bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical aspects of platelet function
Authors: Clemetson Kenneth J
Published: November 2023

1. Introduction

Since the primary function of platelets is haemostasis, or stopping bleeding, platelet dysfunction is generally seen as an under–or over-expression of this, i.e. in bleeding problems or in various haemostatic or thrombotic disorders. The first task of the clinician faced with such symptoms is to eliminate the possibility of the disorder being produced by a lack or defect in plasma coagulation factors such as factor VIII, von Willebrand factor, fibrinogen, factor IX, factor V, etc. which are commonly affected. Having shown that these lie within the normal range it is necessary to check for platelet-related problems. Basic parameters such as platelet count, platelet volume, and granulosity can be rapidly determined by flow cytometry. Thrombocytopenia, or low platelet count can clearly lead to bleeding problems though there is some controversy about what should be regarded as a threshold value. Normal values lie in the range 150000–400000 platelets/µl but bleeding problems may not appear until values below 10000-20000 platelets/µl are reached. There are many causes for thrombocytopenia which will be dealt with later. When necessary, basic treatment is intravenous platelet concentrates to try to restore platelet levels although the recent cloning of thrombopoietin may, when it becomes generally available, make this a preferred therapy.
Platelet procoagulant activity and its measurement
Authors: Bevers Edouard M , Comfurius Paul , Reutelingsperger Chrisp. M , Zwaal Robert F A
Published: November 2023

Platelets serve an important function in the haemostatic process. Vessel wall injury causes exposure of subendothelial structures to the blood stream and initiates the process of platelet adhesion, aggregation, and release of granule contents resulting in formation of the primary haemostatic plug. Tissue thromboplastin, released by the damaged vessel wall, initiates activation of the coagulation pathway, leading to formation of thrombin. The action of thrombin on soluble plasma fibrinogen gives rise to deposition of an insoluble fibrin network which consolidates the primary haemostatic platelet plug to a stable thrombus. There exists a mutual interaction between thrombin formation and platelet activation: while thrombin is one of the most potent platelet activators, its formation is strongly enhanced in the presence of activated platelets. The latter is due to the exposure of procoagulant phospholipids at the surface of the platelet plasma membrane, known as the procoagulant response. In this chapter, we will describe two methods to measure this procoagulant response. One method concerns the direct measurement of a lipid-dependent coagulation reaction, i.e. the formation of thrombin by the enzyme complex factor Xa and factor Va, known as the prothrombinase reaction. The other method is based on the detection of surface exposed procoagulant lipid, i.e. phosphatidylserine (PS) by means of Annexin V, a protein with high affinity for anionic phospholipids.
Imaging of individual cell surface MHC antigens using fluorescent particles
Authors: Smith Patricia R , Wilson Keith M , Morrison Ian E G , Cherry, Richard J , Fernandez Nelson
Published: November 2023

Class I and class II major histocompatibility complex (MHC) molecules are cell surface, transmembrane glycoproteins that have similar overall structure but have readily distinguishable subunit structures (1). The two classes of MHC molecules present peptides, produced from different intracellular compartments, at the cell surface where they are recognized by two major subsets of T cells, CD4 and CDS, respectively. Integral membrane glycoproteins are able to diffuse to varying degrees within the plane of the membrane and so can potentially form protein clusters (2). This clustering of cell surface receptors in response to a particular signal or antibody is important in transmembrane signal transduction (3, 4). The density of class I and class II subsets and their relationship with other cell surface molecules in the correct orientation is thought to be vital for effective T cell recognition.
Approaches to studying MHC expression in mammals other than primates
Authors: Ellis Shirley A
Published: November 2023

Until recently, nearly all studies on MHC expression and function had been carried out in humans and mice. In these species an enormous body of data has now been collected concerning the minute details of polymorphism present in the classical MHC class I and II genes, as techniques have progressed from serology, through biochemistry, to various molecular approaches. Using this information it has become possible to understand how MHC, peptide, and T cell receptor interact (1), to study the mechanisms involved in evolution of the MHC (2), and to speculate on the role of pathogens as a selective force in maintaining the high level of diversity seen in these molecules (3).
Intrinsic (low-level) chemiluminescence
Authors: Vladimirov Yury A
Published: November 2023

The first attempts to measure the intrinsic low-level ultraviolet emission (‘Mitogenetic rays’) of living cells and biological tissues were made by the Russian scientist Alexander Gurvich who used different dividing cells as biological detectors of ultraviolet (UV) radiation. More recently, gas-discharging quartz tubes with inert metal cathodes as UV-photon counters have been used (see ref. 1 for review).
Quantitative analysis of 4-hydroxy-2-nonenal
Authors: Kinter Michael
Published: November 2023

Tissue damage resulting from the production of reactive oxygen species has been implicated in a diverse series of pathologies. One component of tissue damage by reactive oxygen species is the oxidation of cell membrane poly unsaturated fatty acids. Polyunsaturated fatty acid oxidation can proceed by a chain reaction mechanism referred to as autooxidation in which the termination reactions form a variety of products including a series of aldehydes. The aldehydes reported as products of polyunsaturated fatty acid oxidation are listed in Table 1, which shows that one class of aldehydes formed is the 4-hydroxy-2-alkenals, exemplified by 4-hydroxy-2-nonenal (4HNE).
ER-Golgi membrane traffic and protein targeting
Authors: Machamer Carolyne.
Published: November 2023

In eukaryotic cells, the vast majority of proteins destined for secretion or insertion at the plasma membrane follow a common pathway through the cell (1). They are synthesized on ribosomes bound to the endoplasmic reticulum (ER) membrane, cotranslationally translocated completely or partially into the ER lumen (see Chapter 4), and then transported via multiple budding and fusion steps through the Golgi complex to the cell surface. Many co- and post-translational modifications occur to newly synthesized proteins as they move through this exocytic pathway. In the ER, polypeptides must be folded properly: formation of disulfide bonds, core glycosylation, and oligomerization contribute to this process. In the Golgi complex, oligosaccharides are processed and proteolytic processing can occur. The enzymes involved in these modifications are resident proteins of the ER or Golgi complex, and must be retained in the appropriate compartment in the face of substantial membrane flow.
Mass spectrometric detectors
Authors: Chapman John R
Published: November 2023

Mass spectrometry (MS) is one of the most powerful physical methods available for identifying analytes in terms of their molecular mass (Mr) and for elucidating their molecular structures. Furthermore, MS possesses a unique attribute compared to other spectroscopies, namely its high sensitivity, so that it provides one of the few methods that is entirely suitable for the identification, structural elucidation, and for the quantitative measurement of even trace amounts of the various analytes present in a mixture. Generally, however, and certainly for complex mixtures, by combining MS with a chromatographic technique the molecular identity of each of the separated components of a mixture may be more readily ascertained. Historically, the first combination of MS with a separation technique was that achieved with gas chromatography, namely GC-MS, but as will become evident from the present chapter, during the last decade this desirable practice has been extended to HPLC to give the hyphenated technique usually designated as LC-MS.
Ion-exchange chromatography of proteins and peptides
Authors: Henry Michael P
Published: November 2023

The phenomena that occur during protein HPIEC are no different from those seen in the earliest forms of ion-exchange chromatography of these biopolymers. The first ion exchangers were particulate, large diameter (> 50 micron), porous, soft gels derived from polystyrene, polysaccharides, and polyacrylates. Modern high-performance media are microparticulate in nature and are derived from silica, hydroxylated polyethers, polystyrenes, and crosslinked. non porous polyacrylates. The buffers and pH ranges employed in HPIEC are the same as those used in soft-gel chromatography.

On the other hand, the physical ruggedness of modern ion-exchange packing materials has changed certain aspects of the methodology of this technique. For example, since chromatography is so rapid there is less need for low temperatures and the stability they bring. Columns may be used many times (several hundred) before discarding.
Principles of immunodiagnostic tests and their development; with specific use of radioisotopes as tracers
Authors: Edwards Raymond , Blincko Stuart , Howes Iain
Published: November 2023

Immunodiagnostic tests, or immunoassays used for diagnostics, have been used extensively in many scientific disciplines and in many different ways. Such tests encompass any analytical method which use antibodies as reagents, the results from which assist a diagnostic interpretation. The most obvious use has been found in clinical applications, but, the term can be applied equally to other areas of investigation, e.g. food spoilage agents, environmental pollution and illicit drug use. Thus in a wider sense, it would be applied to the assessment of any intrinsic factor associated with a state or condition to be ascertained.
Growth factor receptors in cell transformation
Authors: Heldin Carl-Henrik , Rönnstrand Lars
Published: November 2023

The growth of cells is regulated by polypeptide factors that stimulate or inhibit proliferation. More than 50 such growth regulatory factors are known (Chapter 2). They exert their effects on cells by binding to specific receptors at the cell surface. For factors that stimulate cell proliferation, activation of protein tyrosine kinases appears to be a common and maybe obligatory event. Several growth factors bind to receptors with intrinsic tyrosine kinase domains which are activated after ligand binding. Other growth factors bind receptors which after ligand binding form complexes that contain intracellular tyrosine kinases.
Oncogenic transcription factors: FOS, JUN, MYC, MYB, and ETS
Authors: Baker Suzanne J , Curran Tom
Published: November 2023

Many oncogenes encode important components of the cellular machinery that are responsible for the translation of growth and differentiation signals in response to a diverse array of extra- and intracellular stimuli. Included in this broad range of oncogenic molecules are growth factor receptors (see Chapters 2 and 3), cytoplasmic signalling molecules (Chapters 4 and 5), and nuclear transcription factors. Transcription factors are critical components of signal transduction pathways that can transmit a regulatory signal to altered patterns of gene expression. It is not surprising that the disruption of normal regulation of such important control points can contribute to tumorigenesis. This chapter will focus on several transcription factors originally discovered as transforming genes carried by retroviruses (for a historical review see ref. 1). The intention is to provide a generalized view of the mode of action and regulation of these genes and their relationship to cellular growth and differentiation rather than a comprehensive review of the extensive literature.
Mammalian cell cycle control
Authors: Pines Jonathon
Published: November 2023

A proliferating cell must co-ordinate DNA replication and chromosomal separation to ensure that the genome is replicated completely, and that a single copy is correctly inherited by each daughter cell. The cell cycle is the coordinated series of events that achieves these aims. Our current understanding of the basic mechanism underlying the cell cycle is that many of the key events are initiated by a family of conserved serine/threonine protein kinases, the cyclin-dependent kinases (CDKs), that are activated by the cyclin family of proteins.
The retinoblastoma gene product and its relatives
Authors: La Thangue Nicholas B
Published: November 2023

An overwhelming body of cancer research supports the idea that the integration of growth-promoting and growth-restraining pathways maintains the fidelity of the cell cycle (see Chapter 7). During the genesis of tumour cells, such pathways become aberrantly regulated, culminating in an absence of cell cycle control and uncontrolled proliferation. A key player in negative regulation of the cell cycle is the retinoblastoma tumour suppressor gene product (pRB), historically identified through studies on the rare recessive paediatric eye tumour, retinoblastoma, where predispositior, to the hereditary form of the disease correlates with a loss of heterozygosity at chromosome 13q14 (1, 2). These studies, together with the recessive nature of the disease, suggests that inactivation of the RB gene is important in the generation of retinoblastoma, and implied a negative role for pRB in regulating proliferation. Subsequent molecular characterization of the RB gene indicated that the locus is invariably mutated in both hereditary and sporadic retinoblastoma as well as in a variety of other types of tumour (2, 3).
Integration of control devices. I. Pathogenicity
Authors: Dorman Charles J
Published: November 2023

One of the driving forces behind microbiological research has been the desire to understand how and why some bacteria cause disease in humans, in animals, or in plants. The employment of molecular genetic and cell biological techniques has led to the identification of factors which make key contributions to the ability of bacteria to be pathogenic. These may be classified as dedicated virulence factors (for example, toxins) which one might expect to find exclusively in pathogenic bacteria and, accessory virulence factors (for example, nutrient uptake systems) which might be equally important in the commensal and the pathogenic situation (1).
Antibodies packaged in eggs
Authors: Jensenius Jens Christian , Koch Claus
Published: November 2023

Like mammals, chickens make antibodies in response to challenge with antigen. Numerous publications have described the humoral antibody response in chickens to various antigens given by different routes and with different adjuvants (reviewed in refs 1 and 2). It is well established that chickens possess at least three immunoglobulin isotypes: a high molecular weight IgM type immunoglobulin, and two 7—8S immunoglobulins, an IgG type constituting the major plasma immunoglobulin, and an IgA type in external secretions, e.g. in the gall bladder and oviduct (3). The term ‘IgY’ is often used for the chicken IgG type immunoglobulin. This nomenclature was suggested by Leslie and Clem (4) to reflect some unusual features of chicken IgG heavy chains. Later data, especially recent results from molecular cloning, have shown that IgY has a fourth constant heavy chain domain, which shows homology to the hinge region of mammalian IgG (5,6). These data also suggest that avian IgY may be related to a common ancestor for mammalian IgG and IgE. However, we find that the available information justifies the use of ‘IgG’ to denote the chicken 7S immunoglobulin and we shall do so throughout this text.
Immunofluorescence and immunoenzyme histochemistry
Authors: Brandtzaeg P , Halstensen T S , Huitfeldt, H S , Alnes K N V
Published: November 2023

In 1941 Albert Coons and co-workers for the first time labelled the immunoglobulin (lg) fraction of an antiserum with fluorescein to visualize the corresponding antigen in tissue sections by fluorescence microscopy (1,2). A new morphological discipline, based on probing for structural or functional markers, was thereby founded. The term immunohistochemistry should preferably be reserved for immunological probing in tissue sections, while immunocytochemistry usually refers to cell smears, cytospin preparations, and monolayer cultures. For simplicity, immunohistochemistry will be used as a general methodological term in this chapter.
Flow cytofluorimetry
Authors: Damgaard G , Nielsen, C H , Leslie R G Q
Published: November 2023

The employment of flow cytofluorimetry (FC) in both qualitative and quantitative investigations of cell phenotype and activity has increased explosively in recent years. The strength of the technique lies in the fact that detailed information is acquired on individual cells passing through the detector system: information which, upon acquisition of data from a predetermined number of cells, can be processed to provide a statistical profile for the cell population, or populations, of interest. Thus FC differs fundamentally from the traditional radiometric approach, which only provides information on the cell population as a whole. In addition, the technique has largely supplanted fluorescence microscopy in the study of single cell suspensions, because it is able to provide quantitative data on much greater numbers of cells very rapidly.
Assays for Complement Receptors
Authors: Bartók István , Walport Mark J
Published: November 2023

The complement system is a group of about a dozen cell surface molecules and 20 glycoproteins present in the plasma of vertebrates. The first complement-mediated activity to be described was destruction of bacteria by fresh serum. This lytic activity of serum was composed of a heat stable component, antibody, and heat labile ‘complementary’ proteins. Complement has a complex series of effector activities which fall into three broad categories. The first category of effector activities follow the binding of the major complement opsonins, C3b and C4b and their catabolic products, to three types of complement receptor, complement receptors types 1, 2, and 3 (CRl, CR2, and CR3) located on a wide variety of cell types. The consequences of ligation of these receptors include phagocytosis of opsonized particles, activation of leucocytes and the transport of immune complexes. The interactions of complement with these three receptors forms the major focus of this chapter. The second category of effector activities of complement is the activation of inflammatory mechanisms by the interaction of the anaphylatoxins, C5a and C3a, with specific receptors present on leucocytes. The third category of effector activities results from the insertion of the membrane attack complex of complement into cell membranes. The consequences of this are lysis, particularly of bacteria and non-nucleated cells, and cellular activation by sublytic doses of membrane attack complex.
Identifying proteins for proteome studies: a two-dimensional gel electrophoresis approach
Authors: Sanchez Jean-Charles , Wilkiks Marc R , Appel Ron D , Williams Keith L , Hochstrasser Denis F
Published: November 2023

In the study of biological systems, it is known that genetic analysis alone cannot always predict any particular phenotype. This is because gene expression and protein post-translational modification arc modified by a complex interaction of genes, cellular events. and the environment. This phenomena, known as epigenesis, also encompasses the redundancy of gene and cellular networks that can often accomplish similar tasks and adapt themselves to reach identical end-point’>. For the researcher, genetic analysis of biological systems is therefore not necessarily reliable for the accurate prediction of phenotype or disease in many cases (1).
Ligand binding
Authors: Levitzki Alexander
Published: November 2023

Receptors arc the focus of a number of different research disciplines. Hormones. neurotransmitters. drugs, antibodies, and lections exert their bio chemical effects through an initial interaction with specific receptors. Even lysosomal enzymes reach their target. the lysosome, through a receptor mediated pathway. The binding of the ligand to its specific receptor triggers a cascade or biochemical reaction that eventually leads to the final physiological response.
Affinity labelling
Authors: Colman Roberta F
Published: November 2023

An important approach to identifying amino acid residues within the hiding sites of enzymes is to exploit the specificity of the enzyme for a natural subs1ratc or regulatory ligand to limit chemical modification to the immediate vicinity of that site. In this approach. termed affinity labeling. a reagent is designed that is structurally similar to the normal ligand of the enzyme. but which also features a functional group capable of reacting covalently with many different amino acids. The expectation is that the affinity label will initially form a reversible enzyme-reagent complex analogous to the cyma-ligand complex and, once bound at the specific site, will react irreversibly with an amino acid residue within that site. The application o[ this technique can result in tagging of a particular binding site of a purified protein or in the labelling of one protein within a complex mixture of proteins. Affinity labeling can he used to localize a given site within a protein of knave amino acid sequence and thus to evaluate experimentally a site that was predicted on the basis of an amino acid sequence motif. It allows comparisons to be made between the substrate sites of the cyma in solution and those site localized within the crystalline form by X-ray diffraction. It can be used to test whether a compound bound irreversibly at a substrate site influences the enzyme conformation, subunit interaction, or the reactivity of other ligand site-,. Affinity labels with appropriate chromogenic or fluorescent substituents can be used to introduce spectral probes into specific sites on enzymes to report on the environment. on conformational changes, or on distances between functional sites. Affinity labelling can also provide the knowledge base to make rational choices of target amino acids for its directed mutagenesis. The general principles of affinity labelling have been summari1.ed in books and review articles (1-3).
Protein expression in mammalian cells
Authors: Otter-Nilsson Marlies , Nilsson Tommy
Published: November 2023

Protein expression has become a major tool to analyse intracellular processes, both in vitro and in vivo. The choice of expression system depends entirely on the purpose of the study. For some cases, transient transfection is the most obvious choice because of its relatively short time investment. In other cases, homogeneous populations and large quantities of cells may be required, which involves making cell lines stably expressing the desired protein. It may also be advantageous to express proteins under inducible promotors; this is particularly true if the protein exerts pathological effects on the cell. There are several inducible systems available but none, so far, is easy and straight-forward. Expression through virus infection is also possible. Here, large quantities of cells can be infected at the same time and the protein assayed for shortly after infection. The drawback, however, with this method is the need for special precautions when making and handling virus stocks and likely side-effects exerted by the virus on the cellular machinery upon infection. Protein expression can also be achieved directly via microinjection of plasmid DNA directly into the nucleus of the host cell. This allows for protein expression in single cells which may be desirable when performing video microscopy. Thus the choices of expression systems are multiple and one should carefully consider the range of these available before investing time and other resources in the experiments themselves. It is the goal of this chapter to describe the various expression systems so as to make this choice easier.
Expression in Xenopus oocytes and cell-free extracts
Authors: Matthews Glenn M
Published: November 2023

Adult female Xenopus laevis carry approximately 40000 oocytes at various stages of development. During growth, oocytes accumulate cellular components which carry the developing embryo through many cell cycles. Although much of the content of the fully grown oocyte consists of yolk proteins absorbed from the maternal circulation, there is also a large reserve of translational components required for the high level of protein synthesis which occurs during the period immediately after fertilization. The oocyte also contains a store of each of the three RNA polymerases sufficient to support zygotic transcription up to the 30000 cell stage (1).
Cell-free coupled transcription–translation systems from Escherichia coli
Authors: Kramer Gisela , Kudlicki, Wieslaw , Hardesty Boyd
Published: November 2023

Development of a bacterial cell-free coupled transcription-translation system in which a specific polypeptide was synthesized and could be identified was pioneered by Zubay (1). E. coli genomic DNA or bacteriophage DNA was used as the template for protein expression. Zubay (1) also pointed out that E. coli extracts are unsuitable for translation of isolated mRNA because of their rapid degradation. This statement still holds true despite the development of RNase-deficient E.coli strains.
Imprinting at the mouse and human IGF2R loci
Authors: Neumann Bjorn , Wutz Anton , Smrzka Oskar W , Barlow Denise P , Lyle Robert
Published: November 2023

The mammalian insulin-like growth factor 2 receptor (IGF2R), also known as the cation-independent mannose-6-phosphate receptor (MPR300), is a multifunctional protein, primarily involved in lysosomal transport (1, 2). This transmembrane receptor, hereafter called IGF2R, binds insulin-like growth factor 2 (IGF2) and numerous ligands tagged with mannose-6-phosphate to target them to the lysosomes. Thus, lysosomal targeting mediated through the IGF2R functions as a pathway for the transport of lysosomal enzymes and, in the case of IGF2, as a clearance mechanism (3-5). IGF2R binds other secreted growth factors, such as proliferin (6), and is probably required for the activation of transforming growth factor 13 (TGF-13) (7). The involvement of IGF2R in embryonic growth control (5) and the observed loss of heterozygosity at the IGF2R locus in both hepatocellular carcinomas (8, 9) and breast tumours (10) suggests that IGF2R may function as a tumour supressor gene.
Starch gel electrophoresis of allozymes
Authors: May Bernie
Published: November 2023

Thirty years ago a novel, relatively simplistic methodology revolutionized our ability to study evolution (starch gel electrophoresis of allozymic proteins). Lewontin and Hubby (1) and Harris (2) illustrated convincingly how allozyme electrophoresis permitted a wealth of Mendelian data on populations to be gathered in a relatively short period of time. The availability of these data has revolutionized many fields of evolutionary biology, including systematics, Sociobiology, genomic organization, and population genetics. Prior to the appearance of allozyme electrophoresis only morphological and behavioural mutants were available to study single gene variation. This new technique brought the electrophoretic separation of proteins together with the specificity of histochemical detection of the protein products of single loci (3). Now dozens of simple Mendelian markers are available in most organisms. The utter simplicity of this methodology has permitted thousands of scientists to use this technique in their research.
Cleavage of mismatched bases using chemical reagents
Authors: Giannelli Francesco , Green, Peter M , Ramus Susan J
Published: November 2023

Chemical cleavage of mismatch (CCM) was developed in 1988 as a method for screening cloned pieces of DNA for single base mismatches (1), but in combination with PCR was very soon used for the direct analysis of genomic DNA (2) and, later, for the analysis of mRNA (3). This technique detects mismatches in hybrid DNA. Heteroduplexes are formed by adding labelled normal DNA usually to an excess of unlabelled patient DNA. The heteroduplexes are reacted with either hydroxylamine which recognizes mismatched C bases or osmium tetroxide which recognizes mismatched T bases. Any mismatched or unmatched C or T bases are modified by the respective chemicals so that the modified strand can be cleaved with piperidine at the site of the mismatch. The products are analysed by electrophoresis on a denaturing polyacrylamide gel and the size of the fast running cleavage bands indicates the site of the mismatch. All manipulations involving the chemicals must be performed in a fume-hood.
Selective amplification of specific alleles
Authors: Syvänen A -C
Published: November 2023

A variety of methods have been developed to detect known mutations and polymorphisms. Many of these methods are based on the polymerase chain reaction (PCR) because a segment of DNA can be amplified more than one millionfold for study or analysis. The amplified DNA may be analysed by chemical cleavage, restriction enzyme digestion, single-strand conformation polymorphism, gel electrophoresis, or hybridization to specific oligonucleotides.These methods, however, all require additional steps following PCR. Therefore, one of the simplest and most rapid techniques is to modify the PCR for amplification of specific alleles by using specially designed oligonucleotides. This approach we have termed PCR amplification of specific alleles (PASA) (1, 2). Other laboratories also have developed this method to distinguish between specific alleles (3-6). Synonymous terms for PASA include allelespecific amplification (ASA), allele-specific PCR (ASP), and the amplification refractory mutation system (ARMS). The method is generally applicable for the detection of known point mutations, small deletions and insertions, polymorphisms, and other sequence variations.
The protein truncation test (PTT)
Authors: Luijt Rob B Van Der , Fodde, Riccardo , Dunnen Johan T Den
Published: November 2023

The detection and characterization of point mutations in genes responsible for inherited disorders has become one of the most common practices in human molecular genetics. Several protocols are nowadays available: single strand conformation polymorphism (SSCP) (1), RNase protection (2), hydroxylamine and osmium tetroxide (HOT) chemical cleavage (3), and denaturing gradient gel electrophoresis (DGGE) (4, 5). These techniques are generally aimed at the identification of single base substitutions, insertions, or deletions in relatively small DNA fragments (50-500 bp). However, when dealing with large genes, it is desirable to analyse larger fragments of the coding region to speed up the analysis and pin-point the location of the alteration prior to the nucleotide sequence determination. To this aim, mRNA-based mutation detection protocols are often employed. Moreover, if the spectrum and the type (i.e. missense, nonsense, or frame-shift) of the prevalent mutations at a given diseasecausing gene has already been established, one would prefer to employ a protocol ‘targeted’ for the detection of the most frequent type of mutations.
In situ hybridization of mRNA with hapten labelled probes
Authors: Xu Oiling , Wilkinson David G
Published: November 2023

In situ hybridization with hapten labelled probes offers several advantages compared with S-labelled radioactive probes, including a single cell resolution of the signal, the ability to directly visualize spatial patterns by hybridizing whole tissues, and the ability to visualize different transcripts simultaneously by use of multiple labelling and detection methods. Several haptenized nucleotides are available, including digoxigenin- (DIG), fluorescein-, and biotin-DTP that can be used to make labelled RNA probes. DIG and fluorescein can be detected with antibodies, and biotin with streptavidin, to which enzymes such as alkaline phosphatase (AP), J3-galactosidase, or horse-radish peroxidase have been conjugated. For each of these enzymes, a variety of chromogenic substrates are available that yield different coloured products. Currently, the reagents that have found widespread favour since they give a high sensitivity and low background are DIG labelled RNA probes detected with an AP-conjugated anti-DIG antibody and the chro¬ mogenic substrate mix of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) plus 4-nitroblue tetrazolium chloride (NBT). Fluorescein labelled probes detected with an AP-conjugated anti-fluorescein antibody and BCIP/NBT give similar results. In this chapter, we describe methods for non-radioactive in situ hybridization of whole mount tissues and of tissue sections.
Biological image processing and enhancement
Authors: Horgan G W , Reid C A , Glasbey C A
Published: November 2023

This chapter considers simple ways to enhance digital images, both to make visual interpretation easier and to make the images more amenable to subsequent automatic analysis. It is typically the stage in image analysis that follows image capture. Methods include adjustment of brightness or contrast, edge detection and edge sharpening, removal of small irrelevant features (termed noise), and adjustment for background intensity variation. Image enhancement can only improve and clarify information that is already present in an image: it cannot recover what was never there in the first place! Therefore, at the stage of image capture it is essential to seek the best image quality by suitable sample preparation, adjustment of instrumentation, choice of lighting, etc. We state this as a principle:
Computer prediction of transporter topology and structure
Authors: Hong Rong-I , Sansom Mark S P
Published: November 2023

One of the resources which may be exploited in studying membrane transport proteins is the enormous (and increasing) body of genome sequence data. At the time of writing, complete genomic sequences are known for 21 prokaryotes (1), a yeast (2), and for Caenorhabditis elegans (3). Current estimates suggest that integral membrane proteins comprise about 20–30% of most genomes (4, 5). This provides a strong motivation to exploit this information in terms of trying to understand the relationship between membrane protein sequence and structure (and eventually function). Prediction of protein structure is fraught with difficulties and despite intense efforts, ab initio methods for structure prediction remain elusive (6). However, for membrane proteins (or at least those whose membrane domains are composed largely of (l-helices), the prospects of prediction are somewhat brighter as the lipid bilayer restricts the number of degrees of freedom of the system. Prediction of transmembrane (TM) Cl-helices is widespread and a number of different methods and hydrophobicity scales have been devised to aid such predictions (7). Other methods allow the prediction of preferred orientations of such TM helices, thus placing restrictions on how they may be packed together in the intact protein.
Mapping phenotypic trait loci
Authors: Taylor Benjamin A
Published: November 2023

When a genetic locus is known only by a phenotype, mapping generally re quires analysis of segregating crosses (i.e. meiotic mapping). Phenotypic trait loci vary from major mutations affecting simple qualitative trait differences such as coat colour, to quantitative trait loci (QTLs) that exert small statistical effects on complex traits such as body weight. If the phenotypic trait variant is a new mutation, a new cross must be generated to detect linkage. On the other hand, if the new locus represents a variant that distinguishes inbred strains, special genetic systems, such as recombinant inbred strains, may provide an attractive alternative to a traditional linkage cross.
Characterization of cellular antigens using monoclonal antibodies
Authors: Hynes Gillian
Published: November 2023

Monoclonal antibodies are powerful biological tools due to their ability to distinguish an antigen molecule, typically a protein, from a complex mixture of macromolecules. A number of techniques have been developed to detect cellular antigens using monoclonal antibodies as specific probes. Consequently, a characterization study can be advanced considerably after the generation of a monoclonal antibody reagent to a target protein, and a possible strategy is outlined below:
Analysis of signal transduction pathways using protein kinase inhibitors and activators
Authors: Naito Yasuhito , Kobayashi Ryoji , Hidaka Hiroyoshi
Published: November 2023

A complete understanding of the organization and functioning of the protein phosphorylation system requires the pooling of expertise in several different fields, such as molecular pharmacology, genetic manipulation, biochemistry, and cell biology. The advent of a new class of effective pharmacological agents is always an event of considerable interest, in particular for new types of antagonists, i.e. potentially important groups of compounds that act by specifically blocking one or more of the steps in intracellular signalling systems (1). There is now convincing evidence that intracellular protein phosphorylation systems are of fundamental importance in biological regulation. A large number of cellular mechanisms involving protein phosphorylation have been revealed over the past decade. However, interrelationships amongst signal cascades are complex and uncertainties concerning the cellular responses remain. In order to obtain improved understanding, sophisticated methods for estimating changes in the activities of cellular response elements after extracellular stimulation have to be developed. While progress has been made in the biochemistry and molecular biology of protein kinases, it has proved very difficult to elucidate their function in intact cells. For this reason researchers studying second messenger systems and protein phosphorylation have long sought specific and effective inhibitors that would permit definitive determination of physiological roles of protein kinases (2).
Analysis of signal transduction pathways using molecular biological manipulations
Authors: Sutherland Calum
Published: November 2023

This chapter describes the approaches currently available to generate molecular biological tools for the dissection of intracellular signalling pathways, together with protocols required for their application. Many standard techniques of molecular biology, which have been previously documented, are required. In such cases, specific examples are provided with reference to alternative protocols.
Instruments for fluorescence imaging
Authors: Amos W B
Published: November 2023

Fluorescence microscopy is a highly sensitive method: in the last decade it has been extended to the detection of single fluorophores (1). To do this, as much as possible of the emission must be collected and a low background must be achieved by shielding the detector from the short-wavelength light used to excite the fluorescence.
Functional characterization of co-activators using mammalian cell microinjection
Authors: Rose D W , Mullek T M , Rosenfeld. M G , Glass C k.
Published: November 2023

The past few year; have led to an appreciation that the transcriptional activity of the nuclear receptor family is regulated by binding of the receptor. in a ligand-dependent manner. to a multicomponent complex of co-activator proteins. many of which possess histone acetyltransferase activity. The activity of these co-activators is; also further regulated by a variety of signal transduction pathways. Many of these co-activator molecules have now been isolated, often on the basis of their interaction with the nuclear receptors. The functional characterization of these protein complexes has been facilitated by the use or a series of powerful single cell microinjection assays that employ the use of specific anti-co-activator antibodies used in conjunction with reporter constructs specific for each of the nuclear receptors and expression constructs for wild-type and mutant forms of the co-activators.
G proteins II: Gq, G12, and Gz
Authors: Glick Jennifer L , Meigs Thomas E , Casey Patrick J
Published: November 2023

The ability of the cell to sense and communicate with its environment is critical for its survival, and therefore for the survival of the organism. Simpler life forms such as Dictyostelium sense toxic or unfavourable environments and rely on the process of chemotaxis to steer away from these areas and toward areas where conditions are more suitable. During the development of multicellular organisms, progenitor cells must migrate appropriately and coordinate their differentiation with one another in order to produce a viable embryo. Adult organisms rely on sensory cells such as photoreceptors, the olfactory epithelium, and taste buds, as well as a central nervous system, to guide them out of danger and track down food or potential mating partners. Further still, in multicellular organisms, cells must be able to respond to complex signals that originate from many regions of the body. A classic example of this is the well-known ‘fight or flight’ response, wherein an animal senses imminent danger and the body immediately prepares itself for the necessary action (for instance, with a faster heartbeat to increase blood flow to its musculature). Many of the processes involved in this type of response (vision, memory, smell, taste, reflex) are in large part regulated by heterotrimeric G proteins (G proteins).
Mammalian MAP kinase pathways
Authors: Kyriakis John M
Published: November 2023

Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of cellular regulation. All eukaryotic cells possess multiple MAPK pathways, each of which is preferentially recruited by distinct sets of stimuli, thereby allowing the cell to respond in parallel to multiple divergent inputs. Mammalian MAPK pathways can be recruited by a wide variety of different stimuli ranging from hormones, such as insulin and growth hormone, to mitogens (i.e. epidermal growth factor, EGF; platelet-derived growth factor, PDGF; fibroblast growth factor, FGF), vasoactive peptides (angiotensin-II, endothelin), inflammatory cytokines of the tumour necrosis factor (TNF) family, and environmental stresses such as osmotic shock, ionizing radiation, and ischaemic injury.
ATM and DNA-PK: the sensing, signalling, and repair of DNA damage
Authors: Bristow Robert , Bluyssen Hans , De Klein Annelies , Van Gent Dik
Published: November 2023

A significant and ongoing problem for every living cell is the occurrence of DNA damage caused by exogenous and endogenous agents. For example, DNA base damage and DNA strand breaks are induced as consequences of the normal cellular processes of DNA replication and oxidative stress, or after exposure to exogenous DNA-damaging agents including UV-light or ionizing irradiation. As a result, cells have developed a sophisticated approach to the integration of DNA damage-sensing and subsequent DNA repair as a means of preserving genomic stability. This chapter will mainly focus on the cellular activity and biochemistry of two members of the PI3K-related protein kinase family named ATM (for ‘mutated in ataxia telangiect-asia’), and DNA-PK (DNA-dependent protein kinase), which are thought to play important roles in the sensing, signalling, and repair of DNA strand breaks.
Detection and quantification of heterogeneous viral targets: HIV as a model
Authors: Kwok Shirley
Published: November 2023

The diversity of the human immunodeficiency virus (HIV) genome presents it as one of the most difficult targets for the development of nucleic acid-based assays. The extensive genetic variability of HIV is primarily due to the high error rates of the viral reverse transcriptase (3.4 x 10-5 per base incorporated) and the absence of proofreading mechanisms (1). HIV-1 and HIV-2 comprise the two groups of viruses that cause AIDS. The HIV-ls are divided into three groups: M (for Major), 0 (for Outlier), and N (for non-M, non-0). The HIV-1 group M and HIV-2 viruses have been further divided into subtypes A-K and A-G, respectively, based on phylogenetic analysis of proviral sequences (ref. 2; see Chapter 8). The group O viruses are as heterogeneous as the group M viruses and comprise a number of distinct genetic subtypes that remain to be identified and characterized (3). The recently identified, HIV-1 group N viruses are distinct from group Mand O (4). Within individuals, HIV-1 undergoes continuous genetic changes; patients usually harbour a swarm of highly related but individually distinguishable viral variants, which are referred to as quasispecies. Genomic recombination between two different HIV-1 populations frequently occurs in vivo, which further contributes to HIV genetic variability.
PCR-based methods of HLA typing
Authors: Erlich Henry A , Trachtenberg Elizabeth A
Published: November 2023

Over the last two decades, molecular genetic techniques have been used to isolate the genes encoding the HLA class I and class II molecules and to characterize their genomic organization as well as their sequence diversity. The allelic sequence polymorphism at the HLA class I and II loci, revealed by extensive sequencing studies on a variety of human populations, is far greater than the antigenic variation detected by conventional serological typing (1). For example, there are about 280 DRB1 alleles defined by the second exon sequence but only about 15 different DR serological specificities or serotypes. Similarly, for the HLA-B locus, more than 450 alleles have been reported, compared to about 48 serological specificities. For the DPA1 and DPB1 loci, which encode the DP molecule, no serological typing system has been available and, consequently, most functional investigations of DP polymorphism, such as disease association studies or the analysis of DP mismatching in transplantation, have been possible only since the development of DNA-based HLA typing methods (2). The HLA class I and class II loci are the most polymorphic coding sequences in the human genome. The study of their allelic sequence diversity as well as the development of simple and rapid DNA-based typing methods has been greatly facilitated by the development of PCR amplification in the mid 1980s (3–6).
Fluorescence and fluorochromes
Authors: Ormerod Michael G
Published: November 2023

Fluorescence occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength. The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters. Fluorescence has been used to visualize certain molecules (and hence structures) by light microscopy for many years. It is a sensitive technique because a positive signal is detected against a negative background. It has found widespread application in flow cytometry and, together with light scatter, is the major type of measurement made in most machines. The ability to detect fluorescence simultaneously from two, three, or four com pounds fluorescing at different wavelengths enables several parameters to be measured and opens up the field of multi-parametric analysis.
Signal transduction in flamentous fungi: regulation of protein kinases
Authors: Ye Xiang S , Wu Liping , Osmani Stephen A
Published: November 2023

In eukaryotes, including fungi, protein kinases are key components of signal transduction pathways. Normally a cascade of reversible protein phosphorylation/ dephosphorylation is the major mechanism of signal transduction, which provides both signalling specificity and diversity. Although second messengers, such as Ca2_, IP3, cAMP, and G proteins have pivotal roles in many signalling events, their physiological functions are normally mediated through regulation of protein kinases.