bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactive dosing of filter aid in response to particle assessment
Authors: Hammond R V , Freeman G J , Reed R J R , Smedley S M
Published: November 2023

Variation in solids loading during filtration presents difficulties for the brewer. Our aim is improved efficiency of filter-aid utilization following laser-based measurement of suspended solids in rough beer. Reliable measurement of differently-sized particles has been achieved for unfiltered beer, containing yeast (0-2500 mg/1) and trub (0-300 mg/1). In-line optical measurements are continuously applied to a mathematical model within the process control system. This adjusts feed rates of the filter-aids most suited to filtration of the particles present for optimum solids loading. Extended filter runs and reductions in filter-aid usage and disposal are predicted.
Relation between wort DMSO and DMS concentration in beer
Authors: Leemans Christine , Dupire Stéphane , Macron Jean-Yves
Published: November 2023

By the addition of deuterated DMS - which can be quantified by GC-MS - at the beginning of fermentation on a pilot scale, the evaporation ratio has been determined. As a result of this, the origin of DMS in beer could be precised. It becomes evident that the DMS produced during the fermentation is in the majority of the cases more important than the DMS originating from the degradation of SMM during brewing processes. By the addition of deuterated DMSO in wort at pitching, a direct relation between malt DMSO concentration and beer DMS quantity has been demonstrated.
Cellular and cell-free assays for Tat
Authors: Laspia M F
Published: November 2023

The human immunodeficiency virus 1 (HIV-I) encodes a novel regulatory protein known as Tat. Tat greatly increases the expression of genes linked to the long terminal repeat (LTR) of the virus. It does so by binding to a structured RNA element, called TAR RNA (see Chapter 9), located in the 5’ untranslated region of all HIV mRNAs (1-3). The mechanism of tran activation by Tat appears complex, but the principal level at which Tat acts is transcriptional. Based on results obtained in a model system consisting of COS cells transiently expressing replicating plasmids, Peterlin and colleagues (4) were the first to obtain evidence that Tat stimulates transcriptional elongation. By contrast, Tat was found to increase both transcriptional initiation and the efficiency of transcriptional elongation in a recombinant adenovirus HeLa cell model system (5, 6). In this system, in the absence of Tat the level of HIV-I transcription is low and the density of transcribing RNA polymerases declines as a function of the distance downstream of the promoter. Tat increases the number of RNA polymerases in the immediate vicinity of the promoter and also suppresses transcriptional polarity in downstream sequences leading to a large increase in transcription rates overall (Figure 1). A general trans-activator protein, the adenovirus ElA 13S protein, elevates HIV-I promoter proximal transcription, but does not significantly stimulate elongation. Thus, Tat appears to stimulate both transcriptional initiation and elongation. Recently, a cell-free transcription system has been developed (7) that faithfully reproduces in vitro important aspects of trans-activation by Tat observed in vivo (8).
Analysis of HIV-1 LTR DNA binding proteins
Authors: Ou S -H I , Wu F , Gaynor R
Published: November 2023

The HIV-1 long terminal repeat (LTR) acts as a promoter and contains numerous regulatory elements that are critical for activation of gene expression in both the presence and absence of the trans-activator protein, Tat (1). Mutagenesis of the HIV-1 LTR has been performed and these mutants were used to direct the expression of a variety of reporter genes such as chloramphenicol acetyltransferase or luciferase. These constructs have been used to define critical regulatory domains which modulate HIV-1 gene expression either in the presence or absence of Tat. Some of these regulatory elements are involved in the control of gene expression in a variety of different cells while others appear to be tissue-specific. In addition to DNA regulatory elements, HIV-1 contains an additional element known as TAR (trans-activating region), which is capable of forming a double-strand RNA structure (see Chapter 9) (2, 3). This RNA element, which is critical for Tat activation, serves as the binding site for both Tat and cellular proteins (2, 3). This review will be on cellular factors that bind to DNA regulatory elements in the HIV-1 LTR.
Use of fluorescent indicators to measure intracellular Ca2+ and other ions
Authors: Sage Stewart O
Published: November 2023

Changes in cytosolic calcium ion concentration ([Ca2+]i) play a major role in platelet activation. Elevation of [Ca2+]i alone can stimulate shape change, aggregation, and secretion, although physiologically other second messengers such as diacylglycerol may contribute to these events. As with other nonexcitable cells, platelet agonists stimulate a rise in [Ca2+L by promoting the entry of Ca2+ into the cytosol from two sources. Ca2+ is released from intracellular stores in the dense tubular system, a structure analogous to the endoplasmic reticulum of other cells, and Ca2+ enters the platelet across the plasma membrane.
Preparation of highly purified human platelet plasma and intracellular membranes using high voltage free flow electrophoresis and methods to study Ca2+ regulation
Authors: Authi Kalwant S
Published: November 2023

The ability of experimentalists to harvest and study purified fractions of plasma and intracellular membranes and organelles has contributed tremendously to our knowledge of platelet structure and to our understanding of the mechanisms associated with platelet activation and its control. The platelet is a delicately poised cell capable of rapid activation upon contact with a stimulus. The ease with which a relatively homogeneous population of platelets can be obtained, its importance in the cardiovascular system, and the surprising magnitude of signalling mechanisms that are present has made this cell one of the most thoroughly studied within the body. Its size (being one of the smallest of mammalian cells) and rapid excitability has prompted the development of new strategies in both its handling and disruption. Indeed, these techniques have allowed a study of platelet structure and membrane organization at a level of sophistication that in some respects has not been possible for other cell types.
Study of proteins associated with the platelet cytoskeleton
Authors: Fox Joan E B
Published: November 2023

The platelet cytoskeleton is made up of a network of cross-linked actin filaments that exists throughout the cytoplasm, a microtubule coil that is wound tightly on itself just beneath the membrane, and a membrane skeleton that coats the inner surface of the phospholipid bilayer. There appear to be interconnections between each of these components of the cytoskeleton. Further, the membrane skeleton associates with the cytoplasmic domains of transmembrane receptors. In this way the network of cytoplasmic filaments functions as a scaffold that is linked to the plasma membrane and serves to regulate the contours of the membrane and thus the shape of the cell.
Methods for detecting signalling via MHC class II molecules which can lead to either activation or programmed cell death
Authors: Mooney N , Truman J P , Charron D
Published: November 2023

The transmission of signals by MHC class II molecules has been extensively examined. A number of studies have demonstrated either an augmentation or a decrease in cell proliferation (assessed by 3H-thymidine incorporation) after treatment of B lymphocytes with anti-MHC class II monoclonal anti bodies (mAbs). Initial studies in the mouse described the activation and nuclear translocation of protein kinase C (PKC) and the production of cyclic adenosine monophosphate (cAMP) (1) after stimulation of B lymphocytes with anti-MHC antibodies. Later studies in humans revealed the activation of phospholipase C (PLC), increased intracellular calcium [Ca++L and production of diacylglycerol (DAG) (2, 3, 4). Interestingly, the involvement of PKC in signalling via HLA class II antigens did not necessarily involve translocation, since increased membrane and cytosol associated PKC activity was detected as well as increased levels of PKC a and PKC mRNA (5, 6). A later study revealed that PKC a, and 6 are all increased at the mRNA level following signalling via HLA class II (7).
Analysis of MHC class II-specific T cell clones
Authors: Pawelec Graham , Obata Fumiya , Sansom David , Friccius Hilke , Daikeler Thomas , Adibzadeh Medi , Schaudt Kurt , Pohla Heike
Published: November 2023

The application of T cell cloning in immunogenetics at the end of the 1970s (1) enhanced an early awareness of the complexity of functionally relevant lymphocyte stimulating determinants, mostly associated with MHC class II, and cytotoxic lymphocyte targets, mostly associated with MHC class I, and proved instrumental in establishing the mechanism of T cell recognition of antigen and alloantigen. Techniques for the generation, propagation and utilization of monoclonal strains of lymphocytes-T have become established as vital tools in diverse areas of immunology, but, with the exception of some early necessity-driven studies on the optimization of culture conditions (e.g. see ref. 2), there are relatively few published data on the practical details of establishing and maintaining human T cell clones (TCC). In fact, standard techniques work well enough, if not perfectly, that they have changed little since they were first established (1, 3). However, there seems to remain a widespread assumption, at least amongst those cellular and molecular immunologists with less personal experience in the area, that once T cell clones have been established, and provided that culture conditions are favourable, cultures can be maintained as permanent cell lines. While this may be true of a relatively small number of T cell clones, probably most experienced cloners will agree that, at least in humans, this is not the case for the majority of normal T cell lines (which should therefore be referred to as strains, not lines, according to tissue culturists’ standard nomenclature). Thus, the majority of T cells maintained in culture are found to have finite lifespans, and those examples of immortal lines in the literature may represent rare, not completely normal, variants.
The molecular basis of alloreactivity
Authors: Lightstone Liz , Deacock Sarah , Heaton Tricia , Lechler Robert
Published: November 2023

Allogeneic major histocompatibility (MHC) molecules are capable of stimulating uniquely strong primary immune responses. This is illustrated in vitro in the mixed lymphocyte reaction, and in vivo leads to organ graft rejection, and graft versus host disease in recipients of allogeneic bone marrow transplants. The strength of the allo-response is due to the very high frequency of T cells in the ‘normal’ T cell repertoire which are able to react with MHC alloantigens. Whilst this phenomenon was observed many years ago (1) it is only relatively recently that the tools have become available to characterize the molecular basis of allo-recognition. In this chapter, the methods used by ourselves and others to elucidate the structure-function relationships between allo-reactive T cells and their ligands are described.
Elution and analysis of peptide pools from MHC class I molecules
Authors: Powis Simon J , Butcher Geoffrey W
Published: November 2023

Class I major histocompatibility complex (MHC) molecules are widely distributed polymorphic cell surface glycoproteins involved in the presentation of antigens to CD8+ T-lymphocytes. They function by binding short peptides in the environment of the endoplasmic reticulum (ER): these peptide-class I complexes are then transported to the cell surface where intercellular interaction with T-cell receptors can occur. It is a requirement for recognition by T-lymphocytes that protein antigens are degraded into small fragments prior to MHC binding. The ability to predict what fragment of any given protein antigen would bind to a class I MHC molecule and become a T-cell epitope would therefore be of great use in the development of novel vaccine strategies. In recent years several important features of the class I MHC antigen presentation pathway have been elucidated which make identifying putative epitopes a viable experimental goal.
Sequencing of proteins isolated by one-or two-dimensional gel electrophoresis
Authors: Ferrara Pascual
Published: November 2023

One-dimensional (1D), and especially two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), are generally considered as the highest resolving techniques for the analysis of complex protein mixtures. Both techniques have been extensively used for the study of cell protein changes in biological processes such as proliferation and differentiation (1). In 2D PAGE the power of electrophoresis analysis is enhanced when individual spots on the gel are identified. However, the identification of the spots is not a trivial task; several direct and indirect methods have been developed (2, 3). A straightforward approach is the production of sequence information.
Expression, purification, and characterization of recombinant soluble MHC class I molecules
Authors: Brunmark Anders , Jackson Michael R
Published: November 2023

Functional MHC class I molecules are made up of three components, transmembrane MHC class I heavy chain, soluble rmicroglobulin, and a short peptide which is typically eight to ten amino acids long. This trimolecular complex is assembled within the endoplasmic reticulum and transported to the cell surface to which it is anchored via the transmembrane domain. Initial attempts to solve the structure of this complex utilized proteolysis to release a soluble class I fragment from the surface of human cells. Using this material the structure of the extracelluar domain of an MHC class I molecule containing a heterogeneous mixture of peptides was solved in 1987 (1).
Investigating the effects of oxygen free radicals on carbohydrates in biological systems
Authors: Griffiths H R , Lunec J
Published: November 2023

Carbohydrates are a large family of related molecules defined as polyhydroxy aldehydes or ketones, and their derivatives. They perform a diverse range of biological functions, both in their own right, in the derivation of energy for cellular maintenance and survival, or as conjugates to larger macromolecules such as proteins or lipids. In addition, the 5-C sugar deoxyribose is an integral component of the DNA backbone and its oxygenated form is critical to the integrity of RNA.
Protein transport to the nucleus and its regulation
Authors: Jans David a.
Published: November 2023

Eukaryotic cells by definition possess a nucleus (karyon) and the existence of a nuclear subcellular compartment means that the genetic information, the DNA, is separated from the site of protein synthesis, i.e. the cytoplasm. Separation is effected by a double membrane structure, the nuclear envelope, which is both contiguous with the endoplasmic reticulum (ER) and largely ER-like in terms of lipid and enzyme composition. Since gene transcription and translation take place in separate compartments, specific transport events must occur: first, mRNA must make its way from the nucleus into the cytoplasm in order to be translated into protein; and secondly, the proteins that are required in the nucleus need to be specifically transported from their site of synthesis in the cytoplasm into the nucleus. Nucleocytoplasmic transport processes in both directions are precisely controlled, whereby the regulation of the subcellular distribution of proteins which regulate gene transcription, RNA processing, or other nuclear events is integral to many cellular processes (1). Since nuclear localization of transcription factors (TFs) (2-5), morphogens (6, 7), or oncogene products (8-11) appears to precisely accompany changes in the differentiation or metabolic state of eukaryotic cells, it is clear that nuclear protein import is a key control point in regulating gene expression and signal transduction (1).
Protein sorting during endocytosis
Authors: Ktistakis Nicholas t. , Roth michael g.
Published: November 2023

Movement and sorting of proteins along the endocytic pathway is complex. At most branching points in the pathway movement can potentially be multidirectional. For example, a protein passing through the endosomal membrane system has access to transport routes that could take it ‘back’ to the cell surface, ‘forward’ to the lysosomes, or in a ‘lateral’ direction towards the trans Golgi network (TGN). Secondly, movement along the endocytic pathway involves multiple terminal destinations, most notably the cell surface (either apical or basolateral in polarized cells), the lysosomes, or the TGN. Thirdly, in some instances multiple pathways can be used to achieve the same final destination. This is most evident in the case of some lysosomal proteins where targeting to lysosomes can be either intracellular (directly from the TGN) or via the cell surface following internalization.
Baculovirus expression systems
Authors: Merrington Claire L , King Linda A , Possee Robert D
Published: November 2023

The baculovirus expression system has provided an efficient and effective way to synthesize foreign proteins in eukaryotic cells. Analysis of the baculovirus genome has identified genes which are dispensable for the propagation of the virus in cell culture. Additionally, the promoters associated with these genes are exceptionally active, leading to the production of high levels of protein in virus-infected cells. As a result, baculoviruses have been successfully employed as vehicles for the production of large amounts of recombinant proteins which are structurally, functionally, and antigenically authentic. The material produced using the baculovirus expression system is therefore suitable for a number of applications, such as structure-function studies, therapeutics, and vaccines.
Protein synthesis in eukaryotic cell-free systems
Authors: Clemens Mike J , Pruijn Ger J M
Published: November 2023

During the last two decades cell-free protein synthesizing systems have been used extensively for the translation of eukaryotic mRNAs, for studies on the mechanism and regulation of the translation process, and the factors involved.
Linkage mapping of quantitative trait loci in plants and animals
Authors: Haley Chriss. , andersson leif
Published: November 2023

Much of the variation within populations, or between lines or breeds, is quantitative in nature. That is, variation between individuals does not fall into discrete classes in Mendelian proportions but is continuous, showing a gradation from one extreme to the other. Examples include growth rate and leanness in livestock. hypertension and intelligence quotient in man, and yield and flowering time in plants. To the extent that differences between populations are maintained, and that there is a tendency for offspring to resemble parents, there is evidently some genetic component to this variation. The genetic basis of such phcnotypic variation lies in the combined effects of variation at several or many loci. Environmental influences are superimposed upon (and may interact with) these genetic contributions to variation.
Primed in situ DNA synthesis (PRINS)
Authors: Gosden John R , Speel, Ernst J M , Shibasaki Yoshiro
Published: November 2023

Techniques for visualizing DNA sequences on chromosomes have come a long way since Pardue and Gall (1,2) and John et al. (3) first showed that repeated sequences could be visualized in nuclei or on chromosomes by isotopic in situ hybridization. In those first experiments, the probe (RNA or DNA) was made radioactive with 3H by growing cells in the presence of labelled precursors, purifying the nucleic acid, and hybridizing it in situ to cytological preparations. Later came the use of Escherichia coli RNA polymerase to make a complementary RNA from a known DNA template (4,5). Refinements of this process, and the introduction of cloned DNAs labelled by nick translation, permitted the localization of single-copy genes (6). However, because all these methods used autoradiography of either3H- or 125I-labelled probes, mapping was a tedious process, involving the analysis of grain distribution over large numbers of metaphase spreads before genuine signals could be distinguished from background. It was only with the introduction of non-isotopic labelling methods and, in particular, the use of fluorescence, that cytological mapping of unique DNAs by in situ hybridization became a simple and standard laboratory technique (7), now referred to almost universally as FISH (fluorescence in situ hybridization).
Microsatellites
Authors: Schlotterer Christian
Published: November 2023

Microsatellites are highly polymorphic DNA markers with discrete loci and co dominant alleles. They have a wide range of applications which previously were difficult to address experimentally. Microsatellites are almost uniformly distributed over the entire genome, which is what makes them so useful for genome mapping projects (1,2). Their high variability has made them a marker of choice in behavioural ecology, as they allow the determination of paternity and kinship (3,4). Finally, they are becoming increasingly important in studying the structure of natural populations. This chapter provides information on how to get started with microsatellite cloning and analysis, and provides some back ground information on the interpretation and analysis of the data.
One-dimensional polyacrylamide gel electrophoresis
Authors: Shi Qinwei , Jackowski George
Published: November 2023

With the approach of the post-genome era and the popularity of recombinant DNA technology, there has been a resurgence in the use of polyacrylamide gel electrophoresis to identify and characterize various gene products. Electrophoresis is a relatively simple, rapid, and highly sensitive tool to study the properties of proteins. The separation of proteins by electrophoresis is based on the fact that charged molecules will migrate through a matrix upon application of an electric field usually provided by immersed electrodes. Generally the sample is run in a support matrix such as agarose or polyacrylamide gel. Agarose is mainly used to separate larger macromolecules such as nucleic acids whereas a polyacrylamide gel is widely employed to separate proteins. Polyacrylamide gel electrophoresis can be used to analyse the size, amount, purity, and isoelectric point of polypeptides and proteins. Therefore this technique has become the principle tool in analytical chemistry, biochemistry, and molecular biology.
Growth factor-toxin chimeras and their applications
Authors: Davol Pamela A , frackelton A Raymond , calabresi paul
Published: November 2023

Clinically, the most successful drugs in use today are those which were designed to target specific metabolic pathways. Not surprisingly, growth factor receptors expressed on the surfaces of cells have been recognized as potentially important therapeutic targets. The specific affinity of growth factor ligands for their receptors combined with the observation that many growth factor receptors are rarely expressed at high levels on normal, non proliferating cells have made growth factor ligands attractive molecules for the delivery of toxins for the treatment of neoplastic, autoimmune, cardio vascular, and proliferative eye diseases and transplant rejection (see reviews in refs 1-3).
Drugs and inhibitors as affinity ligards
Authors: Hooper Nigel M
Published: November 2023

With the pharmaceutical industry ‘s search to identify ever more therapeutic agents, numerous drugs and inhibitors are being synthesized. Several of these compounds can be used as affinity ligands for the purification of a particular target protein. However, some of the earliest affinity ligands were those based on the dyes used in the textile industry, such as the synthetic polycyclic dyes Cibacron Blue and Procion Red which bind to a wide range of proteins including dehydrogenases, kinases, and other enzymes requiring adenylyl containing groups (1); the use of such dyes is described in Chapter 6. How-· ever, other proteins, e.g. albumin and interferon, bind in a less specific manner due to electrostatic and/or hydrophobic interactions with the compounds.. More recently numerous drugs and enzyme inhibitors have been used as selective affinity ligands. In some cases the interaction between the ligand and the protein has been mapped in detail, often through co-crystallization of the compound with the protein and subsequent X-ray crystallographic analysis.. However, details of how a compound interacts with a particular protein need not be known for its use as an affinity ligand.
Surface Modification of Liposomes
Authors: Torchiiin Vladimir P , Weissig Volkmar , Martin Francis J , Heath Timothy D , R.C.New Roger
Published: November 2023

Modification of the liposome surface is a powerful approach to control liposome properties and biological behaviour. Attachment of certain specific ligands (such as antibodies, peptides, hormones, sugars) makes liposomes targeted; attachment of contrast agents converts liposomes into efficient diagnostic tools; coating liposomes with polymers, such as poly(ethylene glycol) imparts to them an ability to circulate long without being opsonized and recognized by cells of the reticulo endothelial system, etc.
pH-sensitive liposomes
Authors: Peschka-Süss Regine , Schubert Rolf
Published: November 2023

In 1980, it was suggested that there might be a possible clinical application of liposomes, which are designed to release their contents in an environment such as tumour tissue with a slightly decreased pH of around 6 (1). An advanced approach of pH-sensitive liposomes is the intracellular pH-triggered release of liposome compounds from the endocytotic pathway to the cytosol.
Surface fluorescence microscopy with evanescent illumination
Authors: Axelrod D
Published: November 2023

The interaction of molecules with surfaces is central to numerous phenomena in biology: e.g. binding to and triggering of cells by hormones, neurotransmitters, and antigens; the deposition of proteins upon foreign surfaces leading to thrombogenesis; electron transport in the mitochondrial membrane; cell adhesion to surfaces; concentration of reactants at surfaces and consequent enhancement of their reaction rates with other surface bound molecules; the dynamical arrangement of submembrane cytoskeletal structures involved in cell shape, motility, and mechanoelastic properties; and molecular events preceding cellular secretion from intracellular granules.
Purification of macrophages
Authors: Gessani Sandra , Fantuui Laura , Puddu Patrizia , Belardelli Filippo
Published: November 2023

The investigation of cell populations consisting of a single cell type confers a great advantage over the complicated examination of mixed cell populations in many experimental studies. The achievement of enriched preparations of cells of the monocyte/macrophage lineage is particularly troublesome as these cells display a variety of functional and morphological phenotypes and are readily activated in response to environmental signals. Macrophage purification is further complicated when cells of this lineage represent a minority of the whole mononuclear pool (e.g. peripheral blood).
Analysis of gene expression in mononuclear phagocytes
Authors: Paulnock Donna M , Doan Joyce E S , Hamilton Thomas A
Published: November 2023

Specific gene expression is a hallmark of differentiated cell populations, as well as of cellular activation within specific cellular subsets. This is particularly true in the context of the mononuclear phagocyte, which has long been described on the basis of changes in cellular phenotype and function associated with activation by proinflammatory or stimulatory agents (1, 2). This chapter will provide a discussion of methods used for analysis of gene expression in the context of mononuclear phagocytes. Even in such a restricted context, however, this represents a broad and diverse subject. Furthermore, gene expression may be modulated both quantitatively and qualitatively at multiple levels, which can be broken down into transcriptional, post-transcriptional, translational, and posttranslational (subcellular localization/secretion) categories. In order to maintain reasonable scope, this chapter will be limited to considerations of experimental methodologies which address the early aspects of gene expression, i.e. transcriptional and post-transcriptional regulation.
Image warping and spatial data mapping
Authors: Baldock Richard A , Hill Bill
Published: November 2023

Modifying the geometry of (warping or morphing) a digital image is important for many purposes. D’Arcy Thompson (1) used coordinate transformations to model variations in form with a view to understanding the underlying processes controlling growth. More recently, warping has been required as part of the data capture or reconstruction process, i.e. to restore what is assumed to be the correct geometry. Examples are to correct for spatial distortion in the imaging process, such as perspective transformation or spherical aberration in the optics, or to convert from one coordinate frame to a rectilinear array such as in ultrasound scanning or computed tomography, or to correct for geo-metric distortions introduced in the material processing before imaging such as in microtome sections of tissue. In medical imaging complex distortion of the geometry of images may be necessary to compare images from one scanning modality to another (see Chapter 6), to compare images from the same individual over time, to average between individuals, and to compare an individual with an atlas. Morphing has also been used for special effects, particularly in film and television and for simulating morphogenesis (2). With the development of spatially organized databases, e.g. geographic information systems (GISs) or, more recently, biological databases such as the Brain Map project in the USA (3) or the Edinburgh Mouse Atlas and gene-expression database (4), it is important that data accumulated at different times, with different instruments, and in the biological situation from different individuals, are mapped onto the same standard coordinate frame.
Reconstitution of membrane proteins: the Ca2+-ATPase of sarcoplasmic reticulum
Authors: Lee Anthony G
Published: November 2023

1 Introduction

Biological membranes have complex structures, reflecting the wide range of functions they are required to perform: most biological membranes contain many hundreds of different species of protein and lipid. One approach to understanding this complexity is to purify individual components from the membrane and study their properties in isolation. Purification of an intrinsic membrane protein generally requires it to be first dissolved out of the membrane in some suitable detergent, so that it can then be separated from the other membrane proteins. Some membrane proteins are stable for long periods in detergent solution, but many are not; even those that are stable may show altered properties in the unusual environment of a detergent micelle. For these reasons it is better to transfer the protein, once purified, from the detergent back into a lipid bilayer; this is the process of reconstitution. Reconstitution gives the simplest membrane system that is likely to be a fair representation of the native membrane. Reconstitution can be used to define the minimum number of protein components necessary to carry out some defined function of the membrane, and to study the interactions between lipids and proteins in the membrane.
Spatial analysis of gene expression
Authors: Nonchev Stefan G , Maconochie Mark k.
Published: November 2023

The generation of mRNA is, in most cases, a prerequisite for gene function. Thus the study of _mRNA or transcript generation is important for understanding the potential functions of genes, and many molecular techniques have been used to analyse gene expression. Routine molecular technologies involve either the immobilization of total RNA or poly(A) + RNA on membranes followed by hybridization of DNA or RNA probes, or the use of oligonucleotide primers and reverse transcription–polymerase chain reaction (RT-PCR). However, further molecular methods are being developed, and it is likely that DNA chip and microarray technology will permit more global assays of gene expression. All the above methods are two-dimensional, since spatial information is lost by removal of the cells and tissues to provide the material for assay. It has become clear that the restriction of gene expression to discrete domains in higher eukaryotes is one important method of regulating gene function, and thus gene expression assays which are able to reflect the three dimensions of embryos, tissues and organs are likely to remain important components of genetic analyses for some time to come.
Analysing mouse chromosomal rearrangements with G-banded chromosomes
Authors: Akeson Ellen C , Davisson Muriel T
Published: November 2023

This section describes the preparation of metaphase chromosomes from mouse peripheral blood lymphocytes for chromosomal typing of living mice. Included is a list of mouse strains and their relative response to phyto-haemagglutinin (PHA), a list of the requirements necessary for the production of well-spread metaphase chromosomes with good morphology for banding, a Giemsa banding (G-band) method for these chromosomes, and their classification into a karyotype. The identification of mouse chromosomes is presented with a detailed process for determining aberrant chromosomes and describing the common pitfalls of misclassification. A precise description of the characteristic banding patterns (landmarks) at different stages of chromo some condensation is presented elsewhere (1). The cell-harvesting part of the procedure also can be used to prepare mitotic chromosomes directly from fetal liver or bone marrow.
Generation of transgenic mice from plasmids, BACs and YACs
Authors: Hammes Annette , Schedl Andreas
Published: November 2023

Transgenesis, or the stable integration of foreign DNA into a host genome, has developed into one of the most powerful techniques for analysing gene function and regulation (1). Applications of this technology are numerous and range from investigating the basic mechanisms of gene regulation to the generation of models for human diseases, which can be used for pathophysiological and therapeutic studies. The list of over-expression studies includes the use of cDNA constructs encoding transcription factors, cell surface receptors and structural proteins. In addition constructs encoding antisense RNA, to inhibit endogenous gene expression, can be used in transgenic experiments. Before describing the technical steps involved in the generation of transgenic mice, we will briefly highlight a number of important points to consider in designing a transgenic experiment. Due to space limitations this section cannot be comprehensive and the interested reader is referred to original articles where transgenic technology has been applied.
Quality assurance
Authors: Seth John
Published: November 2023

All analytical laboratories, whether in the field of health care, environmental monitoring or manufacturing industry must be able to demonstrate that their output meets defined quality standards. Basic research laboratories, which generally have operated independently of such controls and standards, are increasingly adopting them in response to the growing emphasis on ‘evidence-based medicine’, and/or as aspects of good management.
Selection of antibodies from phage libraries of immunoglobulin genes
Authors: Osbourn Jane K
Published: November 2023

This chapter explains the various approaches it is possible to take in the selection of phage antibodies with specific binding characteristics from large phage display libraries. The particular selection approach taken will be determined by the form and availability of the starting antigen and the desired properties of the selected antibody. One of the main benefits of the phage display approach to antibody generation is the ability to tailor the selection regime to generate antibodies with particular characteristics such as: high affinity, neutralization potency, ability to recognize specific epitopes, or to recognize specific cell types. A variety of screening regimes can also be employed to identify antibodies that can be utilized for particular applications such as: Western blot reagents, immunocytochemistry reagents, or species cross-reactivity. The phage system provides a route to the generation of antibodies to antigens that are normally inaccessible using conventional immunization techniques, for example mAbs to toxic moieties, carbohydrate-containing molecules, and anti-self antigens can easily be selected using the appropriate conditions. Methods for the generation of phage antibody libraries from immunized rodents are described elsewhere (Chapter 2) and these techniques are equally applicable to the generation of human phage antibody libraries if appropriate primers are used (1). The selec tion methods described here can be employed for immunoglobulin genes dis played in scFv, Fab, or diabody formats. The methods described provide a necessarily brief outline of the possible approaches and it should be stressed that there are no hard and fast rules for selection protocols; investigators should use as much imagination as possible in designing selection procedures which will best suit the final application of the antibody. Possible selection methods such as in vivo selection (2), selective infective phage selections (3, 4), and various deselection methods (e.g. 5) have not been discussed in detail here and reference should be made to the appropriate literature for further ideas on these approaches.
ARM complexes for in vitro display and evolution of antibody combining sites
Authors: Taussig Michael J , Groves Maria A T , Menges Margit , Liu, Hong , He Mingyue
Published: November 2023

Antibody VH and VL region genes are readily obtained by PCR and can be recombined at random to produce large libraries of single chain fragments (1). Such libraries may be obtained from normal or immune B lymphocytes, or con structed artificially from cloned gene fragments with synthetic H-CDR3 regions generated in vitro (2). Libraries can also be generated by mutagenesis of cloned DNA fragments encoding specific Vtt/ VL combinations and screened for mutants having improved properties of affinity or specificity.
lmmunofluorescence microscopy
Authors: Pepperkok Rainer , Girod Andreas , Simpson, Jeremy , Rietdorf Jens
Published: November 2023

Antibodies provide a powerful tool to localize antigens in cells or tissues by immunocytochemistry at the light or electron microscope level. The develop ment of efficient fluorescent dyes which can be coupled to antibodies for their visualization by fluorescence microscopy has pioneered a technology which is known as immunofluorescence microscopy (1PM). IFM is easy to apply to many biological and medical questions, the protocols involved are short, and the development of sophisticated imaging equipment has even made possible the acquisition of quantitative data on the 3D distribution of several antigens in the same specimen.
FACS analysis of clinical haematological samples in transplantation for cancer
Authors: Millar Barbara C
Published: November 2023

The systematic approach to the naming of leucocyte antigens using the cluster of differentiation (CD) designation (1) was made using monoclonal antibodies (mAbs). Direct conjugation of these antibodies or indirectly, via a two-step pro cedure, to fluorochromes permits visualization of individual cell types. The development of automated fluorescence activated cell analysis (FACS) to assess leucocytes in peripheral blood (PB) and bone marrow (BM) samples by their CD designation has provided an accurate and reproducible method, which is relatively easy, quick to perform, and permits far greater numbers of cells to be analysed than can be done using microscopy.
lmmunocytochemical staining of cells and tissues for diagnostic applications
Authors: Dodson Andrew R , Sloane John P
Published: November 2023

Immunocytochemistry can be defined as the in situ demonstration of cellular constituents by specific antibody-antigen reactions. It has its origins in the 1940s with the pioneering work of Coons et al. (1). Since that time many workers have developed and refined the technique, so that today it is an essential part of diagnostic pathology.
Study of protein phosphorylation in intact cells
Authors: Haystead Timothy A J
Published: November 2023

The phosphorylation of proteins on the amino acid residues serine, threonine and tyrosine is generally recognized as a fundamental mechanism by which the regulation of intracellular events is achieved. The process is reversible, enabling cells to respond in a dynamic way to a myriad of signals. The significance of protein phosphorylation is rapidly becoming apparent from the ongoing genome projects, with the discovery that 1–2% of the entire human genome may encode protein kinases (1). Conversely, in the case of protein tyrosine phosphatases, there may be as many genes encoding these enzymes as there are opposing tyrosine kinases. Although not as readily identified by primary structure predictions alone, there may also be numerous genes encoding regulatory subunits which control the activity of the catalytic subunits of serine/threonine phosphatases (2–5). The finding that nature has put so much evolutionary effort into controlling phosphorylation events in the cell is a testament to its importance, as well as to its complexity. The finding that many protein kinases and phosphatases are localized with their substrates in discrete subcellular compartments, and not floating in the intracellular ‘ether’ as previously thought, adds additional complexity. Methods that enable phosphorylation events to be examined directly as they happen in the intact cell in response to a particular stimulus are, therefore, highly desirable. This chapter focuses on state of the art, non-invasive, methods that enable changes in intracellular protein phosphorylation to be measured as they occur in intact cells. One of the great advantages of studying protein phosphorylation, as opposed to other regulatory mechanisms such as allosteric regulation, is that protein phosphate esters are stable at physiological pH and can survive homogenization of the cell.
Fluorescence microscopy of living vertebrate cells
Authors: Pepperkok Rainer , Shima David
Published: November 2023

Fluorescence microscopy of living cells has always been a powerful tool for cell biologists. Presently it is experiencing an unexpected renaissance and is on its way to having a place in almost every branch in modern biology from developmental biology to cellular biochemistry. Labelling of organelles or structures in living cells, compared with work in fixed cells, has the obvious advantage that the dynamics of the labelled structures or molecules can be studied. Also, labelling in living cells does not suffer from possible problems induced by the fixation or permeabilization of the cells as is necessary for immunofluorescence or electron microscopy.
Fluorescence microscopy in yeast
Authors: Hagan Iain M , Ayscough Kathryn R
Published: November 2023

The reductionist desire of the modern biologist to analyse complex problems in the genetically amenable systems which offer the fewest variables has seen yeasts come to the fore in modern cell biology as the leading model eukaryotes. Both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe offer many advantages for the analysis of diverse aspects of cell biology. They are unicellular but also have limited options of differentiation in sexual reproduction and pseudohyphal growth. Their usual mode of growth and the tight correlation between growth and cell division have made them excellent systems for the analysis of the control of cell cycle progression. Moreover the strong classical and molecular genetics have meant that yeasts are not only a favourite of the research scientist but also appeal to the biotechnologist in their desire to produce large quantities of cloned proteins. These factors, combined with its small genome size, made budding yeast the obvious choice for a concerted effort to establish the first complete DNA sequence of a eukaryote (see Table 1 for genome website). This information in turn has heightened interest in yeast as a model system as many genes can be identified in silica and the boundaries of potential complexity of different pathways have been set. It is now known exactly how many members of a particular protein family exist in S. cerevisiae. This defines limits for modelling a particular process as the possibility that perhaps another isoform of a particular protein exists is no longer an acceptable escape clause to consolidate a flagging model.
Initial characterization of new orphan receptors
Authors: Sladek R , Giguère V
Published: November 2023

Nuclear receptors are transcription factors with the intrinsic ability to be regulated by changes in the intracellular levels of small lipophilic compounds. Since the molecular cloning of the glucocorticoid receptor in 1985, over 50members of the nuclear receptor superfamily have been identified (1). Most of these gene products were discovered as a result of their sequence homology with other family members: however, their sequence conservation often reveal<; little about their individual function as transcription regulatory proteins. The ultimate goal for many investigators is to identify ligands for these orphan receptors: basic characterization of receptor functions including their DNA binding mode and transactivation properties provides an important first step towards realizing this goal.
Adopting orphans: finding ligands
Authors: Kersten S , Wahli w.
Published: November 2023

This chapter outlines the strategy for finding natural ligands for orphan nuclear receptors. The first part discusses the overall approach for identifying compounds or a class of compounds that can activatethe orphan receptor. The second part focuses on the methods that can be employed to demonstrate that the previously identified activator is indeed a bona fide ligand, meaning that the compound is able to directly bindto the orphan receptor with high affinity.
In vitroassembly of steroid receptor-Hsp90 complexes
Authors: Smitii D F
Published: November 2023

Receptors for the steroid hormones—oestrogens, androgens. progesterone, glucocorticoids, and mineralocorticoids-typically exist in heteromeric complexes prior to binding hormone (for a recent. comprehensive review s<:C ref. 1 ). After hormone binding or certain non-hormonal signals. the receptor monomer dissociates from complexes and undergoes a series of biochemical changes that lead to its activation as a transcription factor. In early studies of steroid receptors, the un activated receptor was commonly observed to migrate as an 8--10S complex by density gradient centrifugation while the activated receptor migrated at 4-5S, the approximate i/e of a receptor monomer or dimer.