bluuebunny/crossref_metadata_2025 huggingface dataset:
Seeding Techniques
Authors: Stura E A , Wilson I A
Published: November 2023

A seed provides a template for the assembly of molecules to form a crystal with the same characteristics as the crystal from which the seed originated. Seeding has often been used as a method of last resort rather than a standard practice. It has been used in situations when, after the first crystallization event. further attempts at crystallizing the same or subsequent batches of the same protein, under apparently identical crystalization conditions, either failed to yield any crystals at all, or provided crystalline precipitates or very small crystals. The use of seeding in crystallization can simplify the task of the crystallographer even when crystals can be obtained by other means. We will explore the various seeding techniques, and their applications in the growth of large single crystals.
Phase Diagrams
Authors: Ries-Kautt M , Ducruix A
Published: November 2023

Establishing a solubility diagram is one way of quantifying the influence of crystallization parameters (sec Chapter 1, Table 1) on the solubility of biological macromolecules. In this chapter, theoretical concepts of solubility will be developed firstly, to reiterate how a compound dissolves in a solvent, and to point out what i particular to biological macromolecules especially concerning the type of molecular interactions which can he found in a macromolecular solution. Indeed, interactions between solvent or additives and a biological macromolecule, which are necesary for its solubilization, can compete with intramolecular interactions responsible for the tertiary structure. The concepts of salting-in and salting-out will be summarized.
Aerodynamics of flight
Authors: Dudley Robert
Published: November 2023

Flying animals comprise the most morphologically diverse and numerically abundant form of life in the terrestrial biosphere. Animal flight can be described at a variety of phenomenological levels, ranging from the biophysics and ultrastructure of llight muscle to the behaviour of migrating locust swarms. An aerodynamic analysis of flight entails description of wing and body motions, evaluation of the forces associated with these motions, and calculations of the concomitant power requirements during locomotion. Additionally, the implications of wing and body morphology for more general measures of flight capacity, such as characteristic airspeed and manreuvrability, can be assessed quantitatively.
Isolation of sinoatrial node cells
Authors: Brown H F
Published: November 2023

The dissociation of cardiac tissue into single cells opened up new possibilities for physiological investigation which have led in the last decade to major advances in knowledge of heart function at the cellular level. Cardiac cell isolation was pioneered in the 1970s (1) and was quickly adopted by many other groups. The basic principle is simple: cardiac tissue is exposed to enzymes (always collagenase and often other enzymes such as trypsin, protease or elastase) in a Tyrode solution containing a very low concentration of calcium. Under these conditions, the cell junctions arc disrupted and will seal over to give intact but separate cells. In practice, it is not always easy to obtain cells that remain calcium tolerant when returned to physiological levels of calcium and that are neither too fragile nor too ‘tough’ for procedures such as patch–damp recording, while retaining their normal membrane pumps and receptors. Nevertheless, the isolation of ventricular and atrial cells is now successfully carried out in many laboratories.
Pharmacological analysis of synaptic transmission in brain slices
Authors: Henderson Graeme
Published: November 2023

1. Introduction

For the study of synaptic transmission in the mammalian central nervous system (CNS) the in vilro brain slice preparation presents a significant number of advantages over in vivo techniques.

(a) Foremost amongst these is the relative ease with which recordings can he obtained using extracellular, intracellular and whole-cell patch-clamp techniques.

(b) In certain slice preparations (e.g. the hippocampus), one can see under a low-power dissection microscope the presynaptic fibre tracts, the areas of synaptic contact and the cell body layer; thus, the stimulating and recording electrodes can be positioned at the desired site under visual control.

(c) Since the brain slice is maintained in an artificial bathing solution, the ionic composition of the extracellular environment can be manipulated.

(d) With intracellular and whole-cell patch-clamp recording, the ionic composition of the intracellular milieu can also be controlled.

(e) Drugs can be applied to the synapse under study in known concentrations by addition to the bathing solution.
Whole-cell patch-clamp recording from neurones in spinal cord slices
Authors: Pickering Tonye , Spanswick Dave , Logan Steve D
Published: November 2023

1. Introduction

The development of the patch-clamp methodology in the laboratories of the 1991 Nobel prize winners Neher and Sakmann has revolutionized our understanding of ion channels. The patch-damp recording technique was originally developed to allow high-resolution current recording from single ion channels (1). The vital step in this process is the formation of a gigaohm seal between the electrode glass and the membrane. This tight seal electrically isolates the few square microns of membrane in the pipette by reducing the leakage current to a level where picoampere currents flowing through single channels are resolvable.
Mathematical description of neuronal firing patterns
Authors: Hindmarsh J L , Rose R M
Published: November 2023

The purpose of this chapter is to explain in a simple and direct manner how to build and develop mathematical models of the behaviour of the membrane potential of a neurone. We do this by presenting models of increasing sophistication. In Section 2, a model due to FitzHugh is used to illustrate the main ideas. This is followed in Section 3 by a series of models, each an elaboration of the previous one. The models of these two sections are qualitative in nature and independent of the precise details of the various ionic currents present in the cell.

Almost all the ideas we introduce below are represented graphically. The necessary graphs can be easily reproduced using a microcomputer, and the reader is strongly encouraged to do so. Some notes on the BASIC programs that have been used to produce Figures 6a and 6b are to be found in the appendix to this chapter.
Electrophysiological techniques for studying the quantitative pharmacology of the isolated spinal cord
Authors: Wallis D I
Published: November 2023

The spinal cord from the neonate mammal provides a useful in vitro preparation for investigating the physiology and pharmacology of motoneurones and the pathways that converge onto them. This chapter gives examples of isolated spinal cord preparations from the neonate rat and illustrates the types of experiment that can be conducted.
Generating mosaics by site-specific recombination
Authors: Golic Kent G
Published: November 2023

Does the developmental fate assumed by a cell depend on signals that it received from its neighbouring cells? This is the fundamental question addressed by the study of cellular interaction during development. If cell-cell communication can be implicated in the specification of cell fates then a series of further questions can be asked. Which cells are talking and which are listening? What molecules are involved and how do they work? When are these molecules needed? In organisms in which there are mutations available that alter cell fate these questions can be fruitfully investigated by the analysis of mosaics. There are molecular techniques available that can tell when and where a gene is expressed; genetic mosaics can be used to determine which part of this expression pattern is functionally significant (see also Dudley and Poethig, this volume).
Cell transplantation
Authors: Prokop Andreas , Technau Gerhard M
Published: November 2023

Cell transplantation is a powerful tool for studying cellular interactions during development. It allows the behaviour of cells of known origin in various kinds of environments to be studied. Thus, cell transplantation has been used in both vertebrates and invertebrates to address several types of questions. For example, isotopic and isochronic transplantations of cells have been used to study the developmental potential of cells (1-9). Heterotopic transplantations allow one to test for the regional determination of cells and their ability to communicate with the surrounding tissue (1, 10-12). Heterochronic transplantations allow one to test for the reversibility of the determination of the transplanted cells or for the time period in which particular cellular communication processes are active (13, 14). Using heterogenetic transplantations (transplantation of mutant or transformed cells into wild-type hosts or vice versa) it is possible to test for the interrelations between genetic functions and for the autonomy or nonautonomy of phenotypes (15, 16). Interspecific transplantations of cells have been used to study the functional compatibility of mechanisms that involve intercellular communication (17, 18).
Using the two-hybrid system to detect protein-protein interactions
Authors: Bartel Paull , Chien Cheng-Ting , Sternglanz Rolf , Fields Stanley
Published: November 2023

Protein-protein interactions are essential in almost all biological processes, including replication, transcription, secretion, signal transduction, and metabolism. Thus a central question in the study of any protein is to determine what other proteins are in contact with it. The answer, in the case of oncogene-encoded proteins for example, has provided enormous insights into the problems of cell cycle control and differentiation. It is, therefore, clear that intense research efforts will continue to focus on identifying proteins that interact with some protein of interest, referred to here as the target protein. Current approaches to detect interacting proteins include traditional methods such as co-immunoprecipitation, crosslinking, and copurification through gradients or chromatographic columns (see Midgley and Lane, Chapter 6 of this volume). These biochemical methods have the major disadvantage that interacting proteins are generally known only as bands of a particular relative mobility on a polyacrylamide gel. To progress from these bands to cloned genes is often a difficult undertaking, involving such methods as the initial purification of sufficient protein for amino acid sequencing or antibody production, followed by additional work to screen a library for the corresponding.
A brief history of the cell cycle
Authors: Reed Steven I , Hutchison christopher J , macneill stuart
Published: November 2023

A particular feature of research into the eukaryotic cell cycle is the diverse range of experimental organisms in use in laboratories around the world, ranging from unicellular yeasts, filamentous fungi (Aspergillus),marine invertebrates (e.g. clams and starfish), and flies, to vertebrates such as Xenopus,mice, and humans. In this chapter we briefly review the history of the eukaryotic cell cycle in terms of the organisms and experimental approaches used, and show how diverse species have contributed, and will continue to contribute, to our current understanding of the workings of the cell cycle.
Controlling entry into tnitosis in fission yeast
Authors: Macneill Stuart a. , Fantes peter a.
Published: November 2023

Universal control over entry into mitosis

As described in Chapter 1 the timing of entry into mitosis in eukaryotic cells is determined by the timing of activation of a protein complex (called MPF) containing one molecule of a cyclin-dependent kinase (cdc2) and one of cyclin B. The genes encoding the cdc2 and cyclin B (cdc13) proteins in the fission yeast Schizo-saccharomyces pombewere identified genetically as being required for entry into mitosis long before their biochemical function was known or even guessed at. Similarly, two key regulators of cdc2 function, the cdc25 tyrosine phosphatase and the weel protein kinase, were first identified and characterized genetically in fission yeast. At present, many other elements implicated in the mitotic control network are known only in this organism. Here we review recent advances in understanding the biochemical basis of the regulatory network that controls entry into mitosis in S. pombe,in identifying new elements involved in this regulatory network, and in understanding how entry into mitosis is integrated with other cell cycle events. We begin with a brief introduction to the biology of the fission yeast cell cycle.
RNA-catalysed RNA processi•ng
Authors: Jacquier Alain
Published: November 2023

RNA processing reactions, as most chemical reactions occurring in living cells, are greatly accelerated by enzymatic catalysis. Proteins were, for a long time, the only class of molecules known to support enzymatic activity. The discovery, a decade ago, that several RNA processing events could be catalysed by RNA molecules (1, 2) thus revolutionized our views in several areas of biology and, most particularly of course, in the RNA processing field. These catalytic RNA molecules have been called ‘ribozymcs’.
Simple tips for photomicrography of embryos
Authors: Stern Claudio d.
Published: November 2023

Illustrating the results of experiments both appropriately and attractively is an important aspect of embryological (as of other) research. Here we give some guidelines that should help to decide how to photograph embryos and improve the resulting photomicrographs, concentrating on some common errors. Good microscopy is as important as the camera techniques themselves and we will start by quickly reviewing some of the basic techniques, giving their advantages and disadvantages for some common applications. At the end of this chapter we will give some advice on how to visualize and photograph embryos after specific techniques.
Chick Limb Buds
Authors: Tickle Cheryll
Published: November 2023

Since the pioneering work of Harrison, Zwilling, Saunders, and others, the limb has become one of the classical model systems in which to study cell interactions in development. A considerable body of work in the last few decades has established the basic principles of limb development and regeneration. Amphibians are often used for studying limb regeneration, while the chick limb has been the main source of information about limb development. Experiments on chick limb buds have led to the identification of regions such as the apical ectodermal ridge (AER), zone of polarizing activity (ZPA) and progress zone as key players (for reviews see refs. 1-6). Recently, molecular approaches have started to be combined successfully with more classical experiments (e.g. ref. 7), and localized misexpression of genes achieved through the use of retroviral vectors (8) (see Chapter 20).
Nuclear Transplantation
Authors: Gurdon John B
Published: November 2023

Nuclear transplantation is a procedure by which the nuclear material of a fertilized egg is replaced by that of another cell. The purpose of this procedure is twofold. The primary purpose, and that for which the technique was devised, is to ask whether the genetic material in a somatic cell is functionally equivalent to that of the zygote nucleus. If a somatic nucleus can replace the zygote nucleus in an egg by supporting normal development, this shows that there has been no loss or permanent inactivation of genes in the course of development leading to the formation of the somatic cell in question. Once this result has been achieved, a related question is how the nucleus of a specialized cell can be switched back to an embryonic state so as to express all genes required for normal development. The second and more recent purpose of nuclear transplantation is to create substantial numbers of genetically identical individuals from the nuclei of one embryo. This second aim has possible practical value in propagating animals with a valuable genetic constitution.
Laser Ablation of Cells
Authors: Eisen Judith , Warga Rachel M , Edgar, Lois G , Wood William B
Published: November 2023

In principle, almost any cell that can be visualized with Nomarski DIC optics can be ablated by laser-irradiation. But such optical clarity is a property of the embryos of only a few animal species, such as the nematode, Caenorhabditis elegans, some other invertebrates (sea urchin, annelids, etc.), and fish embryos. In this chapter, we give some basic methods for laser ablation of individual cells in fish and nematode embryos, but the methods are broadly applicable to embryos of other species where cells can be resolved sufficiently clearly in the intact embryo by interference optics.
Introduction of Genes Using Retroviral Vectors
Authors: Price Jack
Published: November 2023

Retroviruses are naturally evolved agents of gene transfer. Because they work under normal physiological conditions, they can be used to introduce genes into cells in vivo. Potentially, therefore, they can introduce genes into embryos over a wide range of developmental stages. The principal developmental study to which they have been applied is cell lineage, where the transferred gene is non-functional; a more recent application is the use of retroviruses as a localized agent of transgenesis.
|Site-specific recombinases and their interactions with DNA
Authors: Johnson Reid C
Published: November 2023

Site-specific recombination reactions are associated with a wide range of biological processes, especially where generation of genetic diversity is a principal goal. For example, regulatory mechanisms involving site-specific inversion reactions have enabled bacteria to evade host immunity systems by regulating the expression of multiple surface proteins (1). Viruses have evolved highly regulated site-specific recombination systems to integrate into, and excise from, host chromosomes (2). These reactions also play important roles in the partitioning of chromosomes (3, 4) and in the selective activation of genes during terminal differentiation in at least two different prokaryotic systems (5, 6). In eukaryotes, site-specific recombination reactions are responsible for mediating assembly of immunoglobulin and T-cell receptor gene fragments, though progress on the biochemical characterizations of these reactions has been slow (7). The FLP system in yeast, however, has been extensively studied (8) and will be discussed below.
|The HMG-box domain
Authors: Bianchi Marco E
Published: November 2023

The HMG-box is a new kind of eukaryotic protein domain involved in DNA binding. It has been discovered in a number of chromatin proteins, general transcription factors, and transcriptional regulators which control tissue differentiation and sex determination. The most unusual feature of HMG-boxes is that they recognise and/ or produce primarily conformational features in DNA, even if some members of this family can also bind sequence-specifically to particular sites on linear DNA. The diverse set of HMG-box-containing proteins (which we will call ‘HMG-box proteins’ from now on) appear to be involved in a broad spectrum of DNA transactions, ranging from transcription to DNA packaging in chromatin. Such versatility is uncommon and suggests that the ability of HMG-boxes to manipulate DNA can be used in similar ways in highly different functional contexts.
Measurement of mitochondrial protonmotive force
Authors: Brand Martin D
Published: November 2023

Protonmotive force is the central intermediate in bioenergetics. Mitochondria normally produce it by pumping protons from the mitochondrial matrix across the inner membrane during electron transport. Protonmotive force drives the synthesis of ATP by the ATP synthase as well as several other important bioenergetic reactions such as ion transport, transhydrogenation, and proton leak-mediated heat production.
Identification, functional assessment, and quantitation of receptors and membrane regulatory proteins whose ligands are C3 and C4
Authors: Nickells Michael W , Atkinson John P
Published: November 2023

Activation of the complement system leads to the deposition of C3b and C4b on the surface of activators. C3b and C4b in turn are ligands for complement receptor type 1, CR1 (Table 1) which is primarily expressed on peripheral blood cells and mediates the binding of the activators to these cells. C3b and C4b may then be cleaved specifically by the serum serine protease, factor I. This reaction occurs only in the presence of a cofactor protein such as CR1 or factor H (see Figure 1). The resulting proteolytic fragments of C3b and C4b are now poorly recognized by CR1 but serve as ligands for additional complement receptors, namely CR2, CR3, and CR4. These receptors are expressed on B lymphocytes or phagocytic cells.
Southern blotting of cytokine genes
Authors: Diaz M O , Mckeithan T W
Published: November 2023

Southern blot analysis of genomic sequences (1) is one of the basic tools of molecular genetics. This technique is principally used to determine the physical organization of particular DNA sequences for which a probe is available. Its power of resolution has recently been extended up into the megabase (106 base-pair) level by the technique of pulsed field gel electrophoresis (2). In many cases, the organization of large gene families, and the distance between particular cytokine genes have been determined using this technique.
Polymerase chain reaction to quantitate cytokine Mrna
Authors: Platzer Cornelia , Blankenstein Thomas
Published: November 2023

Cytokines display their function preferentially at local sites, occurring transiently and in low concentrations. For more effective intervention in pathophysiologic processes mediated by cytokines it is important to study their complex cross-regulatory network in vivo. Therefore, it is necessary to quantify cytokines in local sites of expression. Detection of the protein is hampered by usually low production of cytokines, by their short half-life in vivo and by the lack of specificity of certain antibodies for intracellular estimation. Consequently, analysis of mRNA has been widely used as substitute for studying variations in local cytokine levels. However, conventional methods of mRNA analysis such as ‘Northern’ and ‘dot blot’ hybridization, and nuclease protection mapping are not sensitive enough to detect mRNA in samples limited by low cell number or low copy-number per cell. Moreover, these methods permit only crude quantification. Thus, polymerase chain reaction (PCR) strategies have been developed that allow quantification of cytokine mRNA at a very sensitive level. Various quantitative RT-PCR procedures have been reported, most relying on the use of external standards and comparing products generated in separate reactions. However, because of the exponential nature of the PCR reaction small variations in the amplification efficiency may result in considerable changes of product yields. In addition, the amount of product can reach plateaux during later stages of the reaction due to consumption of PCR compounds or generation of inhibitors. These problems can be overcome by the use of internal controls in competitive PCR as has been employed by several groups (1–6).
Two-dimensional gel electrophoresis for purification, sequencing, and identification of cytokine-induced proteins in normal and malignant cells
Authors: Smith Diana M , Tran Huu M , Epstein Lois B
Published: November 2023

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) permits high resolution and purification of individual proteins from complex mixtures of proteins. Proteins are separated first according to their charge, employing isoelectric focusing (IEF), and then in the second dimension, according to their molecular mass. 2D PAGE is unequalled in its ability to simultaneously resolve hundreds of proteins from cellular extracts and at the same time provide data on their isoelectric point (pl), molecular mass (Mr), electrophoretic pattern, and relative abundance (1–3). Thus, a large amount of global information concerning the regulatory effects of cytokines on the expression of multiple individual proteins can be obtained in one experiment. 2D PAGE, computer-based analysis of gels and the more recent advances in protein microsequencing technologies, coupled with the capability of searching protein databases for homologous proteins, permits investigators working in the field of cytokine research to answer basic and complex questions about the actions of these important regulatory molecules. For example, it is possible:
Antiviral activity of cytokines
Authors: Lewis John A
Published: November 2023

A variety of biological materials exhibit the ability to induce an antiviral state in cells. To date the best known antiviral agents are the interferons (IFNs), a family of proteins and glycoproteins which are synthesized by virus infected cells or by cells treated with various chemicals and are capable of inhibiting cell-growth as well as inhibiting virus replication and modulating immune responses. Certain other cytokines have been reported to show antiviral properties (1, 2) but in general these effects have not been investigated in depth and the mechanisms underlying such responses are not understood. Assays for specific cytokines are usually of two kinds: bioassays which measure the potency of a preparation are generally based on some specific response of cells to the agent of interest while enzyme-linked immuno-absorbent assay (ELISA) procedures offer a simple and usually sensitive means of measuring the mass of the substance. This chapter describes general procedures for assaying antiviral responses based on our experience with IFNs. Further procedures are described in Chapter 21 and a companion volume (3). These methods can be readily adapted for other cytokines with suspected or demonstrated antiviral activity.
The generation and quantitation of cell-mediated cytotoxicity
Authors: Grimm Elizabeth , Loudon William
Published: November 2023

Cell-mediated cytoxicity (CMC) is defined as the process by which a cell of the immune system directly kills another cell. CMC is composed of a specific series of events occurring between an effector cell (lymphocyte or monocyte) and a susceptible target cell which ultimately results in the delivery of a ‘lethal hit’ by the effector to the target cell. An obligate event in CMC, which distinguishes it from other modes of immunologically mediated target cell killing (e.g. complement-mediated cytolysis) is intimate cell membrane contract between the effector and the target, referred to as conjugate formation. This brief chapter will be restricted solely to the description of lymphocyte mediated CMC systems. Many excellent reviews cover the complicated processes which encompass effector cell-mediated recognition, conjugate formation, triggering, and delivery of the lethal hit ultimately resulting in the death of the specific target (1, 2).
Cytokine regulation of endothelial cells
Authors: Allavena P , Dejana E , Bussolino F , Vecchi A , Ani A Mantov
Published: November 2023

Endothelial cells (EC) have long been considered a ‘passive’ lining of blood vessels, endowed with negative properties, the most important one being that of representing a non-thrombogenic substrate for blood. As such, EC were viewed to participate in tissue reactions essentially as targets for injurious agents. The possibility of isolating and culturing EC from various tissues gave the tools for studying their complex reactions to a variety of activating stimuli. EC have, in this way, emerged as active participants in many physiological and pathological processes. It is now evident that haemostasis, inflammatory reactions, and immunity involve close interactions between immunocompetent cells and vascular endothelium. In particular, the ontogeny and function of white blood cells require an intimate relationship with vascular EC. Cytokines are mediators of these complex bidirectional interactions between leukocytes and vascular elements (for review see 1, 2).
Biological assays for haemopoietic growth factors
Authors: Testa N G , Heyworth C M , Lord B I , Wynter E A De
Published: November 2023

The aim of this chapter is to summarize in vitro standard assays used to investigate the myeloid growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and erythropoietin (Epo). Such methods are illustrated by experimental protocols using murine cells, and with selected examples using human bonemarrow cells.
Assays for macrophage activation by cytokines
Authors: Doyle Anthony , Stein Michael , Keshav Satish , Gordon Siamon
Published: November 2023

The circulating monocyte may undergo a wide spectrum of functional changes during further differentiation. In the absence of inflammatory stimuli monocytes enter the tissues constitutively to become resident macrophages (1). These cells are relatively quiescent with respect to secretory or microbicidal activity and may have as yet uncharacterized trophic functions. In contrast, inflammatory recruitment causes monocytes to rapidly induce their secretory activity. These cells produce a large variety of enzymes, growth factors and cytokines when appropriately triggered. Lymphokines produced at the site of inflammation regulate the fate of elicited macrophages determining whether they are further stimulated to a heightened state of activation with maximal host-defence function, e.g. by interferon-γ (IFN-γ) or whether their secretory and defence activity is held in check (e.g. by IL-4 and IL-10).
Measurement of proliferative, cytolytic, and cytostatic activity of cytokines
Authors: Burke Frances , Rozengurt Enrique , Balkwill Frances R
Published: November 2023

Most cytokines have proliferative, cytostatic, and/or cytolytic activity which is often dependent on the context in which they are acting. IFN-α, β, and γ, TNF, Lymphotoxin, IL-1, IL-4, have all been shown to have proliferative, cytostatic and cytolytic activity depending on the environment (1). Synergistic interactions between cytokines, can often produce powerful antiproliferative effects (2). There is some experimental evidence to suggest that growth inhibitory cytokines such as IFN-a may act in vivo as autocrine or paracrine growth inhibitors (3). Some of the methods used to assess the above par ameters will be described in the following chapter.
Production of cytokine transgenic and knockout mice
Authors: Pasparakis Manolis , Kollias George
Published: November 2023

Transgenic mice have served for many years as fine tools in the dissection and understanding of complex biological phenomena. Studies on gene function in multifactorial and multicellular systems have most benefited, because they often require experimental settings which faithfully simulate the in vivo situation. Additional technological advances which become accessible to a growing number of investigators, offer the opportunity to inactivate or modify endogenous genes in the mouse and open up new possibilities for research. In general, perturbation of gene expression in transgenic systems often leads to measurable alterations in the physiology of the animal and important functional implications come to light.
Development of antibodies to cytokines
Authors: Poole S
Published: November 2023

Antibodies to cytokines can greatly facilitate research on many aspects of these mediators: including the development of specific and sensitive immuno-assays (see Chapter 22); the detection of cytokines in biological fluids, tissues, and cells using immunochemical and immunocytochemical techniques; immunoneutralization of biological activities, and delineation of external regions (epitopes) of the cytokines.
RIA, IRMA, and EUSA assays for cytokines
Authors: Meager A
Published: November 2023

Instructions for 125I-labelling with Enzymobeads (Bio-rad) and the Bolton Hunter reagent (Amersham) are supplied by the manufacturers. A further alternative is the N-bromo-succinimide (NBS) method of Reay (3) and this has been successfully applied to the radioiodination of interleukin-1β (3 (4).

Generally speaking, most current radioimmunoassays for cytokines employ a competitive inhibition assay method. In brief, this means that variable amounts of cytokine, as serial dilutions of a standard or in samples, are incubated with a fixed amount of diluted polyclonal anti-cytokine antiserum, followed by a further incubation period with a fixed quantity of 125I-labelled cytokine. Finally, antibody-cytokine complexes are removed from solution by addition of a second antibody (to the first species immunoglobulin) or other antibody-binding reagent, e.g. protein A. The amount of 125I-cytokine bound therefore decreases as the concentration of unlabelled cytokine increases.
Measuring parameters of growth
Authors: Baserga Renato
Published: November 2023

This chapter deals with the various parameters of growth and how they can be measured. A tissue can grow by: (i) increasing the number of cells; (ii) increasing the size of the cells; or (iii) increasing the amount of intercellular substance. Since the intercellular substance of a tissue is usually a secreted product of the cell, for example collagen, it can be considered as an extracellular extension of the cytoplasm. We can therefore consider an increase in intercellular substance as a variation of an increase in cell size and thereby reduce tissue growth to two mechanisms, growth in size and growth in the number of cells. This is true regardless of whether we are dealing with normal or abnormal growth, with the intact animal or with cells in culture.
Studying transport processes in absorptive epithelia
Authors: Artursson Per , Karlsson Johan , Ocklind Goran , Schipper Nicolaas
Published: November 2023

Transport processes in epithelia have traditionally been studied in a variety of organ and tissue preparations from different species. However, native epithelial tissues are complex and contain many different cell types. Monolayer cultures from established epithelial cell lines have the advantages of being structurally simple, homogeneous, and easy to manipulate. Transport studies in epithelial monolayers are, therefore, often easier to perform and interpret (1). The major disadvantage of epithelial cell cultures is that in most situations they are unlikely to serve as a complete substitute for the more complex normal epithelium. Moreover, epithelial cells in culture may express carriers and enzymes in a different or more variable manner than seen in vivo. Despite these disadvantages, recent studies in epithelial cell monolayers have brought new insights into the functions of a variety of transport mechanisms of endogenous as well as exogenous molecules such as drugs. The aim of this chapter is to present some of the protocols used in our laboratory to study passive and active drug transport processes across monolayers of the human intestinal epithelial cell line Caco-2 grown on permeable supports (Figure 1). However, before such studies are performed it is important to know how to culture and assess the integrity of the cell monolayers on permeable supports in a reproducible manner. Methods for such characterization are presented initially. The methods for studying integrity and transport processes are general and should, therefore, be applicable to solutes other than drugs and to epithelial cell cultures other than Caco-2.
Reconstruction of human skin epidermis in vitro
Authors: Lenoir-Viale Marie-Cécile
Published: November 2023

Since the pioneering work of Rheinwald and Green in 1975 (1, 2) for establishing keratinocytes in culture, numerous advances have been made in the reconstruction of human epidermis in vitro. The more recent techniques are based on the reconstruction of a substrate resembling dermis on which keratinocytes of different sources can be cultivated. After immersion for a few days, these cultures are raised to the air-liquid interface in order to mimic in vivo conditions.
Design and Optimization of Growth Media
Authors: Greasham Randolph L , Herber Wayne K
Published: November 2023

The primary objective in developing a microbial culture medium is to ensure that the required nutrients are present in appropriate forms and at non inhihitory concentrations. This objective can he quite challenging when considering the tremendous diversity of microorganisms present in nature, exhibiting diverse nutritional requirements ranging from simple chemicals, such as atmospheric carbon dioxide and a few inorganic salts, to complex chemicals, such as lipids, vitamins and nucleotides.
Measurement of Biomass
Authors: Ison Andrew P , Matthew Gavin B
Published: November 2023

The on-line measurement of some physical parameters in fermentation broths, such as pH and temperature, is relatively easy and commonplace (see Chapter 4). This has been made possible by the advanced technology that has now become standard. As yet, on-line monitoring of biomass in the fermenter has not become routine. However, there are many techniques available for biomass determination.. This chapter gives detailed protocols for those techniques likely to be used by microbiologists, biochemists. microbial physiologists. and bio chemical engineers. For those requiring substantial investment and specialist equipment, a strategy for the application of the technique is given. In these cases it is necessary to assume that the operator has gained some experience with the equipment and is familiar with the operating manuals of commercially available equipment. Most techniques will require some investment of time, dependent on the experience of the researcher, to personalize the technique for the specific application and to ensure its reproducibility.
Detection Of Sense: Antisense Duplexes By Structure-Specific Anti-Rna Antibodies
Authors: Lukacs Noemi
Published: November 2023

It is generally assumed that inhibition of gene expression by antisense RNA involves the formation of sense: antisense RNA hybrids (for review see refs 1-3). The formation of such hybrids is well established in prokaryotic systems and has also been shown experimentally in a few cukaryotic systems (4-6). In most eukaryotic organisms, however, attempts to detect sense:antisense RNA complexes have failed, although the biological effects of the antisense RNA were obvious. Since in several cases the simultaneous synthesis of both RNA strands has been confirmed by nuclear run-on transcription assays, and since reduced steady state levels of sense RNA have been detected, it has been proposed that sense:antisense hybrids are very rapidly degraded in vivo (7-9). In addition to the classical duplex formation degradation mechanism other antisense control mechanisms may also exist, and have been discussed (1-3, 7-9).
SV40 DNA replication
Authors: herendeen Daniel , Kelly thomas j.
Published: November 2023

Much of what is currently known about the mechanisms of DNA replication in eukaryotic cells has come from studying SV40 and related viruses. SV40 is a small DNA-containing virus that is indigenous to Asian macaques. It produces a lytic infection in permissive primate cells in culture and it is capable of transforming nonpermissive rodent cells to a tumorigenic state. The virus has served as a power-ful model system for understanding many aspects of the molecular biology of eukaryotic cells, including transcription, DNA replication, and the regulation of growth (1-4).
JJroducing antibodies in Escherichia coli: from PCR to fermentation
Authors: Pluckthun Andreas , Krebber Anke , Krebber Claus , Horn Uwe , Knupfer Uwe , Wenderoth Rolf , Nieba Lars , Proba Karl , Riesenberg Dieter
Published: November 2023

The expression of antibody fragments in Escherichia coli has become a widely used technique for several reasons; it is, in principle, an easily accessible methodology for anyone used to growing E. coli, and it can be advanta geously combined with phage-display technology which also makes use of E.coli (1-3).
Counting nucleosome cores on circular DNA using topoisomerase I
Authors: Clark David J
Published: November 2023

The nucleosome is the basic structural repeat of chromatin. It contains a nucleosome core, a single molecule of histone H 1, and a segment of linker DNA. The nucleosome core is composed of a central octamer of core histones (two molecules each of the four core histones: H2A, H2B, H3, and H4) and about 150 hp of DNA which is tightly wrapped around the octamer, forming nearly two complete turns. A nucleosome core protects one negative DNA supercoil from relaxation by topoisomerase I ( l ). This was shown by counting the average number of nucleosome cores reconstituted on a plasmid in vitro using the electron microscope and then comparing this with the average number of supercoils protected from relaxation in the same samples. This fact is now used routinely to determine the average number of nucleosome cores formed on a circular DNA: the DNA between nucleosome cores is relaxed using topoisomerase I, plasmid DNA is extracted, and the number of negative supercoils is measured in a gel containing chloroquine. The number of nucleosome cores is equal to the number of negative supercoils protected from relaxation.
Purification of RNA
Authors: Wilkinson Miles
Published: November 2023

The most important consideration in the preparation of RNA is to rapidly and efficiently inhibit the endogenous ribonucleases which are present in virtually all living cells. There are several classes of ribonucleases which have been well characterized in eukaryotic and prokaryotic organisms, including both endonucleases and exonucleases. Methods designed for the preparation of RNA depend on speed and/or efficiency of neutralization of ribonucleases for their success. Most methods stipulate the use of ribonuclease inhibitors such as RNasin, vanadyl ribonucleoside complexes, guanidinium hydrochloride, guanidinium isothiocyanate, heparin, and dextran sulphate, to name a few. Proteinase K is sometimes used to degrade ribonucleases, and organic solvents such as phenol and chloroform are used to remove ribonucleases by extraction procedures.
DNA sequencing
Authors: Brown T A
Published: November 2023

The rapid generation oflengthy, contiguous DNA sequences is one of the corner stones of molecular biology. Not every molecular biologist wishes to sequence an entire genome, but most will, at some stage in their careers, sequence a cloned fragment from a genomic or cDNA library or use sequencing to check the construction of a recombinant DNA molecule. This chapter describes the methodology for carrying out these tasks.
Combined B cell and T cell epitopes
Authors: Atabani Sowsan F
Published: November 2023

B and T cell epitopes can either be overlapping or contiguous within a single sequence. Indeed, numerous studies have determined that up to, and within, a synthetic peptide sequence as small as ten amino acids, the B and T cell recognition sites may be distinct (1). Surface immunoglobulins are capable of binding to both linear sequences of amino acid residues and conformational epitopes which represent non-adjacent amino acid sequences brought into close proximity within a native protein, whereas the T cell receptor recognizes linear processed peptide epitopes bound to major histocompatibility complexes on molecules. B cells are among antigen-presenting cells capable of presenting peptide epitopes to CD4+ T cells. T cells produce cytokines and other immunomodulators that provide help for antibody production and induction of memory B cells. A single B and T cell epitope comprises the minimal built-in subunit able to trigger T and B cell co-operation in vivo (2). Moreover, studies have shown that overlapping B and T cell epitopes within a synthetic peptide sequence does not impair its B cell immunogenicity (3). The ability of a free synthetic peptide representing a single amino acid sequence, which varies minimally between strains, to act as a functional immunogen able to induce both long-lasting cellular and antibody responses has major implications for the design of future vaccines. Indeed, there exists numerous examples of peptides which are immunogenic in the free, uncoupled state. These results suggest that a satisfactory single sequence can readily induce full protection of all vaccinated animals against a severe challenge with the virulent parent virus (4, 5). Therefore, the topographical relationship between T and B cell epitopes within a native protein sequence is of great interest. This chapter describes experimental methods by which to identify combined and overlapping B and T cell epitopes within the same amino acid sequence with the aid of synthetic peptides.
Generating monoclonal antibody probes and techniques for characterizing and localizing reactivity to antigenic determinants
Authors: Nelson Paul N
Published: November 2023

This chapter initially provides details of protocols useful for the generation of monoclonal antibody probes. Monoclonal antibodies (mAbs) in general, serve as powerful reagents in diagnostic applications and as tools for the investigation of macromolecules and cells (1, 2). However, it is the further scrutiny of their reactivity to desired target antigens, subsequently pursued in this chapter, which paves the way for the development of secure assay systems and reliable functional studies. The localization of a target epitope may be grossly identified though the combination of enzyme (or chemical) degradation studies, e.g. digestion of IgG to yield Fc and pFc’ fragments, and the analysis of mAb reactivity using techniques such as enzyme-linked immunosorbent assay, haemagglutination, and slot-blotting. Alternatively, in situ localization of cell surface or intracellular antigenic determinants may be achieved by immunocytochemical techniques.
Site-directed mutagenesis in epitope mapping
Authors: Perdue Samuel S
Published: November 2023

The utility of amino acid variants in epitope mapping originated with studies of naturally occurring evolutionary variants of hen egg white lysozyme (HEL) (1, 2) and myoglobin (3, 4). Seven avian lysozymes have been isolated that differ from HEL at 3–10 amino acids (human milk lysozyme differs at 52 positions). The epitopes for several anti-HEL antibodies have been crudely mapped to the lysozyme surface based on their differential recognition of these evolutionary variants (1, 2, 5). Similar studies were also performed using monoclonal antibodies specific for either human or sperm whale myoglobin and a panel of myoglobin variants from a number of vertebrates (3, 4).
Preparation of nucleic acids
Authors: Jowett T
Published: November 2023

This chapter describes protocols for:

(a) Purifying DNA for making libraries, pulsed-field gel electrophoresis (PFGE), Southern blot analysis, amplification.

(b) Purifying DNA and total RNA with and without organic extractions.

(c) Separately extracting both RNA and DNA from the same sample.

The methods described for preparing mRNA directly from whole animals utilize affinity chromatography in combination with spin columns or magnetic bead technology. These methods are suitable for work on a microscale to provide material for Northern blots or RT-PCR. Emphasis is placed on those methods which are easiest and quickest to perform as well as produce high purity of the final products.
Isolation and characterization of lagging strand processing activities
Authors: Bauer Glenn A , Melendy Thomas
Published: November 2023

As a result of the bidirectional DNA replication process, lagging strand synthesis results in the formation of Okazaki fragments, hybrid polynucleotide molecules consisting of a short 5’ RNA primer and a long DNA extension. Lagging strand processing involves the removal of the RNA moiety from these fragments and the completion of DNA replication to synthesize a continuous DNA daughter molecule on the lagging strand at the replication fork.