bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inositol Phosphate Second Messengers
Authors: Kirk C J , Morris, A J , Shears S B 
Published: November 2023

Abstract
The role of inositol lipid hydrolysis in receptor-mediated cell signalling was first suggested by Michell in the mid-1970s (1). However, the current growth in interest in this subject probably stems from the more recent realization that receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] generates two intracellular second messengers. Following receptor activation, this lipid is hydrolysed by a ‘phosphoinositidase C’ to yield diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], which mobilizes Ca2+ from an intracellular pool (see references 2 and 3 for reviews).
Thin-layer chromatography
Authors: Henderson R James , Tocher Douglas R 
Published: November 2023

Abstract
Thin-layer chromatography (TLC) has long been used for the separation of lipid mixtures into their component lipid classes. Despite the fact that the more modern technique of high performance liquid chromatography (HPLC) is finding applications in lipid analysis, several features still make TLC particularly useful for this purpose. For example, although sophisticated and expensive apparatus can be purchased for TLC, good results on a qualitative basis can be obtained relatively simply using the minimum of equipment. Analyses are performed rapidly and many samples can be analysed simultaneously alongside standards. The separated classes can be visualized and the chromatograms kept as a permanent record, or, alternatively, samples can be recovered for further analysis. The present chapter considers the practical aspects of employing TLC for the analysis of lipids. It concentrates mainly on TLC in the ascending mode using glass plates coated with adsorbent because this is the most common form used in lipid analysis. Detailed descriptions of the historical and theoretical aspects of the technique can be found elsewhere (1). TLC with flame ionization detection (TLC-FID) is also considered since this technique is becoming increasingly popular for the quantitative analysis of lipid classes.
In situ hybridization in plants
Authors: Jackson David 
Published: November 2023

Abstract
In situ hybridization techniques provide an exquisitely sensitive method to study tissue-specific or organ-specific patterns of gene expression.
The techniques described in this chapter and based on refs 1--4, have been developed to study the expression of several genes involved in the biosynthesis of pigment (anthocyanin) in different floral organs (petal, style, stamen) of Antirrhinum majus. The methods involve hybridization of tritium-labelled single stranded RNA probes to mRNA in sections of fixed and wax embedded plant material. Hybridization is visualized using photographic emulsion, where radioactive emissions produce latent images in the emulsion which form silver grains when developed. The silver grains are visualized by dark-field microscopy. The techniques are applicable to a wide range of plant tissue and to different probes, including those involved in plant-pathogen interactions. For example, these techniques have been used at the John Innes Institute to study:
(a) storage protein gene expression in developing pea embryos (A. Hauxwell, personal communication);
(b) tobacco rattle virus RNA in and around infection sites on tobacco leaves (V.
Modifications to phosphoinositide signalling
Authors: Conn P Jeffrey , Wilson Karen M 
Published: November 2023

Abstract
Activation of a wide variety of neurotransmitter and hormone receptors results in hydrolysis of membrane phosphoinositides. It is now clear that phosphoinositide hydrolysis is the first step in a multifunctional system employed by neurotransmitter receptors for signal transduction. A number of studies over the last several years have led to a relatively detailed understanding of the nature of this response. These studies have been authoritatively reviewed by many experts in the field (1,2).
Exogenous kinases and phosphatases as probes of intracellular modulation
Authors: Hubbard Michael J , Klee Claude B 
Published: November 2023

Abstract
Protein phosphorylation is a major mechanism of intracellular regulation in neural tissues, modulating a variety of enzyme activities and processes such as those involved in neurosecretion, protein and catecholamine synthesis. Since the phosphorylation state of a phosphoprotein is dynamic, determined by the relative activities of kinases and phosphatases, factors affecting these enzymes must be addressed. Experimental manipulation at the level of kinases and phosphatases is a powerful tool for investigating intracellular regulatory mechanisms involving protein phosphorylation.
The expression of neurotransmitter receptors and ion channels in Xenopus oocytes
Authors: Leonard John P , Snutch Terry P 
Published: November 2023

Abstract
The microinjection of exogenous RNAs into Xenopus oocytes allows the examination of a variety of types of excitability proteins in a standard test environment. These include ion channels, neurotransmitter receptors, pumps, and transporters (1-5). In addition to expression studies, the oocyte system has proven useful for the isolation of cDNAs encoding these molecules [including a kainate receptor/channel (6), the serotonin-lC receptor (7, 8), and a delayed rectifier K channel (9)]. Furthermore, the oocyte system is a popular choice for studying the structural basis of receptor and channel function using mutagenized cDNAs (10-12). The ability of Xenopus oocytes to efficiently and accurately translate foreign RNA has been described in a previous volume in this series (36). This chapter will discuss the necessary requirements to successfully express ion channels and neurotransmitter receptors in Xenopus oocytes.
Molecular approaches to the structure and function of the GABAA receptors
Authors: Stephenson F Anne , Duggan Michael J 
Published: November 2023

Abstract
The GABAergic system is ubiquitous in the mammalian brain. It is estimated that GABA may function at up to 50% of the synapses and thus it is involved in the central regulation of multiple physiological processes. The GABAa receptors are the most abundant mediators of GABAergic neurotransmission. They are GABA-gated chloride ion channels and they are now known to be members of the ligand-gated ion channel super-family. In addition, the GABAa receptors are the site of action for several classes of centrally-acting drugs which include the benzodiazepines, the barbiturates, some steroids and non-competitive antagonists of GABAergic neurotransmission such as the cage convulsant compounts and picrotoxin (1). Indeed, it was the coexistence of the binding site for the benzodiazepines on the GABAa receptor oligomers which led to the isolation of this protein to apparent homogeneity by benzodiazepine affinity chromatography.
Growth and morphology of Fusarium graminearum and other fungi in batch and continuous culture
Authors: Trinci Anthony P J , Robson Geoffrey D , Wiebe Marilyn G , Cunliffe’ Bryan , Naylor T William 
Published: November 2023

Abstract
In 1964, Rank Hovis McDougall PLC (RHM) decided to develop a microbial fermentation to convert wheat starch (a by-product of the production of wheat protein) into high-quality protein foods for human consumption. Filamentous fungi were chosen for this process because of their nutritional and organoleptic properties, and because mycelial biomass, unlike unicellular biomass, can be formulated into a convincing facsimile of fibrous foods such as meat (Figure 1) and poultry. In 1969, Fusarium graminearum (Schwabe) was isolated from soil and this strain (A 3/5) has been used for all subsequent production of mycoprotein. The fermentation process developed for biomass production involved growing A 3/5 in continuous culture at a dilution rate of c. 0.19 h-1. Using a 1.3 m3 stirred-tank reactor (STR), RHM was able to produce c. 50 t of mycoprotein per year. In 1980, after the production of a two-million-word report describing the results of a ten-year programme of toxicology studies, RHM was given clearance by the UK Ministry of Agriculture, Fisheries and Food to market mycoprotein foods for human consumption.
Design of Protein Structures
Authors: Sander Chris , Scharf, Michael , Schneider Reinhard 
Published: November 2023

Abstract
The design of protein structures, whether by variation of natural proteins or from scratch, is an essential first step in the development of newly engineered proteins. Designs based on theoretical and computer methods enter the protein design cycle of repeated steps of experimental testing and improvement, much as in other engineering disciplines. Success in protein design depends on how well we make use of this cycle and on how well we understand the principles of protein folding, both in terms of the underlying molecular physics and in terms of knowledge extracted from the data-bases of macromolecular sequences and structures.
Assay of lipoprotein lipase and hepatic lipase
Authors: Bengtsson-Olivecrona Gunilla , Olivecrona Thomas 
Published: November 2023

Abstract
Lipoprotein lipase (LPL) and hepatic lipase (HL) are two key enzymes in the metabolism and interconversion of lipoproteins (1-3; Table 1). The reactions occur at endothelial surfaces of blood vessels, where the enzymes are attached by interaction with polyanions, probably heparan sulphate proteo glycans anchored in the endothelial cell membrane (4). LPL appears all over the vascular bed of most extrahepatic tissues, though in varying amounts, while HL is localized at the liver and some steroid hormone producing glands. LPL acts mainly on the triglycerides of chylomicra and VLDL. The released fatty acids are taken up by nearby cells or are circulated in blood as albumin bound free fatty acids. LPL is under metabolic and hormonal control. HL acts mainly on smaller lipoproteins (HDL, IDL, chylomicron remnants). Its function is not yet fully understood. Some animal species, e.g. guinea pigs and cows, have little or no HL. HL is regulated by steroid hormones, but does not seem to be metabolically regulated.
Membrane traffic pathways polarized epithelial cells
Authors: Ellis Juliet A , Jackman Mark R , Perez Jorge H , Mullock Barbara M , Luzio J Paul 
Published: November 2023

Abstract
Epithelial cells maintain a selective permeability barrier between extra cellular environments and regulate the composition of the fluids on either side of the cell layer. They achieve this by a diverse array of specialized activities including ion transport, secretion, and transcytosis (1), which are dependent on the polarized distribution of proteins and lipids between the apical and basolateral plasma membrane domains of the individual cells (2). Establishment and maintenance of cell surface polarity requires the formation of intercellular contacts and junctions, correct cell-substrate adhesion, highly ordered cytoskeletal networks, and specialized protein and lipid plasma membrane traffic pathways (3). This chapter confines itself to such membrane traffic pathways in polarized epithelia and, in particular, describes methods to examine the sorting of proteins to the correct plasma membrane domains. We provide protocols to study two different polarized cell types; a human, colon derived, adenocarcinoma cell line with enterocyte-like morphology called Caco-2 (4), which can be grown on filters in vitro as a polarized cell layer, and rat hepatocytes (5), which can be studied in situ in rat liver. We also discuss data from the MDCK (Madin-Darby canine kidney), renal-cortex cell line (6), which can also grow in culture as a polarized cell layer and from which much of our present knowledge of membrane traffic pathways in epithelial cells is derived.
Sorting between exocytic pathways in PC12 cells
Authors: Cramer Louise P , Cutler Daniel F 
Published: November 2023

Abstract
Exocytic protein traffic in neuronal and endocrine cells is complicated by the presence of more than one route to the cell surface. In addition to the constitutive secretory pathway, regulated secretory routes are also present in these cells. Regulated secretion is characterized by the intracellular storage of secretory material which is only released from the cell following the application of an external stimulus. This is in contrast to constitutive secretion in which proteins are not stored inside the cell but are constantly released. The organelles acting as storage compartments in neuronal and endocrine cells are both striking and characteristic of the cell type. In endocrine cells, dense core granules (DCGs) store secretory proteins, peptides, and small molecules such as classical neurotransmitters. In neurons, synaptic vesicles store neurotransmitters but not proteins. The coexistence of both regulated and constitutive secretory pathways in one cell was demonstrated by Kelly and co-workers in a DCG-containing cell line (AtT20) derived from the anterior pituitary (1, 2). This demonstration implied that regulated secretory cells must be able to sort regulated secretory proteins and constitutive secretory proteins so that only regulated secretory proteins are targeted to DCGs during granule formation. Later the coexistence of both pathways was also demonstrated in other regulated secretory cells.
In vitro reconstitution of vesicular transport from the endoplasmic reticulum to the cis Golgi in semi-intact cells
Authors: Schwaninger Ruth 
Published: November 2023

Abstract
Proteins destined for the plasma membrane, lysosomes, and endosomes, or the extracellular fluid are transported between organelles of the exocytic or secretory pathway of eukaryotic cells by vesicles that bud from one compartment and fuse with another. A high degree of specificity for this sequential budding, targeting, and fusion of transport vesicles must be maintained, or the cell would rapidly destroy its highly compartmental organization. Progression of newly synthesized proteins along this pathway can be followed by observing the maturation of N-linked oligosaccharide chains of glycoproteins during their passage through the different organelles. This is possible as individual oligosaccharide-processing enzymes reside in particular subcellular compartments.
Lipid modifications involved in protein targeting
Authors: Masterson Wayne J , Magee Anthony I 
Published: November 2023

Abstract
Several contributions in this volume describe the role of protein sequence motifs in acting as determinants of targeting specificity. In the last 12 years or so a group of post-translational modifications of proteins with various lipid molecules has been identified in eukaryotic cells (1). In many cases these modifications have been shown to be essential for the subcellular localization of the proteins carrying them. Many intracellular proteins are now known to carry fatty acid modifications which serve such a purpose. The 14 carbon saturated fatty acid myristate (C14:0) is amide-linked to the amino-terminus of a range of proteins, exemplified by the pp6osrc (proto) oncogene product. Site-directed mutagenesis has been used to demonstrate that this acylation is required both for membrane localization and transforming activity (2). The myristoylation enzyme has been isolated and is highly specific for acyl chain length (3). Inhibitors which interfere with this pathway provide an attractive approach to specific chemotherapy of cancers and viral infections including HIV (4).
Measurements of sedimentation coefficients
Authors: Steensgaard Jens , Humphries, Steven , Spragg S Peter 
Published: November 2023

Abstract
The sedimentation coefficient (s-value) of a particle is, by definition, its sedimentation velocity in a unit gravitational field (1). It is a commonly-used measure of particle size, especially when a molecular mass cannot be determined or when it is not physically meaningful as in the case of ribosomal subunits or other subcellular components. The sedimentation coefficient (given in Svedberg units, S) is a useful measure of particle size and it is an important parameter for the optimization of centrifugal procedures and the characterization of biological particles. This chapter describes how sedimentation coefficients can be calculated in practice.
Separations in zonal rotors
Authors: Graham John 
Published: November 2023

Abstract
This chapter is primarily concerned with the general design and operation of zonal rotors, but it also illustrates the advantages of zonal rotors by quoting some specific separations. The name ‘zonal’ rotor is unfortunate inasmuch as it is sometimes confused with ‘rate-zonal’ centrifugation. Rate-zonal centrifugation refers to the fractionation of a mixture of different sized particles as they move down (or up) through a gradient under the centrifugal force generated by the centrifuge; each population of particles moving independently of the others as a discrete band or zone at a rate roughly proportional to their size. Zonal rotors were originally designed to maximize the resolution of this process but they can be used for any type of centrifugation except, usually, simple differential pelleting. They also offer the capacity which makes the handling of large volumes of sample relatively easy.
In vivo voltammetric methods for monitoring monoamine release and metabolism
Authors: Stamford J A , Crespi, F , Marsden C A 
Published: November 2023

Abstract
Electrochemical detection (ECD), coupled with high performance liquid chromatography (HPLC), has become the method of choice for the assay of trace amounts of amine neurotransmitters and their metabolites in brain tissue samples since its introduction in the seventies (1-3). In vivo voltammetry essentially involves implanting a miniaturized version of the HPLC electro chemical detector into the brain.
Productivity tools biochemistry
Authors: Beynon Robert J , Easterby Johns. 
Published: November 2023

Abstract
Most commercial software for microcomputers is designed to sell many copies, and thus to be of value to a great many users. In general, this software provides tools that can be shaped or extended to meet user needs. This contrasts with specific software written to perform just a single task; these ‘vertical applications’ are often written by individuals who cannot devote the resources that are needed to generate a fully-supported, powerful, widely applicable tool. Such specific software also has its own advantages, being aimed more closely to a specific task, but its drawback is the relatively high cost of production. General-purpose programming environments, ranging from databases to graphics packages and spreadsheets, offer powerful access to the capabilities of modern microcomputers, and the development of a vertical application within such an environment is often preferable to the development of a task-specific program in a high-level language. Such environments are often overlooked, perhaps because their potential is not immediately apparent. in biochemistry. It is not possible to be comprehensive, and it would be wrong to assume that mention of specific packages implies a general recommendation, although such packages have been of use in our laboratories. At best, we can illustrate some of the potential of such packages, and indicate some of the considerations that are relevant when considering the acquisition of such a package. The scope of the chapter is also restricted to two of the most common types of machine, IBM PC and compatible systems running MSDOS, and the Apple Macintosh series. One of the difficulties attendant upon the introduction of proprietary software is the need to input and use data derived from other packages and instrument control systems, or to output the data in machine-readable form for use by other software. Some discussion of the principles of data exchange is therefore warranted, although there are so many packages on the market that they cannot be covered individually.
Introduction to Risk Calculation in Genetic Counselling
Authors: Young Ian d. 
Published: November 2023

Abstract
Genetic counselling is widely accepted as an integral part of the management of every patient with an inherited disorder. This book describes in detail how the risk can be estimated or calculated for most counselling situations, using nothing more complex than a knowledge of the simple laws of probability and a pocket calculator. All patterns of inheritance are considered. Particular attention is paid to the segregation of balanced chromosome rearrangements and risk calculation in single gene inheritance taking into account factors such as reduced penetrance, late age of onset, consanguinity, and the extended family. Also covered at length is the use of linked markers, an area of rapidly increasing importance in carrier detection and prenatal diagnosis. The text has been written in a straightforward "user-friendly" style with liberal use of worked examples and will be of benefit to everyone involved in the provision and delivery of genetic counselling services.
Identification of a glycosyl phosphatidylinositol anchor on membrane proteins
Authors: Hooper Nigel M 
Published: November 2023

Abstract
The explosion in the number of membrane proteins anchored by a glycosyl phosphatidylinositol (G-PI) moiety is due primarily to the ease with which the presence of such an anchoring structure can be identified. The identification of a G-PI anchor on a protein does not have to rely upon direct structural determination (see Chapter 8). A number of techniques exist whereby the presence of such a structure can be characterized even if the protein is present in low abundance or is not readily purified. The susceptibility of a protein to release from a membrane surface by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) was the original indicator for the presence of a G PI anchor (1). This technique is still the initial method of choice. Purified, recombinant PI-PLC is now available commercially, and simple sedimentation of membranes or whole cells can be used to separate the membrane-bound and released proteins. However, some G-PI anchors are resistant to hydrolysis by bacterial PI-PLC, and alternative methods are required to identify, and/or confirm, the presence of such a structure. These more recent techniques include using the mammalian G-PI-specific phospholipase D (G PI-PLD) recently isolated from plasma as an alternative to bacterial Pl-PLC to demonstrate the release of G-PI-anchored proteins from the membrane. Also, several antisera are now available which recognize the G-PI anchors of unrelated proteins following endogenous or exogenous phospholipase C digestion.
Phospholipases that degrade the glycosyl-phosphatidylinositol anchor of membrane proteins
Authors: Low Martin G 
Published: November 2023

Abstract
An unusual and useful property of the G-PI-anchored proteins is their ability to be released from membranes by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) which degrades the anchor. In fact, as described in Chapter 5, this property provided the first indications for the existence of G PI anchors (1, 2). Because the functions of the G-PI anchor remain obscure there has been growing interest in the possibility that eukaryotic cells might regulate expression of G-PI anchored proteins by activation of endogenous G-PI-specific phospholipases (2). Thus from a practical point of view interest in these phospholipases is of two quite distinct types.
Chemical and enzymic analysis of glycosyl-phosphatidylinositol anchors
Authors: Ferguson Michael A J 
Published: November 2023

Abstract
Glycosyl-phosphatidylinositol (G-PI) membrane anchors are ubiquitous among the eukaryotes and are utilized by a wide variety of cell-surface glycoproteins. The G-PI anchor can be thought of as an alternative to the hydrophobic transmembrane polypeptide domain for membrane protein anchorage. Discussions of the biosynthesis and the known and possible functions of G-PI anchors are to be found throughout this volume and have been extensively reviewed elsewhere (1-3). This chapter will concentrate on structure determination.