bluuebunny/crossref_metadata_2025 huggingface dataset:
The construction and use of cDNA libraries for genetic selections
Authors: Mulligan John T , Elledge stephen J
Published: November 2023

The popularity of the yeast Saccharomyces cerevisiae as a research system is due in large part to its facility as a genetic organism and its usefulness as a model eukaryote. It combines the straightforward collection and analysis of mutations with the ability to isolate the corresponding genes by transformation and complementation. All of the genetic elements necessary for promoting chromosomal replication and segregation reside on small fragments which have been incorporated, along with selectable markers, into small cloning vectors capable of replicating in Escherichia coli or yeast.
Plasmid shuffling and mutant isolation
Authors: Sikorski Roberts. , Keeney, Jill B , Boeke Jef D
Published: November 2023

Genetic analysis of mutants in Saccharomyces cerev1szae offers a unique approach to the study of biological processes. To initiate genetic studies in this organism one can mutagenize a population and screen or select for those mutants which affect the process of interest. Alternatively, one can use techniques selectively to mutagenize a single gene already known to play an important role in this process. The generation of mutant yeast strains starting from a cloned non-essential yeast gene is relatively straightforward. To remove the wild-type gene product, an essential step in analysing recessive alleles, DNA at the wild-type non-essential locus can be deleted entirely from the genome. A collection of mutant alleles can be made in vitro and introduced into the deleted host cell via episomal cloning vectors. The generation of mutant yeast strains from an essential yeast gene is a more complicated task since removal of the wild-type gene in one simple step yields an inviable genotype.
Measurement of transcription
Authors: Piper Peter W
Published: November 2023

This chapter discusses the experimental tricks needed to determine if a cloned yeast gene is transcriptionally active under defined physiological conditions, or if DNA constructs newly inserted into yeast cells by transformation are being efficiently transcribed in vivo. The techniques it describes rely upon the availability of a DNA probe suitable for quantification of the transcript(s) of interest by DNA-RNA hybridization. It should be read in conjunction with standard methods manuals that describe the experimental details of Northern blotting, probe preparation, blot hybridization and the mapping of transcripts on DNA (1).
DNA binding of immobilized proteins
Authors: Flytzanis Constantin N
Published: November 2023

The identification of proteins which have affinity for specific DNA sequences has greatly contributed to our understanding of the transcriptional control of gene expression. A plethora of transcription factors have been purified and characterized by techniques that take advantage of the specificity of recognition that such nuclear proteins exhibit towards relatively small sequences of DNA. In theory at least, having a DNA element guarantees the isolation of the protein which binds to it in vitro, and vice versa.
Cyanobacterial transformation and gene regulation
Authors: Nixon Peter , Jansson Christer
Published: November 2023

The cyanobacteria are a diverse group of prokaryotes that are able to carry out oxygenic photosynthesis using chlorophyll a and phycobilip troteins as their primary photosynthetic pigments. Morphologically, cyanobacteria range from simple unicellular forms to complex filamentous forms that possess several highly differentiated cell types (1). Although cyanobacteria can grow in a variety of modes, including chemoheterotrophy, their ability to perform oxygenic photosynthesis in a similar manner to higher plants, coupled with their relative ease of genetic manipulation, has led to their adoption as model systems for the study of photosynthetic processes. Indeed, unicellular cyanobacteria-like organisms are thought to be the progenitors of higher plant chloroplasts (discussed in Ref. 2). In particular, the photosynthetic electron transport chain is being intensively studied (3-5). However, other important biological problems are being studied using cyanobacteria, including nitrogen fixation, carbon fixation, light-regulated gene expression, chromatic adaptation, and cellular differentiation. In this review we describe techniques used in the genetic manipulation of cyanobacteria and the current status in our understanding of cyanobacterial gene regulation in response to changes in the environment.
Epitope mapping using libraries of random peptides displayed on phage
Authors: Dower William J , Cwirla Steven E
Published: November 2023

In 1985 Young et al. (1) introduced a method for cloning immunoreactive sequences, such as bacterial surface antigens, by expressing the proteins in X. plaques and identifying the clones of interest by probing with the appropriate antibodies. This method relied on the co-localization of the reactive peptide with the cloned DNA in a plaque or colony. This is a powerful technique, but it is difficult to screen more than a few million clones by this approach. Alternative methods, relying on the physical linkage of the protein and DNA, have recently become practical. These methods are known as peptide display techniques, and comprise the cloning of sequences that are expressed as peptides or proteins fused to a carrier protein and displayed on the outer surface of microorganisms. The surface peptide is immunoreactive, and the encoding DNA is carried within the microorganism. The clones of interest are isolated by affinity purification of the exposed peptides on a matrix of immobilized antibody or other binding protein. This approach, because it provides for the efficient isolation of individual microorganisms from a complex mixture, allows the screening of extraordinarily large numbers of clones.
Effect of acetyl substituents on the conformational stability and functional interactions of acetan polysaccharide
Authors: Ojinnaka C , Morris E R , Morris V J , Brownsey G J
Published: November 2023

Acetan is similar in structure to xanthan, and adopts a similar 51 conformation in the solid state, but has longer (pentasaccharide) sidechains, no pyruvate substituents, and an additional site of acetylation (believed to be at C(6) of the branched glucose residues). Acetan solutions show clear evidence (by optical rotation and DSC) of conformational ordering on cooling. The transition is fully reversible on heating, with no thermal hysteresis. The intrinsic viscosity of the ordered form decreases with increasing concentration of salt up to ∼10 mM KCl, yielding an estimated persistence length of ∼60 nm (in comparison with ∼50 nm for ordered xanthan). At higher salt, however, the intrinsic viscosity increases sharply, indicating extensive aggregation, which is reflected in development of xanthan-like ‘weak gel’ rheology in more concentrated solutions. Deacetylation stabilises the helix, but suppresses aggregation.
Some physico-chemical aspects of protein processing in foods
Authors: Tolstoguzov V B
Published: November 2023

To describe the structure formation mechanisms in foods the concept of protein conformational potential is proposed. The production of many structured foods may be regarded as the formation of intermolecular interactions throughout a processed food system using the intrarnolecular bonding potential of its proteins and polysaccharides. The term “conformational potential” describes the capacity of a protein to form the intermolecular junction zones that are required to generate the desired structural, rheological and other physico-chernical properties of a given food system. Two basic physico-chernical characteristics are mainly responsible for the protein conformational potential. These are the co-solubility of a protein with other food ingredients and the denaturation-renaturation behaviour of proteins. This new concept encourages better understanding of food protein functionality at the molecular level. To illustrate this new approach, the structure formation processes of some food systems are considered.
A comparison of the in vitro and in vivo digestibilities of retrograded starch
Authors: Botham R L , Ring S G , Noel T R , Morris V J , Englyst H N , Cummings’ J H
Published: November 2023

The resistance to digestion of different forms of processed starch by mammalian pancreatic a-amylase was examined in ileostomists. Chick peas and chick pea starch were processed by cooking in water at 100°c and allowed to retrograde at 4°C prior to consumption by ileostomist volunteers, the diet of the volunteers being free of other plant polysaccharides. The digestibility of the starch was assessed from analysis of the ileostomy effluent. The polysaccharide in the effluent was subjected to sugar and methylation analysis to determine the monosaccharide composition and the mode of linkage. The main resistant product from feeding the retrograded starch gel was a linear dextrin with a number average degree of polymerisation of 14. The cellular matrix modified the digestibility of the starch with both linear and branched molecules escaping digestion. From the ratio of the cell wall polysaccharides to dextrins in the effluent, it was found on average 15% of the ingested starch had escaped digestion.
Rheological investigation of polysaccharide interactions in mixed systems
Authors: doublier J L
Published: November 2023

The viscoelastic behaviour of polysaccharide mixtures can be investigated using oscillatory shear measurements or transient experiments and compared to that of their individual components. As examples, mixtures of a galactomannan (guar gum, locust bean gum) with another polysaccharide (xanthan gum or K-carrageenan) are described. Depending upon the polysaccharides and the composition ratio, viscoelastic behaviour changes noticeably. At the optimum of the synergistic effect, that is at a polysaccharide/galactomannan ratio of the order of 50/50, strong gels are obtained. However, their viscoelastic behaviour suggests that the molecular mechanisms underlying the properties cannot be limited to a single type of interaction. On the other hand, peculiar viscoelastic behaviour is experienced with ratios as low as 1/99, irrespective of the type of galactomannan (guar gum or locust bean gum). Despite the high content in galactomannan, the properties cannot be compared to those of macromolecular solutions such as displayed by the galactomannan alone. Typically, the behaviour is solid-like as can be seen in oscillatory shear at low frequency as well as at relatively long times in creep measurements. This means that a weak gel has been formed. The effects of temperature on the properties of xanthan/galactomannan mixtures show that those gels melt at a temperature of ∼60°C, well below the conformational transition of xanthan molecules. This clearly suggests that melting of xanthan is not required to induce melting of the mixture as usually believed. All these results are discussed on the basis of current models that have been proposed for the understanding of the mechanisms involved in synergistic interactions between polysaccharides.
The chemistry of iso-alpha-acids: an explanation of their mode of action
Authors: Simpson W J , Fernandez J L , Hughes P S , Parker D K , Price A C
Published: November 2023

The ability of iso-a-acids to bind metal cations is important with respect to antibacterial activity and foam. Divalent cations bind to trans-isohumulone, bringing about a change in its solubility and light absorption properties. Monovalent cations do not bind to isohumulone unless a divalent cation is present. The flavour threshold of trans-isohumulone is not markedly affected by pH indicating that both anion and undissociated molecule are flavour active.
Flocculation of brewer’s lager yeast
Authors: Aar Paul C van der , Straver Marika H , Teunissen Aloys W R H
Published: November 2023

Flocculation behaviour and its mechanism of brewer’s yeast (NewFlo) are studied by integrated research on biochemical, molecular genetically and physiological aspects of floe-formation during beer fermentation. Results show that besides the exposure of a protein (lectin) and its receptor (mannan), hydrophobicity of the cells plays a major role in flocculence regulation. There is a strong correlation between stop of cell division and initiation of flocculence, independent of the factor causing this division arrest.
Die Wahrnehmung des unsichtbaren Riechen und Schmecken: Moleküle als Sinnesreize
Authors: Boeckh Jürgen
Published: November 2023

Behavioral effects and different roles of chemical, especially olfactory sensory stimuli for nutrition, reproduction and social life in humans and their relatives. The importance of learning and valuation of odors; hedonic character, emotional and motivational effects. (2) The process of olfaction as the sensory detection and discrimination of complex chemical patterns, i.e. mixtures of certain components in certain ratios. The sensors for such stimuli in the nose, how they analyze such patterns and convey the result to the brain. (3) Olfactory centers in the brain and their modes of function.
Détection rapide des Enterobacteriaceae
Authors: Laurent F , Leitz R , Husson M O , Drocourt J L , Peladan F , Leclerc H
Published: November 2023

Now, in order to manage his process and the overall quality of his products, the brewer has to detect more quickly microbiological contaminants. We developed a monoclonal antibody against one special epitop of the Cunin antigene, which is common to all Enterobacteriaceae, and now, we are able to detect and count, in 3 hours, all the Enterobacteriaceae, present in a beer or a water sample. This work shows that immunology is probably one of the best ways to set up new, rapid, sensitive and specific methods to detect microbial contaminants encountered in breweries.
Assaying ion channels: design and practice
Authors: Kasai Michiki , Ide Toru , Agnew William S
Published: November 2023

Isolation of the molecule in chemically pure form is an important approach in the characterization of ion channels. This enables determination of molecular composition and size, and may permit functional reconstitution in artificial membranes for biochemical or electrophysiological measurements. Purification requires that the protein be solubilized before separation from other proteins associated with the membrane sample. However, unlike other enzymes, channel molecules typically lack chemical catalytic activity, and quantification in detergent solution must either be performed with a specific biochemical marker, such as a ligand, toxin, or drug that binds stoichio-metrically, or the protein must be returned to a membrane environment for measurement of ion-transporting function. Ligand binding assays constitute the single most important tool for channel and receptor quantification and purification, and the following discussion and examples are aimed at providing insights useful for designing new assay protocols. Some channels will be resistant to this approach, lacking any useful ligand, and we also describe how the progress of an ion channel purification protocol can be followed by bilayer reconstitution.
Channel cloning, mutagenesis, and expression
Authors: Whiting Paul J , Wafford Keith A , Pribilla Iris , Petri Thomas
Published: November 2023

The application of the techniques of molecular biology to the understanding of the structure and function of ion channels has resulted in a quantum leap in our knowledge which 15 years ago would have seemed inconceivable. The cloning of a cDNA encoding a channel protein is now considered only the first step, opening up many areas for detailed study: Computer analysis of the deduced amino acid sequence to predict putative transmembrane topology, secondary structure, and post-translational modification.
Nigral and striatal neurons
Authors: Barker Roger , Johnson Alan
Published: November 2023

This chapter describes simple and reliable methods for the culture of dissociated nigral and striatal neurons. We give an accurate definition of the conditions required for the survival of these cells. These data may have implications for studies on neural development as well as those concerned with neurodegeneration. In this last respect two of the commonly encountered yet major neurodegenerative diseases of the CNS are Parkinson’s disease (PD) and Huntington’s Chorea (HC), which predominantly affect the dopaminergic nigral and GAB Aergic striatal neurons respectively (1,2). This coupled to the use of these cells for intracerebral transplantation (3), has led to a large number of in vitro studies involving these neuronal populations (see Table 1 and 2). There is, however, no universally adopted technique although the methods described in this chapter are used in several laboratories and routinely produce reliable results.
Cranial sensory neurons
Authors: Davies Alun M
Published: November 2023

Cranial sensory neurons have many advantages for studying the different phases of neuronal development. These neurons and their progenitor cell populations are well defined and can be easily obtained for in vitro experimental studies and are accessible to experimental manipulation in ova in avian embryos from the earliest stages of their development (1,2). The peripheral target fields of several cranial sensory ganglia, such as the trigeminal ganglion, are clearly circumscribed, and this has facilitated detailed studies of the influence of the target field on various aspects of neuronal development (3-6). In contrast to the functional heterogeneity of dorsal root ganglia, cranial sensory neurons are for the most part segregated into functionally distinct populations. Perhaps as a consequence of this functional segregation, populations of cranial sensory neurons have quite distinct neurotrophic factor requirements. This has facilitated studies of the function and co-operation of different neurotrophic factors in regulating neuronal survival (7–9).
HIV infection of dendritic cells
Authors: Knight Stella C , Patterson Steven
Published: November 2023

Introduction

Dendritic cells (DC) are crucial for stimulation of T cells. They are derived from the bone marrow and share a common stem cell with macrophages. Granulocyte-macrophage colony stimulation factor (GM-CSF) and tumour necrosis factor alpha (TNFα) (1) promote the differentiation of CD34-positive stem cells into mature DC. Small numbers of stem cells are also present in the blood (1-2 per 105 mononuclear cells) as well as more mature DC which may constitute 0.5-2% of the cells.
Sequence analysis of virus variability based on the poymerase chain reaction (PCR)
Authors: Brown Andrew J Leigh , Simmonds Peter
Published: November 2023

During most stages of HIV infection, the viral population contains a large number of closely related variants that have diverged within the indvidual over the course of HIV infection. For example, env sequences of human immunodeficiency virus in lymphocytes of infected individuals may show variability of up to 10% within the same sample (1,2). Under these circumstances, sequence analysis of PCR products amplified from such samples is often unreliable due to ambiguities in the sequencing gel. While there are methods for measuring the relative frequencies of alternative nucleotides at these polymorphic sites (see section 3), this does not distinguish the linkage relationships of the variant nucleotides so it is impossible reliably to reconstruct the constituent sequences.
CD4-positive T cell responses to HIV-1 infection
Authors: Siliciano R F
Published: November 2023

By producing cytokines that modulate the activity of B lymphocytes, macrophages, CD8-positive T cells, and other types of cells, CD4-positive helper T cells play a central role in most immune responses, including the immune response to HIV-1. Analysis of CD4-positive T cell responses in HIV-1-infected individuals is complicated by the fact that CD4-positive cells are selectively infected by HIV-1. Following seroconversion, infected individuals experience a progressive decline in circulating levels of CD4-positive T cells (discussed in Chapter 13). In addition, a state of hyporesponsiveness of the surviving CD4-positive T cells is observed, particularly in later stages of the disease (1). Thus, in analysing the CD4-positive T cell response to HIV-1 and to opportunistic pathogens in infected individuals, the superimposed effects of HIV-l-induced immunosuppression must be considered as well. By the same token, the study of CD4-positive T cell responses in infected individuals can provide important information about the degree of immunologic impairment induced by infection. This chapter will consider some of the theoretical problems with and experimental approaches to assessing CD4-positive T cell responses in HIV-l-infected individuals. We will consider methods for measuring the overall functional integrity of the CD4-positive T cell compartment as well as methods for analysing specific CD4-positive T cell responses to HIV-1.
Ribonuclease H
Authors: Hostomska Z , Hostomsky Z
Published: November 2023

The ribonuclease H (RNase H) is an essential activity associated with HIV reverse transcriptase (RT). RNase H cleaves the RNA in hybrid RNA-DNA intermediates during minus-strand DNA synthesis initiated from the tRNALys, 3 primer. In the course of this ordered degradation of the genomic RNA template, the enzyme also generates a specific RNA fragment in the polypurine tract (ppt) region, which is then used as a primer for initiation of the plus-strand DNA synthesis. RNase H is also involved in the specific removal of both tRNA and ppt primers so that synthesis of the double-stranded DNA of the provirus can be completed. These well-defined functions of RNase H during reverse transcription can not be substituted by cellular enzymes. Since inhibition of any of these functions would block virus replication, RT-associated RNase H represents a valid target for antiretroviral therapy (1).
Expression of HIV proteins using recombinant baculoviruses
Authors: Jones Ian
Published: November 2023

Why express viral proteins in foreign systems? After all, the expression of a viral product in isolation may overlook an essential factor in the host cell environment. A variety of reasons are commonly proffered: The level of expression of any given gene product may be improved to allow antibody generation or commercial development. This is particularly true of non-structural proteins involved in control rather than virion assembly. The expressed viral product can be manipulated by mutagenesis to determine the interplay of structure and function. The function of individual viral proteins can be distinguished from those of the virion background. Experimental work with the ‘real’ virus is difficult or dangerous. These factors provide the driving force behind the sometimes difficult search for suitable expression systems for HIV genes. However, it is important from the outset to realize the limitations of what can be achieved with expression systems alone. The study of one particular gene product can only partially represent its complete role in the viral life cycle since it is the interaction of viral proteins together that results in virus replication and associated pathogenesis. Reservations aside, however, expression of a single viral product can provide much basic information on the way in which a protein functions.
Envelope expression and purification
Authors: Opstal O Van , Fabry L , Francotte M , Thiriart C , Bruck C
Published: November 2023

The envelope glycoproteins of human immunodeficiency viruses (HIV types 1 and 2) play a central role in the transmission of acquired immunodeficiency syndrome (AIDS) (1). The viral envelope mediates attachment of the virus to the CD4 receptor present on target cells (2-4) and the spread of virus by cellto-cell fusion (5, 6). In the infected cells, the viral envelope of HIV-1 is synthesized as a precursor glycoprotein (gp160) which is subsequently cleaved into an external surface protein gp120 and a transmembrane protein gp41, upon maturation in the Golgi (7-9). After cleavage, the two glycoproteins (held together by non-covalent interactions) are transported to the cell surface.
Cellular assays for Rev
Authors: Malim M H
Published: November 2023

The human immunodeficiency virus type-1 (HIV-1) Rev trans-activator is a nuclear phosphoprotein absolutely essential for virus replication. It is expressed from fully spliced (∼ 2 kb) viral mRNAs and is required for the expression of viral proteins, e.g. Gag, Pol, and Env, from the unspliced ( ∼ 9 kb) and singly spliced (∼ 4 kb) mRNAs. The cis-acting target for Rev, the Rev response element (RRE), is an elaborate RNA stem and loop structure that is located within the env gene and is therefore present in all 9 kb and 4 kb transcripts. Although Rev has been the focus of considerable research effort in recent years, its precise mechanism of action still remains elusive; specifically, models invoking the inhibition of splicing, the activation of nucleocytoplasmic RNA transport, the stabilization of RNA, and the induction of translation in the cytoplasm have all been proposed (reviewed in refs 1 and 2).
Vpu
Authors: Cohen E A , Gottlinger H G
Published: November 2023

The vpu gene in the central region of the HIV-1 genome partially overlaps the env reading frame and is predicted to encode a protein of 81 amino acids with a hydrophobic N-terminus and a hydrophilic C-terminus. Vpu is unique to HIV-1 and is absent from HIV-2 and SIV (1). Biochemical studies have shown that Vpu is a phosphorylated type I integral membrane protein and is capable of forming homo-oligomers (2, 3). Immunofluorescence and immune electron microscopy studies have demonstrated that Vpu is concentrated in the Golgi apparatus and in endosomal vesicles (4, 5). Vpu can also be localized to the cell membrane, but does not accumulate there, suggesting that it may be recycled between different cellular compartments (5). Vpu is not incorporated into the virion.
Generation of HLA-specific human monoclonal antibodies
Authors: Pistillo Maria Pia , Ferrara Giovanni Battista
Published: November 2023

HLA class I and class II antigens encoded in the major histocompatibility complex (MHC) are polymorphic membrane glycoproteins whose function is to bind immunogenic peptides inside the cell and transport them to the cell surface, where they can be presented to specific T cells. The interaction between T cell receptor, HLA molecules, and the presented peptides is essential for self and non-self discrimination and for the generation of immune responses. The earliest approaches used to define the polymorphism of HLA molecules were essentially serological based on the use of conventional (polyclonal) antisera derived from polytransfused patients, multiparous women, skin graft recipients, or from planned immunization of healthy volunteers (1-3). Such sera frequently reacted not only with cells carrying the immunizing HLA molecule, but also with cells carrying antigens controlled by other HLA alleles at the same locus. This finding was mainly due to the antibody complexity for the presence of several antibody populations that recognized different epitopes of the HLA molecule and (or) the same epitope with different affinities.
Assembly of MHC molecules: requirements of glycosylation, oxidized glutathione, and peptides
Authors: Ribaudo Randall K
Published: November 2023

The ordered assembly of multisubunit proteins such as MHC molecules from their component chains is a fundamental process in the generation of functional complexes such as receptors, signalling complexes, and presentation molecules. Typically, these processes involve not only the constituent chains which make up the mature complex, but also rely on the action of chaperones, and other accessory proteins. Assembly of the two chains of class I MHC molecules occurs in the endoplasmic reticulum and is dependent on the simultaneous presence of the polymorphic heavy chain, and the nonpolymorphic light chain, 132-microglobulin. Careful pulse chase analysis has demonstrated that the two chains rapidly associate in the endoplasmic reticulum (1), and remain there associated with the molecular chaperone calnexin until they acquire an appropriate peptide (2-5). At this point the trimeric complex can exit the ER and traverse the golgi to the cell surface. From the time of initial translocation of the independent chains to the ER to their final appearance on the cell surface, domains fold, disulfide bonds form, carbohydrate residues are added, and peptide is acquired.
Derivation and analysis of MHC transgenic mice
Authors: Sponaas Anne-Marit , Mellor Andrew
Published: November 2023

Transgenesis is the term given to the process whereby cloned DNA is introduced into the germline of a host organism so that it becomes stably integrated into host chromosomes and is inherited from one generation to another. Procedures making transgenesis possible in mice were worked out in the early 1980s. Immunologists have been particularly active in exploiting the potential of this new genetic approach for studying immunological phenomena.
Deuterated vitamin E: measurement in tissues and body fluids
Authors: Burton Graham W , Daroszewska Malgorzata
Published: November 2023

Vitamin E, being the major (≥90%) lipid-soluble, chain-breaking antioxidant in mammalian tissues and body fluids (1–3), is important for the in vivo protection of lipid membrane and lipoprotein structures against free radical lipid peroxidation. The most abundant forms of vitamin E in foodstuffs are n- and γ-tocopherols (α-T and γ-T; Figure 1). α-T, the most biologically active form of vitamin E, is not only the most efficient radical-trapping scavenger (i.e. chain-breaking antioxidant) among the tocopherols but also one of the best in the entire class of simple phenolic antioxidants (4, 5).
Measurement of antioxidant gene expression
Authors: Bingle Colin D
Published: November 2023

As the reader will have already seen in the rest of this volume, the measurement of levels of antioxidant proteins using a variety of functional assays can yield valuable information to the researcher. To gain a better understanding of the processes regulating levels of antioxidant proteins within a particular experimental system it is becoming clear that studies aimed at investigating regulation at the genetic level are increasingly important. The present chapter details a number of basic protocols which should allow the researcher new to molecular biology to begin to study the steady-state levels of antioxidant gene expression. Such protocols have been used for example to study developmental regulation of antioxidant proteins (1) and their induction by hyperoxia (2), ozone (3), and inflammatory mediators (4). These protocols are by no means exhaustive and in depth discussion of the methods and theory of RNA analysis are given in references 5 and 6. Measurement of steady-state RNA levels also opens the way to further studies of the transcriptional status of antioxidant genes using techniques which allow direct analysis of the genes of interest.
Quality assurance in immunodiagnostics
Authors: Bullock David g. , mackenzie finlay
Published: November 2023

The primary function of immunodiagnostic investigations, as with any healthcare laboratory investigation, is to provide the clinician with analytical data obtained from examination of specimens submitted from patients and to assist in interpreting these results, in order to assist in diagnosis and control of therapy for the individual patient, for research or for public health purposes. Quality assurance is a global term to describe those means of ensuring that the results being issued from a laboratory are dependable and sufficiently accurate and precise to allow decisions to be taken with confidence, i.e. that the service is fit for the purpose. If such confidence is lacking, additional costs will accrue, not only in terms of inappropriate resource utilization through repeat testing etc., but also suffering of individual patients and, in some cases, the entire community. QA measures are not a luxury or optional extra, but must be an integral part of the investigational process in providing an appro priate and effective clinical service.
Introduction
Authors: Smith Deborah F , Parsons Marilyn
Published: November 2023

The eukaryotic parasites are a diverse group of organisms that range from some of the most ancient single-celled species (e.g. Giardia lamblia) to the cytologically complex metazoan helminths. Despite this extreme diversity in body form, all endoparasites share the common features of living within and exploiting a host organism. This exploitation has resulted in the evolution of some remarkable adaptations at the biochemical level: many parasites have dispensed with whole metabolic pathways, while developing novel strategies for the evasion of host defences. In addition, complex mechanisms have evolved to facilitate transmission of parasites to new hosts. Typically, parasites take advantage of the relationships between two species to facilitate transmission: for example, the mosquito takes a bloodmeal from a malarious individual and thereby becomes infected with the parasite. Transmission is not passive, but rather involves parasite development. Therefore most parasites undergo complex, multiphasic life cycles that can involve extracellular and intracellular stages and, for unicellular parasites, maintenance as actively dividing populations or as dormant, non-dividing cysts.
Protein recognition of specific DNA and RNA binding sites
Authors: Stockley Peter G
Published: November 2023

Facetiously, it has been said that there are only two substantive problems in biology. These are: one, to understand how protein molecules recognize and interact with nucleic acids, and, two, to understand the consequences thereof! This is obviously a massive over-simplification, although it does contain a grain of truth. This chapter will address the first of these problems whilst the remainder of this book will deal with aspects of the second. Nucleic acid recognition will be illustrated by reference to specific examples drawn from the regulation of prokaryotic gene expression. This will necessarily not be a comprehensive review given the wealth of knowledge now available in this area. Some discussion of eukaryotic examples will be introduced for comparison purposes. In some cases, the examples chosen to illustrate particular features of nucleic acid recognition are also the topics of separate chapters elsewhere in this book, and in those cases the reader should follow the cross-referencing instructions to obtain more detailed discussions of the biology of the systems being discussed.
Repressors and activators
Authors: Busby Stephen J W
Published: November 2023

The study of adaptive responses in bacteria led to the discovery of a panoply of gene regulatory proteins (1). These are mostly DNA-binding proteins and their role is to modulate promoter activity, thereby coupling the expression of specific genes to changes in the environment. Gene regulatory proteins are ubiquitous: they are essential for all processes of adaptation and differentiation in all living cells. At first sight this is a very complex topic, made incomprehensible by the sheer number of different factors and different modes of operation. The aim of this chapter is to tackle the central question of how gene regulatory proteins function, and to describe a small number of key examples from the microbial repertoire, that reveal principles applicable to most systems.
Post-transcriptional control
Authors: Gu Zhiping , Lovett Pauls.
Published: November 2023

The regulation of gene expression can potentially occur at several stages in the stepwise transfer of information from a gene to its protein product. While transcription initiation appears to be pivotal in the control of expression of many genes, modern methodologies for gene and RNA manipulation have identified various forms of post-transcriptional control in bacteria and eukaryotes. Post-transcriptional control formally refers to all regulation that occurs after transcription. However, this chapter will focus on forms of control that specifically affect translation.
Integration of control devices. II. Sporulation and antibiotic production
Authors: Yudkin Michael D , Chater Keith F
Published: November 2023

An earlier chapter described the specialized characteristics that E. coli cells adopt during stationary phase, and the complex regulation that establishes this differentiated state. In equivalent situations, some other bacteria of diverse taxonomic groups show more obvious physiological and morphological differentiation, to give rise to antibiotic production and to resting cells called spores that are somewhat (or in some cases very much) tougher and longer-lived than stationary phase E. coli cells.
Genetically engineered antibodies
Authors: Owens Raymond J
Published: November 2023

Monoclonal antibody technology has produced immunoglobulins with specificities to a wide range of clinically relevant antigens, including tumourassociated markers, viral coat proteins, and lymphocyte cell surface glycoproteins. However, the use of rodent antibodies in human diagnosis or therapy poses a number of problems. Foremost amongst these is the immunogenic nature of the immunoglobulins (1). This in turn limits the use of repeated doses of antibody essential for chronic treatment. The availability of human monoclonal antibodies would greatly reduce this problem but such antibodies are generally restricted in their range of specificities. Further, human hybridomas are often unstable and/or poor producers preventing the production of large amounts of the human immunoglobulin.
Enzyme amplification systems in ELISA
Authors: Self Colin H , Bates David L , Cook David B
Published: November 2023

Enzyme amplification was devised to allow the development of highly sensitive and rapid immunoassays (1).Enzyme labels were amongst the earliest alternative labels to radioisotopes introduced to immunoassay. With the limited specific activity of radioisotopes, it had been realized that, theoretically, enzymes had a much more sensitive detection capability because of their capacity for continuous catalysis of transformation of substrate to deliver some form of signal; for example colour, fluorescence, or electric current. The original concept of enzyme amplification was developed in response to the fact that, in routinely used tests, enzyme labels were not able to generate sufficient signal to compete with the then much more widely employed radioactive labels. Increasing substrate incubation times to days rather than minutes or hours simply served to increase background signals. Enzyme labels capable of much greater powers of signal generation were required to enable the development of not only more sensitive, but faster immunoassays. The major advantage of employing a sensitive enzyme measurement even in a competitive method with its inherently limited sensitivity (2), was seen to be the ability to deliver a very rapid assay system (3).
Enzyme immunoassays for detection of cell surface molecules, single cell products, and proliferation
Authors: Pearce N W , Sedgwick J D
Published: November 2023

For more than 25 years, immunoenzymatic techniques have been applied extensively to the localization of antigens and antibodies in tissue sections as well as to the quantitation of immunoglobulins (lg) and specific antibodies present in body fluids or in cell culture supernatants. The enzyme-linked immunosorbent assay (ELISA) is a sensitive, rapid, and versatile assay system for the quantitation of antigens and antibodies and is now used extensively for the measurement of soluble molecules. However aside from an early report by Avrameas and Guilbert in 1971 (1) on the application of immuno-enzymatic technology to the analysis of cell surface molecules on isolated lymphoid cells, it is only in the past 15 years that the principles have routinely been applied to the analysis of the varied immunological and biochemical properties of cells in suspension. It is these latter procedures that will be the subject of this chapter.
Antibody affinity measurements
Authors: Saunal Hélène , Karlsson, Robert , Regenmortel March. V Van
Published: November 2023

In order to assess the biological activity of antibody (Ab) molecules, it is necessary to measure the strength of their interaction with the antigen (Ag) in terms of the equilibrium constant KA (1). This constant corresponds to the inverse of the Ab concentration which is needed to obtain 50% of complexed species. Since formation and dissociation of complexes continuously occur the equilibrium state is dynamic. The rates of association and dissociation are of considerable interest since they give information on the way the equilibrium is reached. Changes in reaction rate and affinity can be used to probe structure-activity correlations in one of the reactants (2–4) and to select appropriate reagents for the immunodetection of biological substances.
Analysis of soluble adhesion molecules
Authors: Laan M G Bouma,M P , Dentener, M A , Buurman W A
Published: November 2023

Adhesion of leucocytes to endothelial cells, and their subsequent transendothelial migration into target tissues are crucial events in the development of an inflammatory reponse. This leucocyte-endothelial interaction is mediated by specific adhesion molecules, expressed on the endothelial cell surface, that recognize and bind to their specific ligands on leucocytes. During the last decade, a number of endothelial adhesion molecules have been identified, including E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), which mediate the successive steps of the adhesion process (1). Expression of these adhesion molecules can be induced by a wide range of inflammatory mediators, thereby tightly controlling leucocyte recruitment during inflammatory responses. Also, adhesion molecules are believed to play an important role in the invasive and metastatic cellular processes in the development of cancer (2).
Developtnental regulation of imprinting by DNA methylation
Authors: Chaillet J Richard
Published: November 2023

Genomic imprinting is an unusual and fascinating form of gene regulation in which the two alleles of a gene are distinguished by their parental origin (1-3). Besides potential genotypic (sequence) differences, the two parental alleles of most mammalian genes are indistinguishable in the somatic tissue of the organism. The difference between these non-imprinted genes and the extraordinary imprinted ones originates in characteristic epigenetic marks (imprints) placed on parental alleles. The imprints are established in the germlines of the parents, are transmitted to the embryo following fertilization, and henceforth distinguish the parental alleles of an imprinted locus. One discernible manifestation of this phenomenon is a difference in expression between the maternal and paternal alleles. This role of genomic imprinting in governing the level of allelic activity is an absolute requirement for normal mammalian embryogenesis (2, 4). Fundamental disturbances in the imprinting process, which preclude the 'balanced' inheritance of differentially marked maternal and paternal alleles, frequently result in abnormal embryonic development (5-11). The consequences of many such deviations from biparental (biallelic) inheritance have emphasized the profound importance of the process and have encouraged attempts to understand its molecular mechanism.
Intracellular transport of secretory proteins within intact transient heterokaryons and homokaryons
Authors: Valtersson Conny , Mizuno Masahiro , Dutton, Anne H , Singer S J
Published: November 2023

The overall intracellular pathway taken by secretory proteins inside eukaryotic cells, from their synthesis to their exteriorization, was laid out some time ago (Palade 1975). Initially, proteins to be secreted are synthesized on ribosomes that become attached to the cytoplasmic surfaces of the membranes of the endoplasmic reticulum (ER), and as the proteins are translated they are translocated across the membrane into the lumen of the ER. Following this, the secretory protein (suitably modified) is transferred from the ER to the cis-faces of the Golgi apparatus (GA). After passage through successive Golgi saccules and the trans-Golgi network, the secretory protein is deposited in granules which ultimately fuse with the plasma membrane to release the protein to the exterior of the cell.
PCR in situ hybridization (PCR ISH)
Authors: O’leary John J
Published: November 2023

A major limitation of solution-phase PCR is the inability to visualize and localize amplified product within cellular and tissue specimens. In situ hybridization (ISH) does permit localization of specific nucleic acid sequences at the individual cell level. In conventional non-isotopic in situ detection systems, most protocols do not detect single-copy genes, except for those incorporating elaborate sandwich detection techniques (1).
Reverse transcriptase in situ PCR for RNA detection
Authors: Embleton M Jim
Published: November 2023

Most published applications of in situ PCR for amplification of specific nucleic acid sequences in cells or tissues have been for the detection of genes or viruses which are present in low copy numbers or in a minority of the cell population. Detection may be either indirect, for example by hybridizing a labelled oligonucleotide probe to the amplified DNA, or direct, where the product is amplified with labelled oligonucleotide primers. A variety of labels for oligonucleotides have been used in different situations, including fluorescent dyes for direct microscopic visualization (1,2), and ligands such as biotin or digoxigenin (3-5), which can be used for visualization by other means involving enzyme-substrate systems or binding of labelled antibodies. An application of in situ PCR which so far has been less well exploited is the production of cDNA for cloning. For most cloning applications amplification from nucleic acid templates extracted from cells or tissues is perfectly adequate, and is technically easier to achieve than in situ PCR. However, there are some situations in which it is desirable to co-amplify two separate gene sequences within the same cell, and to maintain them in some form of spatial or physical linkage. The options for this are to carry out PCR on single cells isolated from each other (e.g. in single wells of a microtitre plate), or intracellular PCR on a population of cells. Where the target genes comprise a very large heterogeneous pool, it is impractical to establish huge numbers of single-cell PCRs. A method was therefore devised to perform the PCR in situ on a population of fixed and permeabilized cells which maintained their integrity throughout the process, and to arrange for the amplified sequences to be linked physically within the cells so that they could be recovered in the combinations originally expressed by the cells (1). The specific purpose of this technique was the rescue and linkage of immunoglobulin variable region (V) genes from a population of B lymphocytes containing a multitude of different rearranged immunoglobulin genes, in order to prepare libraries encoding recombinant antibody fragments representative of the original repertoire.