bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA typing reagents: alloantisera and monoclonal antibodies
Authors: Middleton Derek , Boomer Julia , Heyes, Judith , Marsh Steven
Published: November 2023

In order to determine an individual’s HLA type two important ingredients are required. A suitable cell suspension, especially a B lymphocyte preparation when typing for class II antigens, and quality reagents containing specific antibodies against HLA antigens. The reagents are of two forms—alloantibodies produced by various sensitization procedures which are described in the first part of this chapter, and monoclonal antibodies which are described in the latter part of the chapter. Regardless of the source of alloantibodies the normal policy is to screen sera from small amounts of blood (<1 ml) for suitable antibodies and then to obtain large volumes of suitable sera for use as reagents. Many laboratories screen and procure their own reagents and then exchange these with other laboratories. Other laboratories rely on obtaining their reagents from National Tissue Typing Reference Laboratories or by purchase from commercial companies.
Paternity testing
Authors: Toit Ernette Du
Published: November 2023

The primary objective of paternity testing is to determine whether or not a man who is falsely accused of paternity can be excluded from being the biological father of a given child. Examples of disputed parentage and methods for resolving them can be traced back to biblical days. In the Old Testament in I Kings 3 verse 16-28 Solomon made the choice of maternity. Although in Solomon’s case maternity was at issue, more commonly the question is one of paternity. The role of modern science in problems of disputed parentage began with the discovery of the ABO blood groups by Karl Landsteiner in 1900. The recognition that the inheritance of the A, B, and O blood groups followed the genetic rules observed by Gregor Mendel became the cornerstone of all that followed. Until the 1950s the only blood group systems used in paternity studies were ABO, Rh, and MN. Together they provided an exclusion of parenthood in about 50% of disputed cases.
Mapping techniques used to isolate novel major histocompatibility complex genes
Authors: Campbell R Duncan , Trowsdale, John , Dunham Ian
Published: November 2023

The HLA class I and class II regions each encode highly polymorphic families of cell surface glycoproteins involved in immune regulation. The class I region contains at least 17, and possibly as many as 40, highly related genes which include those encoding the classical transplantation antigens (HLA-A, -B, and -C). The class II region (HLA-D) is arranged into subregions HLA-DP, -DQ, and -DR, each containing at least one A and B pair of genes encoding the a and polypeptide chains of the class II molecule. A number of other class II related sequences (including HLA-DO, -DN, and -DM) have also been defined within the HLA-D region. The class I and class II regions are separated by the class III region which also contains genes with immune related functions such as the complement genes C2, C4, and Bf, the tumour necrosis factor (TNF) A and B genes, and the major heat shock protein HSP70 genes. In order to characterize the HLA-A to -DP region in detail, the two techniques of pulsed-field gel electrophoresis (PFGE) and chromosome walking are especially useful.
Signal transduction
Authors: Owen P J , Evans C A , Whetton A D
Published: November 2023

Haemopoietic growth factors and cytokines act on a range of haemopoietic stem and progenitor cells to regulate their survival, proliferation, and differentiation. They also modulate the activities of mature cells, e.g. GM-CSF can prime neutrophils to display enhanced cytotoxic activity. These factors exert their effects by binding to specific cell surface receptors, eliciting various transmembrane signalling events (1). The molecular cloning of many of these growth factors and receptors has allowed their mode of action to be studied. Some of these receptors are members of the same receptor families since they share common structural features. For example the M-CSF receptor and the SCF receptor are both members of the type II receptor/tyrosine kinase family. Similarly IL-2, IL-3, GM-CSF, G-CSF, and other cytokines share some common structural motifs in the alpha chain of their receptors (and are therefore members of the so-called cytokine receptor family). In some cases these receptors share a common (beta) subunit, (e.g. the IL-3, IL-5, and GM-CSF receptors). The IL-6 receptor has some structural similarities to the cytokine receptor family but is apparently unique in that it binds to a second membrane bound subunit (gp130) which elicits the intracellular effects of this cytokine.
Autologous bone marrow transplantation using cultured marrow cells in patients with leukaemia
Authors: Coutinho L H , Chang J , Testa N G , Dexter T M
Published: November 2023

Lately, autologous bone marrow transplantation has been increasingly used as a therapeutic modality in acute and chronic myeloid leukaemias for patients without a suitable HLA matched donor (1, 2). The advantages of using the patient’s own marrow are a lack of graft-versus-host disease (that is commonly seen in patients receiving allogeneic bone marrow cells), the low procedural morbidity and mortality permitting application to older patients, and the possibility of administering highly ablative and potentially more effective doses of chemoradiotherapy to larger numbers of leukaemic patients.
Quantitative autoradiography: a tool to visualize and quantify receptors, enzymes, transporters, and second messenger systems
Authors: Sharif N A , Eglen R M
Published: November 2023

The ditribution and relative concentration of endogenous neurochemicals and their receptors/acceptors has traditionally been determined by immunohistochemical, radioimmunological, and ligand binding techniques. Whilst ligand binding procedures conducted on tissue homogenates have permitted determination of receptor affinities for neurotransmitters and drugs, and gross receptor distribution analysis, they lack the high level of anatomical resolution and sensitivity that can be obtained using autoradiographic technique .
Non-radioactive labelling and detection of nucleic acid probes
Authors: Kessler Christoph
Published: November 2023

Non-radioactive indicator systems are based on the detection of various biological target molecules (analytes) by selective interaction with specific binding partners (probes). Appropriate detector systems are coupled to these probes, either directly by covalent binding or indirectly by additional specific high-affinity interaction. Most of the recently developed non-radioactive systems are based on the enzymatic, photochemical, or chemical incorporation of a reporter group (1, 2) which can be detected with high sensitivity by optical, luminescence, fluorescence, or metal-precipitating detection systems (3-5). Electrochemical detection systems using pH electrodes or sensor technology have also been described (6, 7). In addition, attempts are being made to use specific labelling and detection systems not only for the detection of different target molecule specificities, but also for the detection of a large variety of different kinds of biomolecules such as proteins or glycans (universal detection systems). The various non-radioactive systems can be classified as direct or indirect. These assays differ in the number of components and thus the number of reaction steps used for the detection reaction. Whereas direct systems are mostly used for the detection of standardized target biomolecules, the more flexible indirect systems are often applied to detect different target bio molecules of variable specificity.
Probe preparation by the polymerase chain reaction
Authors: Taylor Graham R , Noble Jayne S
Published: November 2023

The polymerase chain reaction (PCR) can provide an alternative to the use of nucleic acid probes in many cases, for example the detection of restriction fragment length polymorphisms (RFLPs) (1), variable number of tandem repeats (VNTRs) (2), minisatellite polymorphisms (3), and deletions (4). However, there are still many situations that require the use of hybridization probes, including pulsed-field gel electrophoresis (PFGE), in situ hybridization, simple methylation studies using methylation-sensitive restriction endonucleases, and the detection of alleles larger than 2 kbp (for example in the expansions associated with fragile X and myotonic dystrophy, refs 5, 6). In such cases, PCR can be used to generate probes from a variety of tem plates, circumventing the need to perform large-scale cultures and purifications. This chapter reviews some of the PCR-based methods for producing probes and describes protocols for the production, purification, labelling, and use of probes made by PCR.
Screening human recombinant libraries with nucleic acid probes
Authors: Fisher Gabrielle M
Published: November 2023

A recombinant library is a collection of nucleic acid fragments representing all or part of a genome. Libraries can be divided into two major types, genomic and complementary DNA (cDNA), based on the methodology underlying their construction. Genomic libraries are constructed from the entire genome, and, therefore, contain clones representing all genomic sequences including control elements, structural genes, intron sequences, repetitive sequences, etc. In contrast, cDNA libraries are constructed from only the expressed regions of the genome, and, therefore, reflect the sequences present in the mRNA population of the cell of origin and their relative abundance. Recombinant genomic libraries are a particularly valuable tool in the study of large and complex genomes. Genome sizes can range from less than 5 megabases (Mb) in Escherichia coli, to over 23 000 Mb in pines and other plant species, easily exceeding the 3000 Mb in the human genome (Table I). Thus, the amount of DNA in the haploid genome of different organisms does not correlate with the evolutionary complexity of the organism.
Genomic mapping and gene structure analysis
Authors: Hall Len
Published: November 2023

Nucleic acid hybridization with specific gene probes is an important part of many aspects of genomic mapping and gene structure analysis. Using the highly sensitive technique of Southern blotting, gene-specific probes (in the form of cloned cDNAs) can be used to obtain useful preliminary information on the size and organization of the corresponding genomic sequences, the number of gene copies, etc., thereby facilitating subsequent genomic cloning and initial clone characterization. Once genomic clones are available, further characterization will depend to some extent on the size of the gene and the reasons for studying it. Smaller genes may be sequenced in their entirety without too much effort. However, this may not be appropriate for very large genes where only the exons and regulatory regions may need to be identified and fully sequenced (probably after extensive restriction mapping of genomic clones involving Southern blotting with probes from different parts of the corresponding cDNA). In both cases, important structural features, such as the transcriptional start site and the precise location of exon/intron boundaries, will almost certainly need to be established. These can be determined with a high degree of accuracy using techniques such as primer extension analysis (for mapping transcriptional start sites) and nuclease SI mapping (for determining both transcriptional start sites and exon/intron boundaries), both of which involve the use of sequence-specific probes.
Ligation-mediated PCR
Authors: Garrity Paul A , Wold Barbara , Mueller Paul R
Published: November 2023

Using PCR, a DNA fragment can be exponentially amplified with two flanking primers of known sequence. However, for many applications it is desirable to amplify a DNA fragment containing only one end of known sequence or, for genomic sequencing and in vivo footprinting, a family of fragments with one end in common. In ligation-mediated PCR (LMPCR), such substrates are made suitable for PCR by the ligation of a sequence of defined length and sequence content to the undefined end. PCR is then performed using one primer specific for the gene of interest and one primer specific for the ligated sequence (1). LMPCR differs from other single-sided exponential PCR methods in that the relative lengths of the starting DNA fragments are retained with base-pair resolution in the final product, making it suitable for use in genomic sequencing and in vivo footprinting (1, 2).
Amino acid sequence analysis of blotted proteins
Authors: Cook Richard G
Published: November 2023

In the past 10 years, our ability to obtain amino acid sequence information on proteins and peptides has improved dramatically. The amount of protein required for sequencing has been decreased by approximately three logs from 1 nmol to 1 pmol. The major reason for this increased sensitivity has been the development of automated gas and pulsed-liquid-phase sequencers with in-line HPLC detection systems. While the chemistry utilized in sequencing remains the classic Edman degradation (with slight modifications), the protein sequencer instrumentation has been vastly improved at several levels.
Molecular evolution of plants: more genomes, fewer generalities
Authors: Wolfe Kenneth H
Published: November 2023

Studies on the molecular evolution of plant genes have long lagged behind those in other taxa, such as mammals, for a variety of reasons. The result has been a tendency among students of molecular evolution to assume that what is true for mammals will also be true for plants. In this review I attempt to dispel this notion by focusing on the differences between the two taxa. It is not difficult to spot the differences.
Chloroplast transformation
Authors: Hutchison Ronald S , Roffey robin A , Sayre Richard T
Published: November 2023

The chloroplast genomes of most higher plants and eukaryotic algae have been highly conserved throughout evolution. Chloroplast genomes from different plants are not equivalent, however. The size of the genome MAY range between 400 kb, for some algae, to 73 kb for the non-photosynthetic plant, Epifagus virginiana (1, 2; Chapter 3). However, most plants have intermediate-sized chloroplast genomes, of approximately 160 kb, which are characterized by large inverted repeats with intervening large and small single-copy regions.
Epitope mapping using synthetic peptides
Authors: Worthington Jane , Morgan Keith
Published: November 2023

It is of vital importance to researchers in many fields to identify the specific sequences on proteins (epitopes) that are recognized by antibodies or by T-cells. Three basic strategies for epitope identification are currently used. One approach is to use predictive algorithms to identify possible epitopes, to synthesize representative peptides, and then test them (see Chapter 2). This approach is limited by the ability of such programs to identify candidate sequences and the amino acid sequence of the protein must be known. A second approach is to make fragments of proteins by enzymatic methods or by recombinant DNA techniques (see Chapter 8) and to use these to localize an epitope to a particular region of a protein. The sequence of the whole protein need not necessarily be known at this stage and the sequence of the region containing the epitope(s) can subsequently be identified. A third strategy (epitope scanning), which again requires knowledge of the protein sequence, is to synthesize a number of small peptides (usually overlapping in sequence), each representing a small stretch of the protein, which together represent the complete amino acid sequence of the protein (or that part of it which is of interest). These small peptides are then tested by the appropriate assay system and specific epitopes identified.
Combined differential light scattering with size exclusion chromatography techniques for monitoring conformational changes in x-carrageenan
Authors: Chambers S J , Langdon M J , Williams, P A , White R J
Published: November 2023

Light scattering has been used for many years to characterise water soluble natural polymers. Average molecular properties have been derived from time consuming static Zimm Plot techniques using laser goniometers, while molecular weight distributions have been obtained by combining Low Angle Laser Light Scattering (LALLS) with Size Exclusion Chromatography (SEC). However, problems associated with each has limited their widespread application. Traditional static light scattering is very slow, typically taking half a day to generate a single Zimm Plot.
Phillipine natural grade carrageenan
Authors: Tye R J
Published: November 2023

Commercially, carrageenan is produced by two technologies giving rise to “Traditionally Extracted Carrageenan” and “Natural Grade Carrageenan”. The traditional extraction processes (alcohol, gel press and roll dried) involve boiling the seaweed in alkali to dissolve the carrageenan. Depending on concentration, the solutions may be subjected to viscosity control so that the liquor can be pumped and filtered and then coagulated in alcohol or potassium chloride solution giving rise to two distinct precipitated products known as alcohol and gel press carrageenan. Natural Grade Carrageenan is produced by steeping freshly harvested weed in alkali at moderate temperature. The process does not involve dissolution of the carrageenan which is protected by the seaweed’s inert cellulose matrix. The traditional process is energy intensive and produces considerable quantities of pollutants which have to be dealt with at great expense. Natural Grade Products (or Alternatively Refined products as they are often called), do not carry this overhead and are thus much more cost effective. The only chemical difference between alcohol precipitated products and the Philippine materials is the presence of inert cellulose. The active polysaccharides cannot be distinguished by any known chemical or spectroscopic techniques. Gel press materials are readily identifiable by their high potassium chloride content.
Processing optimization and physico-chemical characteristics of flaxseed polysaccharide gums
Authors: Cup W , Mazza G , Biliaderis C G
Published: November 2023

Response surface methodology was used to optimize the aqueous extraction of polysaccharides from flaxseed. Temperature, pH and seed/water ratio were the major parameters affecting yield, chemical composition and rheological characteristics of the crude gum extract. Optimum conditions for extraction were a temperature of 85-90°C and a neutral pH (6.5-7). Seed/water ratio had a smaller effect on yield and properties of flaxseed gum than temperature and pH. Flaxseed gum extracted under optimum conditions gave relatively high yield (7.9%) and contained over 80% carbohydrates. Using ion exchange chromatography, on a DEAEcellulose column, the crude flaxseed gum was dialyzed and further separated into a neutral and an acidic fraction. Protein content, minerals, monosaccharide composition and uronic acid levels of flaxseed gum fractions were determined. Further structural information and molecular size distributions of the gum and its fractions were obtained by methylation analysis, 13C NMR and gel filtration chromatography. Flaxseed polysaccarides exhibited shear thinning and weak gel properties at concentrations above 1% (w/w). The neutral flaxseed gum fraction (NFG) was of higher molecular size and showed similar shear thinning behavior as the crude and dialyzed gums. The acidic fraction, on the other hand, consisted of low molecular size polymers and exhibited Newtonian-like flow behavior.
Computer optimisation of parameters for the preparation of low viscosity guar gum by controlled thermal and acid hydrolysis
Authors: Mathur V , Kanoongo N , Mathur N K
Published: November 2023

Galactomannan polysaccharides when treated with acid catalysts at elevated temperatures, are depolymerised to products of lower viscosities and lower molecular weights. The extent of depolymerisation has to be controlled to obtain a product of predetermined characterstics. Various factors which control the extent of depolymerisation include, besides the reaction time, the temperature of reaction and the concentration of the catalyst, certain other factors e.g. the design of the reactor, rate of mixing/stirring and the gum itself (powder or split). For a given set of experimental conditions, computer optimisation can be done to find suitable sets of conditions for actual working.
Nutritional aspects of difficult-to-digest carbohydrates
Authors: Livesey Geoffrey
Published: November 2023

The chemical composition, structure and intakes of diets tl maintain optimal physiological function, and health, is st ill-defined. Reference requirements of the UK population over 40 nutrients have been suggested and nutritional guides: can be based on such values (1)he attributes of an opted diet for one individual may, however, differ from that another. Individuals who are predisposed by genetics lifestyle to disease may need to be more concerned about th diet than non-predisposed individuals. In such individuals may well be that therapeutic levels of specific nutrients well above those needed by the general population. Most evide relating diet to health is concerned with general populations individuals in disease, whereas it is likely that information.
Influence of galactomannan addition on the viscoelastic properties of pectin-calcium systems
Authors: Silva J A , Lopes Da , Gon<Alves M P , Doublier J L , Axelos M A V
Published: November 2023

The influence of galactomannan addition on the formation of low methoxyl pectin/calcium networks, with different density of crosslinks, was studied through the changes in the mechanical spectrum obtained from dynamic shear measurements. The mixed gels showed an increase in the storage and loss moduli with the increase of galactomannan concentration, especially at high frequency. The pectin/calcium network remains the continuous gel matrix controlling the viscoelastic behaviour of these systems at low frequency. The effect of galactomannan addition on the viscoelastic behaviour of the mixed gels at “equilibrium” depends on the structure of the pectin/calcium network. We suggest that there is no specific interaction and that the changes in the mechanical properties of the pectin gels are due to the entrapment of the galactornannan macromolecules in the pectin-calcium network. One interesting aspect is the decrease of the tendency for the pectin/calcium gels to undergo syneresis, by the addition of the galactomannan.
Technological impact on foam stability: analysis of quality assurance data with multivariate analysis
Authors: Lebouille J L M , Moonen H A J
Published: November 2023

The use of multivariate data analysis techniques, applied to quality assurance data of fermentation and lagering, to elaborate technological aspects of foam stability is described. In multivariate data analysis, co-linearities between variables are extracted and suppressed. During the fermentation the addition of more than one brew in a tank contributes negatively to the foam stability. In the lagering process comparable negative aspects were found.
Patch-clamp recording
Authors: Gibb Alasdair J
Published: November 2023

Patch-clamp recording is now widely used in the biological sciences. The technique was originally developed with the very specific intention of observing ionic currents flowing through single ion channels in cell membranes (1). From this origin the technique has developed into one of the most versatile and precise methods of measurement in biology. For example, no other technique allows the observation of the functioning of a single protein molecule in a lipid bilayer, yet can also allow monitoring of synaptic transmission between neurones in the brain, or permit monitoring of the change in cell membrane area during secretion. Underlying all the applications of patch clamping is the single principle that it provides an extremely sensitive means to measure ionic currents flowing through cell membranes. For their discovery and development of this technique and its application to a range of critical questions in biology, Neher and Sakmann were awarded the 1991 Nobel prize in Physiology or Medicine (1, 2, 3). Two excellent sources of detailed information on the patch-clamp technique are available: the ‘blue book’ guide to patch-clamp recording (4, a new edition of this book is due in 1995) and the recent ‘Ion channels’ volume of Methods in enzymology (5). In recent years various aspects of the patch-clamp technique have been reviewed in several different articles and books (6, 7, 8, 9). This chapter is intended to provide an overview, concentrating on the more practical aspects of the patch-clamp technique.
Avian ciliary and Ium bar sympathetic ganglion neurons
Authors: Nishi Rae
Published: November 2023

Chick parasympathetic and sympathetic ganglia provide a convenient source of readily isolated and cultured neurons that have a variety of uses: bioassays for trophic factors by quantifying survival of neurons in response to defined or undefined activities bioassays for neurite outgrowth promoting activities bioassays for neurotransmitter plasticity In particular, the chick ciliary ganglion is a relatively homogeneous source of cholinergic parasympathetic neurons that innervates striated muscle very readily, hence it has been invaluable for studies of molecular changes associated with neuromuscular synapse formation.
Mouse olfactory epithelium
Authors: Calof Anne L , Guevara Jose L , Gordon Melinda K
Published: November 2023

The olfactory epithelium is unique in mammals in that proliferation of neuronal precursor cells and differentiation of their progeny into olfactory receptor neurons (ORNs) continue throughout the lifetime of the organism (1,2). Many of the studies in our laboratory are concerned with understand ing how specific polypeptide growth factors and cell interactions regulate olfactory receptor neuron generation and survival (3-6). For these studies it is crucial to obtain and culture explants of olfactory epithelium that are free from stromal cells, since the stromal cells may provide signals that affect the development of olfactory receptor neurons. Thus, an entire section of this chapter is devoted to purification of the epithelium from its underlying stroma. For studies of growth factor effects on isolated ORNs, and for studies on olfactory receptor neuron adhesion, migration, and axon outgrowth, we prepare a neuronal cell fraction from embryonic mouse olfactory epithelium, as well as using explant cultures. This neuronal cell fraction consists solely of olfactory receptor neurons and their precursors, which we have called the immediate neuronal precursors (INPs) of olfactory receptor neurons, and does not contain basal cells or supporting (sustentacular) cells of the epithelium (6–8). Using this cell fraction allows us to work solely with neuronal cells in dissociated cell culture, which is a great advantage for quantitative studies.
RNA binding assays for the regulatory proteins Tat and Rev
Authors: Karn J , Churcher M J , Rittner K , KELLEY A , Kelley P J G , Mann D A , Gait M J
Published: November 2023

The replication of the human immunodeficiency virus (HIV) is controlled by two viral proteins, Tat, the trans-activator protein and Rev, the regulator of virion expression. The two proteins play complementary roles in the HIV life cycle. In permissive cells, transcription of the integrated proviral genome is initiated by host cell transcription factors (1-3). Initially, only small, multiply spliced mRNAs including the mRNAs for Tat and Rev are produced (4). Tat synthesized during this primary round of transcription then establishes a ‘positive feedback loop’ which boosts mRNA production 200- to 1000-fold (5- 7). Once a critical Rev threshold is surpassed, HIV gene expression switches away from the production of the mRNAs for the regulatory proteins towards the production of the unspliced or partially spliced mRNAs encoding the virion proteins (8-10).
Vif
Authors: Trono Didier
Published: November 2023

The HIV-1 vif open reading frame (previously called ‘sor’ or ‘A’) encodes a protein of 192 amino acids, with an approximate molecular weight of 23 kDa. Vif is highly conserved among different HIV-1 isolates. Vif is also present in HIV-2 and simian immunodeficiency virus, and in non-primate lentiviruses, such as feline and bovine immunodeficiency viruses, caprine arthritis encephalitis virus, and visna virus, a cytopathic lentivirus of sheep only distantly related to HIV. HIV-1 Vif is translated from a singly spliced viral mRNA, together with other late viral proteins whose expression is induced by Rev. Initial studies showed that HIV-1 strains defective in vif generated virions that infected cells approximately a 1000 times less efficiently than wild-type viruses, hence the acronym Vif, for virion infectivity factor.
Inhibition of HIV by ribozymes and antisense oligonucleotides
Authors: Castanotto D , Bertrand E , Rossi J
Published: November 2023

Introduction

Since the early 1980s, when the immunodeficiency virus (HIV) was shown to be the etiological agent of AIDS, a considerable amount of information on its life cycle has been collected. Although this growing body of knowledge has allowed investigators to evolve rational drug designs, the complexity of HIV has so far made it impossible to develop a cure or a vaccine. The variety of tissues and cells which HIV is able to infect, along with the high mutation rate of the viral genome and the secondary diseases associated with AIDS, further restrict the possibilities for achieving any effective therapy. Moreover, agents that block viral function often have adverse effects on the host, especially after prolonged use. A relatively new approach in the treatment of viral infections is the use of antisense and/or ribozymes in the attempt to bind and block expression of viral transcripts. To date, they have been reported to be effective in cell cultures against viral targets, oncogenes, and transfected genes. RNA enzymes or ribozymes are receiving considerable attention because, unlike antisense, ribozymes are able to turn over multiple substrates and inactivate them irreversibly. The ribozyme technology is based on the design of RNA molecules in which the antisense and enzymatic functions are combined into a single transcript, which can specifically pair and cleave any target RNA. Because of their high specificity and the possibility of being endogenously synthesized within a cell, it is likely that ribozymes will be major players in the development of new drugs during the next several years.
Gene therapy
Authors: Smith C , Marasco W A , Bohnlein E
Published: November 2023

Somatic gene therapy was independently proposed in 1988 by D. Baltimore and J. C. Sanford as an alternative treatment of AIDS (1, 2). This new approach is based on the stable introduction of genetic material into cells of HIV-I-infected patients. The therapeutic genes can be delivered directly into the target cells of HIV in the peripheral blood (CD4-positive lymphocytes, macrophages) using techniques analogous to those used for the first human gene therapy trial for the treatment of AD A deficiency. Differentiated peripheral blood cells have a limited life expectancy and repeated treatment would be required. Alternatively, the antiviral genes can be introduced into the haematopoietic stem cell from which all HIV-1 target cells are derived. In this case, a single treatment could suffice to obtain the therapeutic effect.
Labelling methods for analysis of class II MHC endocytosis and peptide turnover
Authors: A.Reid Pamela , Watts Colin
Published: November 2023

MHC glycoproteins are peptide binding and transport proteins which give a continual update, in peptide form, of the cellular and environmental protein composition on class I or class II MHC molecules respectively (see ref. 1 for a review). Cells expressing these molecules can therefore be screened by T lymphocytes for the presence of foreign peptides. A response will only be elicited if the level of a particular peptide/MHC complex is above the threshold needed for activation of the responding T cells. Whether this threshold is reached will depend on the relative rates of production and loss of peptide/MHC complexes from the cell surface. Production rates will be governed by variables such as the rate of MHC biosynthesis, level of antigen expression or capture and efficiency of antigen processing etc. (2). Rates of loss will depend on (i) the absolute lifetime on the cell surface of MHC molecules and (ii) the lifetime of particular peptide species on MHC molecules. Clearly, if the lifetime of peptides on MHC molecules is much longer than the absolute lifetime, loss rates will simply reflect how long MHC molecules reside on the cell surface. On the other hand, if peptide lifetime is much shorter than absolute MHC lifetime on the cell surface then the rate of loss of complexes will be largely independent of absolute lifetime. This latter scenario raises the possibility that the ‘empty’ MHC molecules generated might be re-used to present additional peptides during their tenure on the cell surface. Since peptide production for MHC binding is not thought to occur on the cell surface, an endocytic cycle of some kind would seem to be necessary to permit re-utilization.
Peptide translocation into the ER
Authors: Neefjes Jacques , Momburg Frank , Ha.Mmerling Gunter , Roelse Joost
Published: November 2023

A crucial step in antigen presentation by MHC class I molecules is the translocation of peptide fragments of protein antigens from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, peptides are bound with high affinity by freshly assembled MHC class I H-chain/l32m heterodimers and are then intracellularly transported to the cell surface and presented by class I molecules to cog+ T cells (1, 2). An ER-located peptide pump termed TAP (Transporter associated with Antigen Presentation) has been genetically defined and demonstrated to translocate peptides from the cytosol to the lumen of the ER (3, 4). Essential to the characterization of TAP were mutant cell lines with inactivated genes for one or both of the two subunits of TAP. In the absence of TAP activity, MHC class I molecules are not loaded with peptides. This results in retention of the class I heterodimer in the ER and decreased cell surface expression of class I molecules.
The interaction of superantigens with MHC class II molecules
Authors: Horgan Carol , Fraser John D
Published: November 2023

Superantigens are specific viral or bacterial products that simultaneously bind to class II MHC molecules and T cell receptors (TCR) without requiring proteolytic processing. They can activate up to 20% of peripheral T lymphocytes, as compared to conventional antigens which activate only about 0.001% of peripheral T cells. The binding occurs at sites on the MHC outside of the peptide binding groove and on the chain of the TCR, away from the conventional antigen binding site.
Methods for the generation of T cell clones and epithelial cell lines from excised human biopsies or needle aspirates
Authors: Libero G De
Published: November 2023

The effector mechanisms and the cellular interactions that regulate the immune response can be studied by taking advantage of defined in vitro systems. These studies can be performed using cloned reagents, such as T cell clones and monoclonal antibodies (mAbs). Studies performed with T cell clones have allowed the identification of surface molecules involved in cellular interactions (1-7), of lymphokines responsible for immunoregulation (8), and of different functional properties of T cells (9, 10). T cell clones have also facilitated the analysis of the molecular requirements for antigen presentation (11-13), antigen recognition (14), and T cell activation (15), and are a unique source of nucleic acids for the characterization of the genes that are expressed only in rare lymphocytes (16, 17).
Techniques to identify the rules governing class II MHC-peptide interaction
Authors: Hammer J , Sinigalia F
Published: November 2023

Major histocompatibility complex class II molecules (MHC) are highly polymorphic membrane glycoproteins that bind peptide fragments of proteins and display them for recognition by CD4+ T cells. The binding capacity of any given peptide to an MHC class II molecule depends on the primary sequence of the peptide and on allelic variations of residues in the binding site of the MHC receptor. Advances in our knowledge of the structure of MHC molecules, and a large number of peptide and MHC class II structure-function studies have provided an increasingly detailed picture of how allelic variations influence peptide binding and presentation to CD4+ T cells (1).
Introduction
Authors: Mason Ronald P
Published: November 2023

A free radical is defined as any atom or molecule that possesses an unpaired electron. It can be anionic, cationic, or neutral. In biological and related fields, the major free radical species of interest have been those of oxygen, that is oxygen free radicals (OFRs).
Detection of free radical reaction products by high-field nuclear magnetic resonance spectroscopy
Authors: Naughton D P , Lynch E , Hawkes G E , Hawkes J , Blake D R , Grootveld M
Published: November 2023

Nuclear magnetic resonance (NMR) spectroscopy uses the absorption of energy from the radiofrequency region of the electromagnetic spectrum to detect changes in the alignment of nuclear magnets during exposure to a powerful external magnetic field. The absorption frequency is dependent on the magnetic (and, therefore, chemical) environment of nuclei. Furthermore, the appearance (multiplicity) of a resonance in the 1H (proton) NMR spectrum of a particular chemical component is influenced by neighbouring hydrogen nuclei in a well-characterized way. Hence, much useful information about the molecules present in a biological sample can be obtained from NMR spectroscopic techniques.
Pulse radiolysis
Authors: Butler J , Land E J
Published: November 2023

The technique of pulse radiolysis was introduced in 1960 simultaneously by Keene (1) (in these laboratories), Boag and Steel (2), Matheson and Dorfman (3), and McCarthy and McLaughlan (4), ten years after the invention of its light equivalent, flash photolysis, by Porter (5).
Visual assessment of oxidative stress by multifunctional digital microfluorography
Authors: Suematsu Makoto , Schmid-Schönbein Geert W , Ishimura Yuzuru , Tsuchiya Masaharu
Published: November 2023

There has been an increase in the understanding of the potential role of oxidants, such as the superoxide anion (OD, hydrogen peroxide (H202), hydroxyl radicals COH), as mediators of cell injury in a wide variety of physiological and pathophysiological conditions. These active oxygen metabolites can interact with cellular membrane structures thereby inducing lipid peroxidation, which has been implicated as one of the major mechanisms of ‘oxidative stress’ in tissue injury. By contrast, the nitric oxide radical (NO”), which has been identified as an endothelium-derived relaxing factor, plays an indispensable role in the maintenance of blood perfusion and antithrombotic properties of vascular endothelium, and contributes as a natural antioxidant to scavenging superoxide anion. Under some pathological conditions, the interaction between NO” and 02 may yield peroxynitrite (ONOO-) and thereby promote cell injury.
Protein hydroperoxides, protein hydroxides, and protein-bound DOPA
Authors: Dean Roger T , Fu Shanlin , Gieseg Steven , Armstrong Sharyn G
Published: November 2023

Other chapters in this volume describe the determination of stable, relatively unreactive products of protein oxidation, such as protein carbonyls. How ever, we have recently shown that radical attack on proteins can generate two kinds of reactive moieties that are fairly long-lived under appropriate conditions: protein hydroperoxides (oxidizing species) and protein-bound DOPA (3,4-dihydroxyphenylalanine) (a reducing species (1, 2); reviewed in ref. 3).
Selective and sensitive measurement of vitamin C, ubiquinol-10, and other low-molecular-weight antioxidants
Authors: Mohr Detlef , Stocker Roland
Published: November 2023

Inadvertent modification of biological macromolecules by free radical-mediated oxidation is implicated in the pathogenesis of a number of human diseases, such as cancer and atherosclerosis. The latter in particular has received much attention recently. Since oxidation of lipids carried in the circulation by lipoproteins can precede that of the apoprotein, considerable interest has focused on the oxidation of lipoprotein lipids and its inhibition by endogenous antioxidants. In particular, the antioxidative action of ascorbate, a-tocopherol, ubiquinol-10, carotenoids, and bile pigments have been investigated in more or less detail. The selective and sensitive detection of endogeneous antioxidants in human plasma and lipoproteins has, therefore, become increasingly important. Although numerous, simple and mostly colorimetric assays are often employed for the measurement of many of the antioxidants, their suitability for the selective detection of antioxidants in complex biological mixtures such as human plasma, is limited. Also, many of the extremely labile (i.e. autoxidizable) antioxidants are present in small concentrations which renders colorimetric assays particularly problematic. Lengthy derivatization procedures required to increase sensitivity can lead to artificial underestimation or even complete loss of the compounds of interest. In this situation, high performance liquid chromatographic (HPLC) methods are useful alternatives and can guarantee a high degree of selectivity and sensitivity. In particular, with the availability of simpler and more reliable electrochemical cells, HPLC with electrochemical detection (HPLC-EC) has become a frequently used detection method. This detection method provides high sensitivity and additional selectivity, due to the different and characteristic redox activity of different antioxidants.
Targeting and import of peroxisomal proteins
Authors: pure Christopher j. Dan
Published: November 2023

Progress in our understanding of the mechanisms by which proteins are targeted to and imported into peroxisomes has been slow compared with the rate at which the processes involved in mitochondrial and endoplasmic reticulum (ER) protein translocation have been elucidated (see Chapters 1 and 4, respectively). This has been due partly to the greater technical difficulty of studying peroxisomes (especially in vitro) and partly to a general ignorance of the importance of peroxisomes to cellular and organismal viability. However, in recent years there has been a considerable increase in interest in, and a parallel increase in understanding of, the way peroxisomes and related organelles (i.e. glyoxysomes and glycosomes) are formed. This has been spurred on largely by the desire to explain the molecular basis of the human genetic disorders of peroxisome biogenesis, such as Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease CIRO), and rhizomelic chondrodysplasia punctata (RCDP) (Table 1) (1), and the mechanism by which certain hypolipidaemic drugs and xenobiotics cause phenomenal expansion of the peroxisomal compartmental volume (2-5). This increased interest is well reflected in the numerous reviews written in recent years on various aspects of peroxisomal, glyoxysomal, and glycosomal biogenesis in general and protein targeting in particular (6-27).
Enzyme-labelled tests with colorimetric, fluorimetric and chemiluminescent detection systerns
Authors: Howes Iain , Blincko Stuart , Little John , Edwards Raymond
Published: November 2023

Enzymes are widely used as labels in immunoassay procedures. They demonstrate great versatility and have been applied to all types of immunoassay. Originally employed in immunostaining and gel-precipitin techniques, enzymes subsequently demonstrated particular utility when used with solid-phase separation systems such as antibody coated plastics (e.g. ELISA) and reagent linked particles (e.g. magnetic cellulose). Enzymes are also used in non separation assays in which enzyme activity is altered by antibody binding. See Chapter 1 for details of nomenclature and classification.
Light scattering techniques
Authors: Newman David j. , Thakkar Hansa , Price christopher P
Published: November 2023

Quantitative immunoaggregation assays are very widely used due to their inherent simplicity and, in the liquid phase anyway, their ready automation, excellent precision and very fast reaction times. Gel-based immunoaggre gation assays are certainly simple and robust but this chapter will focus on the various liquid phase techniques. Amongst the various homogeneous immunoassay technologies available, immunoaggregation assays are one of the few that are available to research scientists who wish to develop their own assays, as they can be carried out not only in sophisticated automated instruments but also in virtually any good quality spectrophotometer. They can be used for both haptens and proteins in most biological matrices and for analytes across the pM to mM concentration range; furthermore, they can use monoclonal and polyclonal antibodies both intact and fragment. Thus, using the appropriate detection system, immunoaggregation assays can compete very effectively, in terms of detection limits and convenience, with many enzyme and radioisotopically labelled immunoassays, and perform significantly better in terms of precision and ease of automation. The commonest techniques are immunoturbidimetry and immunonephelometry with and without the use of particle enhancement. These involve different aspects of light scattering; however, as turbidity can be measured on any spectrophotometer and nephelometry requires specialized equipment that may not be available in every laboratory, the main focus will be on the use of turbidimetric light scattering.
Tumour suppressor genes and inheritance of cancer
Authors: Black Donald M
Published: November 2023

Several genes have been identified which confer an inherited predisposition to cancer. Although the susceptibility is usually inherited as a dominant trait, at the cellular level the susceptibility mutations are almost always recessive. Therefore, for the cancer to develop, the dominant wild-type copy of the gene must be mutated or deleted. Such genes are called tumour suppressors. They encode proteins of diverse function which need to be inactivated for the initiation and progression of malignancy. The existence of families with susceptibility to cancer are important in two ways. First, they give us additional insights into the molecular events which underlie the transformation from normal to tumour cells; and secondly, they allow the possibility of genetic testing for cancer risk.
Trypanosoinatid genetics
Authors: Swindle John , Tait Andrew
Published: November 2023

The genetics of the trypanosomatid group of protozoa have been the subject of considerable research over the last 15 years. The trypanosomatids include a number of highly pathogenic parasitic species which infect a very wide range of hosts including man and domestic livestock. In terms of molecular genetics, the most intensively studied are Leishmania and trypanosomes. This chapter will primarily focus on Trypanosoma brucei, Trypanosoma cruzi, and a few Leishmania species. Two fundamental aspects of the genetics of these parasites will be considered: first, a basic description of their genomes and genome organization together with a review of our current state of knowledge on classical genetics; and second, a review of modern transfection-based molecular genetics. The lack of readily available classical genetic systems, which have been used so effectively in yeast, Drosophila, and Caenorhabditis elegans, has been a major restraint, although the availability of reverse genetics has circumvented some of these limitations.