bluuebunny/crossref_metadata_2025 huggingface dataset:
Force platform and kinematic analysis
Authors: Btewener Andrew a. , Full rorert j.
Published: November 2023

Animals exert forces to move objects in their environment and to move themselves from place to place. Body segment motion, in turn, produces inertial forces that must also be countered by the muscles controlling motion at related joints. Kinetics involves the study of the forces acting on rigid bodies. Kinematics is the study of the motions produced by those forces. Linking the two analyses together, provides a powerful non-invasive approach for studying the hiomechanics of animal locomotion, and motor activity in general. For instance, measurements of the forces applied externally to an animal’s body (such as ground reaction forces exerted on an animal’s limbs when it runs), coupled with kinematic and anatomical data of the limb, allows an analysis of the internal forces that limb muscles must produce to counter externally applied forces. The data obtained from such studies are fundamental to our understanding of how the design of muscu\oske\ctal structures is related to the physical demands placed upon them during various functional activities of the animal in question. In addition, kinematic analysis alone can provide estimates of the inertial loads associated with body segment motion that must be controlled by musdes. Such analyses are particularly important in relation to studies of nervous system control of motor function.
Mechanical work in terrestrial locomotion
Authors: Blickiian R , Full Robert J
Published: November 2023

The amount of metabolic energy required to travel a given distance by an animal using legged, terrestrial locomotion exceeds the costs of swimming by a factor of more than eight and of flying by more than a factor of four (I). Since terrestrial locomotion is expensive, it is reasonable to assume that the relatively high metabolic power input should generate a considerable, and therefore easily measurable, amount of mechanical power output. Surprisingly, a simple and direct link between metabolic and mechanical power during terrestrial locomotion has proved to be elusive (2). Fortunately, the challenge of investigating the link has been rewarding because these studies have and will continue to increase our knowledge of musculoskeletal function for all modes of locomotion.
Assays of cell adhesion
Authors: Hoffman S
Published: November 2023

Classic embryological studies suggested that cell adhesion along with cell migration, cell proliferation, cell death, and differentiation are the primary cellular processes of development. Later studies demonstrated that cells pre pared from embryonic tissues would reaggregate in a relatively tissue-specific manner, but little was known about the molecular mechanisms involved. The study of cell adhesion became a prominent topic in modern cell biological research in the 1970s and 1980s with the identification of specific adhesion molecules. It then became possible to study and compare the functions, distributions, and structures of these molecules (1-5).
The Xenopus oocyte cell-cell channel assay for functional analysis of gap junction proteins
Authors: Dahl Gerhard
Published: November 2023

The paired Xenopus oocyte expression system was originally designed as a functional assay to identify mRNAs involved in the formation of intercellular channels, the functional units of the gap junction (4). Subsequently, the paired oocyte assay was used to demonstrate that the expression of a single exogenous gene, rat connexin32, could induce intercellular communication (3). Thus, a functional criterion for the identity of a gap junction or cell-cell channel-forming protein was established. More recently, a number of genes encoding proteins highly related to connexin32 have been cloned (1). This proliferation of potential gap junction proteins has increased the utility of functional expression systems designed to examine cell-cell channels.
Compartmented culture analysis of nerve growth
Authors: Campenot Robert B
Published: November 2023

The compartmented culture was initially designed as a model system to study the formation and maintenance of neuronal projections. In compartmented cultures, axons (neurites) originating from neurones plated in a proximal compartment grow across a silicone grease barrier and enter into a separate fluid environment within a distal compartment, analogous to axons from neuronal cell bodies in one location in the nervous system projecting their axons through nerves or tracts to targets in another location (Figure 1). The first experimental use of the system was to show that axons of sympathetic neurones will enter into and grow within a distal compartment only if it is supplied with nerve growth factor (NGF) (1). Among the important principles established by this work is that NGF promotes growth of the neurite at its site of application to the neurite and not elsewhere, which is exactly what would be expected of a factor whose function is to direct nerve growth to or within a target location. Thus, NGF locally produced in vivo within the target tissue (and by Schwann cells and fibroblasts in the nerve after injury) would be expected to locally promote nerve growth (and regeneration). Work with compartmented cultures indicates that the immediate mechanisms by which NGF promotes growth are localized to the site of growth and do not involve the cell body or gene expression.
Crossed and hierarchical analysis of variance
Authors: Iles Terence c.
Published: November 2023

This chapter describes the analysis of experiments where measurements are made of the response to several factors, extending the one-way ANOVA where just one factor is investigated. Experiments where the response is measured at all combinations of levels of several factors are called factorial experiments. The value of such experiments is in the opportunity of assessing the interrelationships or interactions between factors; it is possible that the response to one factor may differ according to the levels of a second factor. Also, a single well-designed factorial experiment is usually more efficient than a series of single-factor experiments. Texts that describe details of the analysis of variance of factorial experiments for non-mathematicians are Winer (1) and Sokar and Rolf (2). Further information, though in a more mathematical style, can be found in Miller (3) and Milliken and Johnson (4).
Atlas Protein Interact V 1 C
Authors: Singh Juswinder , Thornton Janet M
Published: November 2023

Amino acid side-chain packing arrangements are of major concern to any scientist interested in modelling protein interactions, including protein engineers, drug designers, molecular modellers, and crystallographers. Despite their important role in protein structure and function, no guide yet exists which describes and analyses the different packing geometries.

These two volumes provide a unique and comprehensive reference to the 400 possible types of amino acid side-chain interactions observed in proteins of known three-dimensional structure. Each type of interaction has been substantially analysed to provide valuable data relating to its favourability and stability. The data is presented both statistically as histograms and in the form of stereo plots, providing an invaluable source of information on the nature of the forces which hold proteins together.
Format of the atlas
Authors: Singh Juswinder , Thornton Janet M
Published: November 2023

This section describes the information shown in the atlas for each of the possible four hundred types of side chain interactions. This information is presented on two pages per pair-type (statistics page and stereo plots page) (Fig. 15 a and b).
Cellular Interactions in Development
Authors: Hartley David A
Published: November 2023

As progress in cell developmental biology carries on at a breakneck speed, new techniques constantly arise to plug the gaps left by traditional strategies. Cellular Interactions in Development provides detailed discussion and protocols of some of these new techniques, which allow the manipulation of developing organisms such as Drosophila or plants, when and where cells interact with each other to influence their development. The book looks at the really exciting innovations of the identification and functional test of molecules which control these cellular behaviours. The book also describes a number of new ways of hunting for these important proteins involved in cellular communication. A fully comprehensive manual which will prove indispensable to researchers in the fields of cell, developmental, and molecular biology.
Clonal analysis in plants
Authors: Dudley Mark , Poethig R Scott
Published: November 2023

Little is known about the nature of cell-cell interactions in plants as many of the most useful methods for studying this problem are difficult, if not impossible, to employ in plants. One of the most significant technical problems is the tendency of plant cells and tissues to dedifferentiate in new environments. As a result, methods that are routinely used to examine cellular interactions in many other systems, such as tissue grafts, cell mixing experiments, or microsurgical manipulations, are not generally useful for studying cellular interactions in plants (although they have been used to study interactions between tissues or organs). In order to study the cellular interactions that regulate cellular and multicellular patterns of cellular differentiation, these interactions must be studied in situ, with a minimum of experimental manipula tion. The ability to juxtapose cells with different developmental potentials in genetic mosaics is one of the few methods by which this can be achieved in plants.
Cloning cell surface molecules by transient expression in mammalian cells
Authors: Simmons David L
Published: November 2023

Since the mid 1980s, there has been a rapid increase in our knowledge about the specific cell surface molecules mediating cell-cell interactions and adhesion events. This has been largely due to the success of molecular cloning techniques, allowing the isolation of functional cDNA clones, encoding these glycoproteins.

There are a number of approaches that can be adopted for cloning cDNAs but one technique in particular, developed by Aruffo and Seed in 1987 (1-3), has been central to this enterprise. It is based on the transient expression of cDNA libraries in mammalian cells and the rescue of specific cDNA clones by antibody capture and panning. The efficacy of this procedure has transformed the field of clone isolation to such an extent that once a suitable antibody or ligand or cell line has been identified which recognizes a cell surface molecule, molecular cloning the cDNA encoding it is now an essentially trivial process.
Purifying and assaying mesoderm-inducing factors from vertebrate embryos
Authors: Smith Jim c.
Published: November 2023

The first cell-cell interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the embryo induces overlying equatorial cells to form mesoderm rather than ectoderm (see Figure 1). The last five years have seen a great increase in our understanding of mesoderm induction, largely due to the discovery of several ‘mesoderm-inducing factors’-molecules which induce prospective ectodermal tissue of the amphibian embryo to differentiate as a variety of mesodermal cell types such as muscle, notochord, mesenchyme, and mesothelium. Most of these inducing factors are members of the transforming growth factor type (TGF ) or fibroblast growth factor (FGF) families (1), and one of the most potent, activin, has been shown to induce expression of mesoderm-specific genes, or of mesodermal cell behaviour, in mouse (2), bird (3), and fish (4), as well as amphibian embryos. This indicates that mesoderm induction occurs in all vertebrates and the process is thus of considerable interest and importance.
S phase and its regulation
Authors: blow J Julian
Published: November 2023

During S phase of the eukaryotic cell cycle, chromosomal DNA is replicated precisely once as a prelude to its segregation to the daughter cells at mitosis. Replication is achieved by between 103 and 105 pairs of replication forks initiating at sites scattered throughout the genome, the number varying with the size of the genome and the length of S phase. These initiation events must be carefully regulated in at least three different ways. First, since most cells do not arrest in the G2 phase of the cycle, it is important that entry into S phase only occurs in cells that are legitimately committed to undergoing the complete cell division cycle. Second, it is important that initiation is coordinated with other cell cycle events so that the ploidy of the cell is maintained by the alternation of S and M phases. Third, it is important to regulate initiation events within each S phase, so that all the chromosomal DNA is completely replicated but no DNA is replicated more than once.
Nematode embryos
Authors: Edgar Lois G , Wood William B
Published: November 2023

The free-living soil nematode Caenorhabditis elegans has many advantages for investigation of development and physiology in a simple animal. Its three- day life cycle, simple growth requirements, hermaphroditic mode of development, and indefinite survival at liquid nitrogen temperature are superbly suited to genetic analysis. Moreover, its transparency throughout the life cycle, small size, simple anatomy, and invariant cell lineage have made possible detailed descriptions of normal and mutant development at the level of individual cells (reviewed in ref. 1). This chapter describes methods for handling and cultivating this nematode and for collecting and studying its embryos.
Embryos and larvae of invertebrate deuterostomes
Authors: Holland Nicholas d. , Holland Linda Z
Published: November 2023

The recent information explosion on pattern-determining genes has reawakened interest in the nature of segmentation, the universality of developmental mechanisms, the phylogenetic relations between animal phyla, and the origin of the vertebrates in particular. Much insight can be expected to come from developmental studies of invertebrate deuterostomes: a group which includes the closest relatives of the vertebrates. All invertebrate deuterostomes are marine, and they comprise (discounting the enigmatic arrow worms) echinoderms, hemichordates, tunicate chordates, and acraniate chordates.
Avian embryos
Authors: Stern Claudio d.
Published: November 2023

The avian embryo offers a very accessible system in which molecular studies can be combined with ‘classical’ embryology. Transplantation, cell labelling, immunocytochemistry, and chemical treatments can be combined with in situ hybridization and Northern analysis to study changes in gene expression after experimental manipulations. Moreover, with new techniques for producing transgenic birds now becoming available, the developmental effects of targeted mutations can be studied in cellular as well as molecular detail.
Transplantation in Avian Embryos
Authors: Stern Claudio D
Published: November 2023

This chapter briefly introduces two techniques for microsurgery. In the first, embryos are operated whilst in modified New culture (Chapter 6, Protocol 3). This is suitable for operations on embryos of up to 1 day’s incubation, and embryos can be cultured for up to 36 h. As an example, an operation in which the anterior tip of the primitive streak (Hensen’s node, the organizer of the amniote embryo) is grafted into an ectopic site in a host embryo is described (see ref. 1). The second technique is for operations in ovo, through a window in the shell, suitable for embryos of 1-3 days’ incubation. The example given is that of a graft of segmental plate (see ref. 2). With some care and a few precautions, embryos operated in this way can be grown to hatching. Older embryos can be operated as described in Chapter 13. A histochemical method for revealing the quail nucleolar marker in chimaeras is also given (adapted from ref. 3).
Immunocytochemistry of embryonic material
Authors: Stern Claudio d.
Published: November 2023

The localization and characterization of protein and carbohydrate molecules in embryos is an essential part of any study on the molecular bases of developmental processes. Methods for immunocytochemistry and for the characterization of antigens are in wide use and well established, and have been reviewed extensively elsewhere (1, 2). Embryonic material, particularly at early stages of development, presents special problems. First of all, it is sometimes difficult to find the correct compromise between antigen preservation, sensitivity of the method of localization, and good structural preservation. Second, when characterizing the antigen(s), it is sometimes difficult to obtain sufficiently large amounts of antigen for detection by conventional immunoblots (Western blots). The following small set of protocols work particularly well for the localization of embryonic antigens in tissue sections at the light microscope level and in whole mounts. A simple, reliable, and sensitive method for the characterization of antigens from small amounts of embryonic material is also included.
In Situ Hybridization
Authors: Wilkinson David G
Published: November 2023

The visualization of patterns of gene expression in embryos is an essential technique for the molecular analysis of development. The detection of mRNA by in situ hybridization has found widespread use because of the speed with which specific probes can be produced from cloned DNA or by the synthesis of oligonucleotides.
Oligosaccharides
Authors: Kennedy John F , Pagliuca Giampiero
Published: November 2023

Oligosaccharides are traditionally defined as polymers of monosaccharides containing from two to ten residues. However, since the naturally occurring polysaccharides rarely contain less than 25-30 residues, it is possible to consider the range of polymers having between two and about 20-25 residues as oligosaccharides, particularly when considering the analytical methods involved. As oligosaccharides fall into the classification between mono saccharides and polysaccharides it is not surprising that the methods used for oligosaccharide analysis are extensions of those used for either mono saccharide or polysaccharide analysis. This chapter uses a slightly different format to that of Chapter 1 in order to give the maximum information, within the limitations of available space, on the modifications which have to be made in adapting the methods described in detail for the analysis of mono saccharides and polysaccharides. Rather than repeat descriptions of practical details etc. this chapter concentrates on the types of methods which can be applied and their relative merits and shortcomings.
Glycolipids
Authors: Morrison I M
Published: November 2023

Glycolipid is the general term applied to a wide range of compounds. The most important and complex are of biological origin, being distributed throughout animal, plant, and microbial cells, but some synthetic examples have industrial applications. Along with glycoproteins, they form the class of glycoconjugates. The carbohydrate, or ‘glyco-components are oligo saccharide chains which are frequently branched. They can also carry covalently-bound, non-carbohydrate substituents such as acetate or sulfate groups. It is the precise carbohydrate structure which confers the biological specificity to a particular glycolipid.
Techniques for transformation of E. coli
Authors: Hanahan Douglas , Jessee Joel , Bloom Fredric R
Published: November 2023

The transfer of plasmid DNA into E. coli, so-called ‘plasmid transformation’, is a cornerstone of modern molecular biology. The original observation that calcium ions induced a state of competence, whereby E. coli cells would take up DNA and establish it in the cell (1, 2), set the foundation for a number of techniques for inducing competence toward plasmid transformation. One class of techniques is founded on this original observation that Ca2+ and other divalent and multivalent cations can induce competence. More recently, an alternative technique, electroporation, involving application of a transient high voltage electrical field to a mixture of E. coli cells and plasmid DNA, has proved to be remarkably efficient. Electroshock transformation techniques currently produce the highest efficiencies of any known methodology. In this chapter we present a spectrum of techniques that together produce reliable transformations at differing efficiencies, and for distinctive purposes. These range from low efficiency colony transformations, which can be performed in a few minutes without advance preparation of competent cells, to electro shock transformations whereby high complexity plasmid libraries can be produced with minimal amounts of starting material. We also describe methods for properly storing E. coli strains that will be used for preparation of competent cells, as well as methods for producing frozen competent cells, and discuss other parameters which are important for reliable production of competent cells. This chapter is adapted from a more comprehensive presentation on methods for and genetic factors influencing plasmid transformation of E. coli and other bacteria (3).
The utilization of cloned DNAs to study gene organization and expression
Authors: Parry Huw D , Alphey Luke
Published: November 2023

In this chapter we present a collection of techniques for the analysis of gene expression. The methods described are those most commonly employed in studies of eukaryotic gene regulation. They utilize cloned genes as the sub jects of study and as a means of generating nucleic acid probes. Southern hybridization is a powerful technique for providing a physical map of a gene on the chromosome. Information on the number of copies of the gene in the genome and the degree of similarity of the gene under study to other known homologues may also be obtained. Similarly, northern blotting provides in formation on the size and abundance of specific mRNA molecules in preparations of poly(A) +RNA.A range of techniques are available to determine the 5’ and 3’ termini of an RNA molecule and the positions of any splice sites within them. RNase and Sl mapping provide both qualitative and quantitative information about messenger RNA (mRNA) structure. Many eukaryotic genes are regulated at the transcriptional level. Sequencing of a promoter region often reveals elements having homology to known transcription factor binding sites. The gel retardation and foot printing methods provide useful information on the binding of specific transcription factors to the promoter regions of genes. Once sequence-specific DNA binding proteins have been identified they may be isolated by DNA affinity chromatography (1). Purified proteins may then be used for in vitro transcription studies to build up a complete understanding of the molecular interactions driving gene expression.
DNA sequencing
Authors: Alphey Luke
Published: November 2023

There are two principal methods for determining the sequence of DNA. These are based either on specific chemical degradation of a DNA molecule labelled at one end (the Maxam and Gilbert method) or on the enzymatic synthesis of labelled DNA, using dideoxynucleotides to terminate the elongating strand (the Sanger dideoxy method). Although there are specific applications which require the Maxam and Gilbert method, the vast majority of DNA sequence is today determined by use of the dideoxy chain termination method.
Use of vectors based on gene amplification for the expression of cloned genes in mammalian cells
Authors: Bebbington Christopher
Published: November 2023

The ability of cloned genes to function when introduced into mammalian tissue culture cells has proved to be invaluable in studies of gene expression. It also provides a means of obtaining, in large quantities, proteins which are otherwise scarce or which arc the completely novel products of gene manipu lation. The great advantage in obtaining human or other mammalian proteins from mammalian cells is that they are generally correctly folded, and arc more frequently appropriately modified and completely functional than when expressed in microbial or insect cells. Secretion of proteins containing signal peptides may also be more efficient in mammalian cells than insect cells.
Retroviral vectors
Authors: Brown Anthony M C , Dougherty Joseph P
Published: November 2023

“The exploitation of retroviruses as genetic vectors provides an important and versatile method of introducing and expressing cloned genes in eukaryotic cells, both in culture and in vivo. Several features of retroviruses favour their choice for this purpose, as an alternative to DNA-mediated gene transfer methods or other viral vector systems. First, the relatively small genomes of retroviruses can be easily manipulated to introduce foreign genes in such a way that any resulting defects in viral replication can he complemented in trans. Secondly, the viruses can be grown to high titres in cell culture. Most importantly, the efficiency of infection of susceptible cells is extremely high. Together with high titres of virus, this can permit infection of almost 100% of the target cells in a homogeneous culture. After infection, retroviral DNA becomes stably integrated in the host cell genome in a precisely defined manner such that virtually all of the infected cells can express the genes carried by the virus. Moreover, retroviruses contain powerful transcriptional enhancer elcmcnls that allow high levels of expression in a wide range of cell types.
Genetic manipulation of embryonic stem cells
Authors: Mohn Amy , Koller Beverly H
Published: November 2023

Embryonic stem (ES) cells are pluripotent stem cell lines established from mouse embryos cultured in vi1ro to the early post-implantation stage of development (1, 2). ES cell lines are established by transferring blastocysts to tissue culture plates seeded with a feeder layer consisting of either primary embryonic fibroblasts or cells of an established embryonic fibroblast line. Under these conditions, the embryo continues to grow, hatching from the zona pellucida and attaching to the feeder layer. As the trophectoderm cells spread out, the cells of the inner cell mass, which would normally give rise to the embryo proper, are exposed to the feeder layer. In many cases, the cells of the inner cell mass begin to proliferate upon contact with the feeder layer, forming: a small dump of cells, which can be picked away from the trophectoderm cells and expanded. After several weeks of growth in culture, these cells often reach a point where they can be propagated using established tissue culture techniques (3). With proper tissue culture conditions, an ES cell line established by this technique can be maintained in a pluripotent, undifferentiated, and euploid state. This maintenance of pluripotency is critical to the applications of ES cells in biological research.
Genomic and expression analysis of transgenic animals
Authors: Waller Sarah Jane , Funkhouser Judith Mcniff , Chooi Kum-Fai , Murphy David
Published: November 2023

Analysis of transgene expression is a cardinal part of any transgenic study. Transgenic pups born from microinjected eggs need to be identified (section 2), and then bred to produce a line (see Chapter 6, section 8.2). Expression of the transgene in the transgenic animals can then be analysed at the RNA (section 3) and protein (section 4) levels. It is very important to examine for transgene expression in as many tissues as possible, including those in which one does not expect to find its expression. Most transgenic animals that express their transgenes do so in a manner that is appropriate to the regulatory elements present. However, a number of transgenes are expressed ectopically, due either to influences of the adjacent cellular DNA or to unforeseen interactions between cA-acting elements in fusion genes. Such ectopic expression may produce unexpected and inappropriate results, including ill health, which should be taken into account when interpreting the results of the study. A thorough post-mortem examination is therefore also recommended for all animals expressing new transgenes (section 5).
Cloning into yeast artificial chromosomes
Authors: Anand Rak Esh
Published: November 2023

The analysis of large and complex genomes requires both mapping and cloning of DNA. Until a few years ago the largest fragment of DNA that could be cloned was - 40 kb in length using a cosmid vector (see Chapters I and 2). The development of yeast artificial chromosome (YAC) cloning vectors has greatly extended this cloning range. Following the description of the YAC vector system (1) the size of DNA fragment that could be maintained as a YAC has increased by almost one order of magnitude. This has greatly facilitated the study of complex genomes and has been instrumental in the efforts to construct the first generation physical map of the entire human genome (2). titre plates provides a valuable long-term resource which can be simultaneously accessed by several researchers. Consequently, it is important to plan carefully before embarking on this exercise that requires substantial time and resource. This chapter will mainly concentrate on the construction of a YAC library. For completeness, determination of the average YAC size within the library and a PCR-based library screening method have also been included.
Redox states and potentials
Authors: Cammack Richard
Published: November 2023

This chapter describes methods for controlling the oxidation-reduction, or redox, state of electron transfer proteins, or membrane bioenergetic systems. There are two principal objectives:
Control and regulation in bioenergetics
Authors: Brown Guy C
Published: November 2023

Control and regulation have traditionally been studied in a non-quantitative way, in order to determine where and how metabolic rates and levels are changed. The analysis of ‘control’ is concerned with where within a system the rates of pathways or levels of intermediates can be changed, i.e. which enzymes or transporters limit the system rates or levels. The analysis of ‘regulation’ is concerned with how a physiological change in rate or level is brought about. A variety of quantitative approaches are now available to help estimate the extent to which different metabolic components control rates and levels, and the extents to which different mechanisms are involved in producing a response to some effector or physiological change. These quantitative approaches have in turn changed our concepts of control and regulation, and how we should talk about them. Bioenergetics has had a traditional emphasis on quantitative analysis, and thus these approaches have rapidly been applied to a variety of bioenergetic systems, including bacteria, mitochondria, chloroplasts, and proteoliposomes.
Patch-clamping of mitochondrial membranes and of proteoliposomes
Authors: Ballarin Cristina , Catta Sorgato M
Published: November 2023

The electrophysiological analysis of mitochondrial membranes developed in recent years has shown the presence of several high conductance ion channels (1). The physiological roles played by these channels are not yet clearly established. However, the continuous increase of information and the contributions of different expertise render the topic an exciting focus of interest.
The complement system: an introduction
Authors: Sim Robert B , Dodds Alister W
Published: November 2023

The complement system is a very important component of immune defence against infection. It is part of the innate (non-adaptive) immune system, and can respond to challenges by microorganisms before an adaptive immune response has developed. Hereditary deficiencies of complement proteins are rare, but individuals who lack part of the complement system are generally at increased risk of recurrent infection. Complement promotes and regulates the phagocytosis or lysis of foreign cells, particles, or macromolecules and host tissue breakdown products. The human complement system is composed of more than 30 proteins, both soluble (in blood plasma and other body fluids) and membrane bound (on blood cells and other tissues), which interact with each other when the system is activated by various stimuli. The properties of the proteins are summarized in Tables 1-3. The cell-surface proteins act either as receptors for fragments of soluble complement proteins, or as regulatory proteins, which control the activities of soluble complement proteins, and protect the cells on which they are situated from attack by complement.
Immunochemical methods for the detection of complement components and complement activation
Authors: Svehag Sven-Erik , Graham R , Leslie Q
Published: November 2023

Complement activation plays an important role in the inflammatory response and in the pathogenesis of many infectious diseases. Simple assays such as radial immunodiffusion and nephelometry are available for measuring concentrations of native complement components in biological fluids. These assays have focused on C4, C3, and factor B as participants in different phases of the complement cascade reaction. However, since hypercatabolism and increased synthesis of complement components such as C3 and factor B may occur concurrently, the information related to in vivo complement activation which may be obtained from measurements of native complement components is limited. In addition, measurements of native complement components may be affected by the presence of activation products in the sample if not properly collected and stored. On the other hand, direct measurement of generated activation products by newer techniques can provide a reliable indication of ongoing complement activation. Such measurements may be used to monitor disease activity and the effect of therapeutic treatment. They do not, however, provide information about the functional state of the complement system. When such information is desirable, functional tests, such as haemolytic assays (see Chapter 2) or assays measuring the opsonic capability (1) of the system, should be used.
C1-inhibitor deficiency and angio-oedema
Authors: Jackson John
Published: November 2023

Tissue swelling or oedema may occur for a variety of reasons. Most frequently, it occurs as a normal response to tissue damage and is a direct consequence of an increase in the vascular permeability that accompanies all inflammatory processes. Because this form of oedema evolves from enhanced blood vessel permeability, it is referred to as angio-oedema. Angio-oedema is an essential component of the inflammatory response, as it allows access to damaged tissues by migrating cells such as those of the immune system. These cells are the vectors of the early critical response to tissue damage and they orchestrate the initiation of tissue repair.
Formation and regulation of the membrane attack complex
Authors: Morgan B Paul , Van Den Berg Carmen W
Published: November 2023

Complement was originally discovered more than a century ago because of its haemolytic and bacteriolytic activities, both consequences of the poreforming capacity of the membrane attack complex (MAC). Following cleavage of CS by the convertase of either activation pathway, the five terminal components of complement (C5b through to C9) become tightly associated in the hetero-oligomeric MAC which inserts deeply into the membrane bilayer and forms a transmembrane pore. Control of MAC lysis is provided by a battery of fluid-phase and membrane protective factors and by active defence mechanisms.
Complement and immune complexes
Authors: späth Peter J , Schifferli Jürg A
Published: November 2023

The antibodies generated by B lymphocytes are found in various body compartments. A large fraction is secreted directly into the gastrointestinal and respiratory tracts where they play an important role in preventing cell-surface binding and invasion of microorganisms. High antibody concentrations are also found in the extravascular space, particularly in plasma. These antibodies react with their specific antigens, whether the antigens are exogenous (e.g. bacteria, viruses, proteins, carbohydrates) or of self-origin. The formation of immune complexes (IC) with soluble or fixed antigens (cell membranes or basement membranes) is often followed by the activation of the complement cascade. Whereas locally high antibody density is essential for complement activation by IgG, only one IgM molecule may be sufficient to fix and activate Cl, the first component of complement. Twenty years ago, it became possible for the first time to measure IC and fragments of complement proteins in plasma. This led to the hope that the monitoring of these parameters would permit definition of therapeutic strategies.
Non-human complement
Authors: Nonaka Masaru
Published: November 2023

Early phylogenetic studies of the complement system detected a ‘complementlike’ haemolytic activity in most vertebrate species tested, as well as in some invertebrate species (1). As will be discussed below, however, functional assay of complement haemolytic activity is problematic, and recent molecular studies have not yet been successful in identifying a genuine complement component or its gene in invertebrates. In contrast, the presence of the complement system has been established at the molecular level, that is, complement proteins and/or cDNA clones have been identified which show primary structure homology with human counterparts, from all major classes of vertebrates, including the most primitive extant vertebrates, jawless fish.
Preservation and handling of stock centre clones
Authors: Scholl Randy , Davis Keith , Ware Doreen
Published: November 2023

The responsibility for preservation and distribution of DNA clones for molecular genetic research and the genome project in Arabidopsis is shared by two centres, the Arabidopsis Biological Resource Centre (ABRC), Ohio State University, which was established in September 1991, and the European Community DNA Stock Centre at the Max-Planck Institute in Kñln, Germany. In addition to DNA resources, the ABRC shares responsibility for seed stock preservation and distribution and dissemination of information for the Arabidopsis community. Hence the DNA laboratory is integrated with the centre’s other services, allowing these complementary efforts to be fully co-ordinated. While preservation of biological resources including DNA is a primary function of the stock centres, they also strive to serve the rapidly changing needs of plant molecular biology research. This review will address the practical problems associated with all aspects of the ABRC DNA operation. Protocols utilized for clone characterization, preservation, and dissemination will be featured. Other topics which will be discussed are the philosophy of DNA centre operation, organization of information relating to DNA stocks, and the future of DNA centre operations.
Genetic mapping using recombinant inbred lines
Authors: Lister Clare , Anderson Mary , Dean Caroline
Published: November 2023

New markers can be positioned on a genetic or molecular map by comparison of the segregation of parental alleles in a population containing recombinant chromosomes, with the segregation data of other markers in the same population. Marker order is determined by identifying which markers (e.g. i-vii) co-segregate in a number of recombinant chromosomes (schematically illustrated in Figure 1). The fewer the recombination events that separate two markers the closer together they map on a chromosome. If one recombination event separates two markers in 100 chromosomes, then the two markers are considered to map 1 centimorgan (cM) apart.
Assays for human B cell responses to cytokines
Authors: Callaro Robin E , Kotowicz Karolena T , Smith Susan H
Published: November 2023

B lymphocyte responses are determined by complex interactions between antigen, cell-surface ligands, and soluble growth and differentiation factors (cytokines) binding to specific receptors on the cell surface. To date, at least 17 of more than 40 recombinant cytokines are known to stimulate B cell activation, proliferation and/or differentiation (Table 1). Many of these cytokines are derived from T cells and are involved in T cell-B cell collaboration, but some are made by B cells, monocytes, and a variety of non-lymphoid cells such as endothelial cells and fibroblasts.
T cells and cytokines
Authors: Cohen S B A , Clayton J , Londei M , Feldmann M
Published: November 2023

The functions of T cells and cytokines are intimately interlinked. It was not until T cell growth factors were defined and subsequently purified, and the cDNA encoding it cloned as ‘interleukin 2’ (1), that abundant non-malignant T cells could be cultured. The isolation of this and other T cell growth factors, now included in the broader category of cytokines, has permitted the development of long-term T cell-lines and clones. T cells can be isolated from normal healthy individuals or patients with disease, according to their antigen specificity and/or phenotype, and then perpetuated in long-term culture by cytokine stimulation. IL-2 is the major T cell cytokine used but certain subsets of T cells will proliferate in response to other cytokines, some independent of IL-2. For example IL-3 will stimulate most α β T cell receptor expressing CD4™cos−Tcells to proliferate, in man and mouse (2, 3).
Assays of cell growth and cytotoxicity
Authors: Skehan Philip
Published: November 2023

This chapter describes non-radioactive assays commonly used to measure changes in cell growth and cytotoxicity induced in a cell population by treatment with agents such as drugs, hormones, nutrients, and irradiation. It focuses specifically on mammalian cell cultures, but the methods can be used with a wide variety of cells, tissues, and microorganisms. Because of the ease and speed with which large numbers of samples can be processed by optical plate readers, most of the protocols which follow are designed specifically for 96-well plates, but are easily adaptable to other culture vessels.
Culture and characterization of human endothelial cells
Authors: Hinsbergh Victor W M Van , Draijer Richard
Published: November 2023

Endothelial cells, the lining cells of all blood vessels, are involved in many physiological and pathophysiological processes, including haemostasis, vasoregulation, inflammation, angiogenesis, and the extravasation of fluid, macromolecules, hormones, and leucocytes. The recognition that endothelial cells are involved in all these processes went in parallel with the ability of investigators to study these cells in culture. Endothelial cell culture was first established in the early 1970s (1, 2). Nowadays, a whole repertoire of molecular and cellular approaches are available to study the activation and metabolic regulation of endothelial cells in vitro and in vivo. Although one has to realize that endothelial cells from different types of vascular beds have distinct features, much information regarding these cells can be obtained using cell culture methods. This chapter describes the culture and characterization of human umbilical vein and microvascular endothelial cells, and simple techniques to study the permeability of human endothelial cell mono-layers in vitro.
Working with Laboratory Fermenters
Authors: Collins Stephen
Published: November 2023

Shake flasks are commonly used to grow microbial cells in small quantities at relatively low cell densities (up to 5-10 g/litre). However, the experimenter has little control over the environment experienced by the microorganisms. Process variables such as pH or oxygen concentration are likely to change during the course of the experiment. Terms such as late log phase are commonly used to describe such cultures when there is, in fact, no data to support the assumption of constant growth rate implied in the term. Indeed. it is likely that changes in the environment will affect the physiology of the cells.
Separation of cells using free flow electrophoresis
Authors: Eggleton P
Published: November 2023

The principal physical characteristics that have been exploited for cell separation techniques are their size (volume), density, or surface electrical charge. If cells differ sufficiently in density, then density gradient sedimentation techniques should be used (see Chapter 2). Cells which differ in size or volume can be separated by countercurrent centrifugal elutriation (see Chapter 3). As all mammalian cells carry a net surface negative charge at physiological pH, free flow electrophoresis (FFE) is able to maximize subtle differences in the charge of cell surface molecules leading to the separation of cells from one another. As the electrophoretic separation normally occurs in a continuous flowing buffer which exceeds 1 gsedimentation, within certain limits, the size or density of the cell is not normally influential in the separation.
Evaluation of Antisense Effects
Authors: Nellen Wolfgang , Lichtenstein Conrad
Published: November 2023

Antisense-mediated gene silencing by expression of antiscnse RNA in transfected cells has become a complementing alternative to gene disruption or gene ‘knock-outs’ by homologous recombination. Though powerful and often successful. there are unknown mechanisms which may sometimes hamper the effectiveness of the experiment. As with experiments with antisense oligonucleotides used to transiently interfere with gene expression, there may also be unexpected side-effects leading to the wrong interpretation of the results (see Chapter 11).
Selecting, Preparing, and Handling Antisense Oligodeoxyribonucleotides
Authors: Toulmé Jean-Jacques , Cazenave Christian , Moreau Serge
Published: November 2023

Combined developments in DNA sequence analysis and in oligonucleotide (oligo) chemistry make the antisense oligo approach a very attractive one to regulate gene expression. Knowing the primary sequence of the gene one wishes to turn down allows the design of a complementary antisense oligo sequence which, upon binding to its cognate RNA, may selectively prevent the function of the target gene. Since this very simple idea was first proposed by a Russian team in the late 1960s (1), numerous examples of successful inhibition of gene expression by antisense oligos have been published in the scientific literature. This ranges from pioneering work in cell-free extracts and cultured cells (2, 3) to recent in vivo experiments (4, 5). Even therapeutic trials in human beings are under way for AIDS and cancer (6). See Chapter 10 for medical applications.
Non-Antisense Effects of Oligodeoxynucleotides
Authors: Stein C A , Krieg Arthur
Published: November 2023

A conceptual problem in the application of oligodeoxynucleotides (oligos) as antisense, anti-gene, or anti-protein agents is that there is no essential difference in the oligo synthesized for either of these three applications. The designation of an oligo as ‘antisense’, or ‘triplex’, or ‘aptamer’ is actually frequently based more on the intention of the investigator than on any scientifically determined mechanism of action. In principle, any oligo might have any of these activities, depending on the relative values of KJ for different oligo binding sites and on the intracellutar localization of the oligo. As an example, a 16 base oligo synthesized to be a triplex-forming oligo was shown to have fortuitous complementarity to an mRNA sequence at 12 non-contiguous bases; this was sufficient to hybridize and activate RNase H, causing degradation of that RNA (1). In addition, many ‘antisense’ oligos have been found to show non-sequence-specific effects; this may perhaps be due to their polyanionic backbones (reviewed in ref. 2).