burke Frances 
Published: November 2023

Abstract
There are various methods used to detect cytokine mRNA in tissues and cell lines and the commonly used ones are described in this chapter. The varying degrees of sensitivity and suitability for use are discussed.
Northern analysis of RNA allows the quantitation of specific messenger RNA (mRNA) sequences and the determination of their size. The RNA sample is separated by electrophoresis through a denaturing agarose gel followed by capillary transfer to a membrane to which the RNA is subsequently covalently linked. The mRNA of interest is located by probing the membrane with a labelled complementary nucleotide sequence which, in the case of a radioisotopic label, is revealed by autoradiography. Reprobing for a suitable housekeeping gene allows quantitative comparison between samples on the same gel.
Purification, sequencing, and synthesis of cytokines and chemokines
Authors: Struyf Sofie , Wuyts Anja , Damme Jo Van 
Published: November 2023

Abstract
This chapter describes the purification strategy routinely used in our laboratory to isolate and characterize novel cytokines from natural sources (cell cultures, body fluids) (1-3). The subsequent steps that constitute this strategy can easily be modified for the isolation of a particular cytokine by adapting the characteristics of the chromatography (type of columns and buffers) to the biochemical properties of the protein to be purified. The identification of cytokines through the isolation of natural proteins is a valuable approach in basic cytokine research. Based on relevant biological assays, new molecules can be isolated or new biological properties for a known cytokine might be revealed.
Cytokine signalling
Authors: Foxwell B M J , Hunt A E , Lali F V , Smith C 
Published: November 2023

Abstract
The study of cytokines as a discrete topic has existed for approximately two decades. Initial studies focused on the identification of new cytokines, a process that is still continuing, with approximately 150 having been identified to date. The subsequent characterization of cytokine receptors led to the identification of several supramolecular cytokine receptor families (see Figure 1). As a consequence of these studies the complex nature of cytokine receptor structure also became apparent, with receptors often composed of multiple components. These observations gave an early indication of the complexity of cytokine signalling mechanisms. A general overview of cytokine and cytokine receptor structures can be found in the Cytokinefacts book (1).
Ria, Irma, and Elisa For Cytokines And Their Soluble Receptors
Authors: Meager A 
Published: November 2023

Abstract
The lack of absolute specificity of cultured mammalian cells for the activities of individual cytokines and the often relatively poor reproducibility of cytokine bioassays has created the need for more specific and reproducible, alternative assays. This need has largely been filled by the development of immunoassays for cytokines. Such assays are based on antibodies to each different cytokine and are generally reasonably sensitive and reliable and are quick and easy to perform. Homogeneous cytokines of known protein content can be used to calibrate immunoassays in terms of cytokine concentration (e.g. pg or ng/ml), since the extent of antibody binding is related in a dose-dependent manner to the im­munoreactive mass of cytokine present. While the correlation of immunoassay results with biological activity appears possible, such correlations are dependent on a number of variables, including the specificity of antibodies, immunoassay type, and cytokine preparation used for calibration. It is therefore inadvisable to express immunoassay results in units of biological activity (1).
Quantitative biological assays for individual cytokines
Authors: Wadhwa Meenu , Bird Christopher , Dilger Paula , Mire-Sluis* Tony , Thorpe Robin 
Published: November 2023

Abstract
Cytokines have often been discovered in complex mixtures by observing their effect on a biological system. This usually becomes the definitive property ( or one of several properties) of the cytokine and can become the basis for its bioassay. Cytokine bioassays are based on various biological effects such as cell proliferation, cytotoxic/cytostatic activity, kinetic effects, antiviral activity, colony forma­tion, or induction of secretion of other cytokine/non cytokine molecules (1).
Gap junction-mediated interactions between cells
Authors: Becker David , Green Colin 
Published: November 2023

Abstract
Gap junction channels are expressed in almost all types of cell. Their roles are diverse and are crucial for normal embryonic development and the functioning of adult organs. Point mutations, in a growing number of members of the gap junction family, appear to underlie several human diseases including profound non-syndromic deafness, heart disease, peripheral nerve myopathy, skin disorders, and cataract promotion. This has led to a rapid increase in the number of researchers investigating gap junctional communication in these disease conditions. In order to study gap junctional interactions between cells, there are three basic approaches which, when integrated, provide a rounded picture of communication in any particular system.
Oocyte–granulosa cell interactions
Authors: Vanderhyden Barbara C 
Published: November 2023

Abstract
Oocyte development occurs largely within the context of a complex and continuously changing environment, involving proliferation, differentiation, and apoptosis of the surrounding somatic or granulosa cells. Decades of investigation have resulted in protocols that enable the replication of many aspects of this process in vitro, some of which are able to maintain or enable to some degree the developmental capabilities of the oocyte. While the acquisition of developmental competence relies most heavily on adequate communication between the oocyte and granulosa cells, the process is complicated further by the fact that these cell–cell interactions are strongly influenced by hormones and growth factors generated by the hypothalamic–pituitary–ovarian axis. This chapter describes some of the methods currently used to investigate oocyte–granulosa cell interactions in pre-antral and antral follicles as well as the procedures that enable the study of the influence of each cell type on the other.
Cell–cell interactions in early mammalian development
Authors: Fleming Tom P , Eckert Judith J , Kwong Wing Yee , Thomas Fay C , Miller Daniel J , Fesenko Irina , Mears Andrew , Sheth Bhavwanti 
Published: November 2023

Abstract
Cell–cell interactions during early development prior to implantation play a key role in the morphogenesis of the blastocyst. The essential processes in blastocyst formation that are dependent upon intercellular contact and signalling and which occur concurrantly are the differentiation of the trophectoderm epithelium and the segregation of the inner cell mass (ICM) as a separate cell lineage from the epithelium. The trophectoderm forms the wall of the blastocyst, is responsible for generating the blastocoel cavity by transport processes, and, later in development, gives rise to trophoblast and most placental cell lineages. The ICM is a non-epithelial cell cluster located on the inner surface of the trophectoderm at one pole of the blastocyst from which the entire fetus forms after implantation (Figure 1).
The Importance of Closure
Authors: Walsh Joseph 
Published: November 2023

Abstract
Building on our review of the many types of endings that occur in clinical practice, I focus in this chapter on how positive endings are significant to the wellbeing of both the client and practitioner. I elaborate on this topic with discussions of eight case vignettes. All of them have been provided by clinical practitioners as examples of situations in which the ending turned out unexpectedly to be the most important aspect of the intervention. The closure that was achieved served to resolve the clients’ and practitioners’ work on problem issues, work that had been unsatisfying during the earlier stages of intervention. The chapter concludes with general principles for practitioners and supervisors to follow in maximizing the chances for positive endings.