bluuebunny/crossref_metadata_2025 huggingface dataset:
The three genomes of Plasmodium
Authors: Feagin Jean E , Lanzer Michael
Published: November 2023

The Greek physician Hippocrates, who lived in the fifth century BC, was the first to describe the characteristic symptoms of a malaria infection: intermittent and often relapsing fever attacks that are accompanied by drenching sweats and followed by shaking chills. Centuries later, the medical almanac of 1888 referred to malaria as one of the great scourges of humanity, wreaking havoc in human civilizations throughout history and claiming more lives than any other infectious disease. Today, malaria is still a major health problem, causing an estimated 300 million cases of illness and killing 1-2 million people-mostly infants-every year. About 2.2 billion people (41% of the world’s population) are now in danger of contracting malaria. The prospect for the future is bleak. The number of malaria cases is expected to rise due to the emergence and spread of drug-resistant malaria parasites and pesticide-resistant malaria-transmitting mosquitoes. Furthermore, a widely effective anti-malarial vaccine is not expected in the near future although several candidate vaccines are now in clinical trials.
Integration of control devices: A global regulatory network in Escherichia coli
Authors: Hengge-Aronis Regine
Published: November 2023

Until the present day, the fundamental concepts developed by F. Jacob and J. Monod in the 1960s during their studies of the lac operon (1), have mainly shaped our view of the regulation of gene expression. What is the structure and function of regulatory proteins, how do they bind to DNA, and how do they influence the rate of transcription of this DNA? Especially with Escherichia coli, these questions can be at least partially answered for many regulatory proteins. Based on this knowledge, we can now start to determine the roles of these single regulatory systems within the physiology of a cell. As single-cell organisms, bacteria process a multitude of environmental signals and use this information to optimally balance their metabolism, growth rate, DNA replication, and cell division. This requires the coordination and interaction of a large number of single regulatory systems in regulatory networks.
Chemically engineered antibodies
Authors: Stevenson G T , Kan K S
Published: November 2023

The chemical engineering described in this chapter will utilize just two immunoglobulin (lg) modules: mouse monoclonal Fab’’Y from individual antibodies, and human Fc-y from normal lgG1. These can be combined in various ways to provide antibody derivatives exhibiting combinations of bi-specificity, chimerism, and multiple Fe regions. Our derivatives have been designed to kill neoplastic cells, in vitro or in vivo, usually by the recruitment of natural effectors such as complement and cells bearing Fc-y receptors. After describing the preparation of the lg modules we shall give three examples of derivatives which may be prepared from them. Once the principles of construction are understood the range of accessible derivatives will encompass far more than these examples, and may be extended to other purposes such as immunohistology. The technology offers a rapid and economic prediction of the performance of some constructs, such as chimeric antibodies, which could then be engineered genetically to avoid the need for repeated chemical preparation. But it is capable also of yielding derivatives with geometries virtually impossible to obtain by genetic means.
Generation and selection of human monoclonal antibodies against melanoma-associated antigens: a model for production of anti-tumour antibodies
Authors: Thomas M D , Melamed, M D , Newton J
Published: November 2023

Melanomas are generally resistant to chemotherapy and radiotherapy, and often give rise to metastases (1). However, the neoplastic transformation of melanocytes induces qualitative and quantitative modulation of cell mem brane antigens (2,3). These tumour-associated immunogenic markers suggest the possibility of a strategy for selective diagnosis and even therapy for melanoma (4—8), e.g. by the production of tumour-specific human monoclonal antibodies. The melanoma tumour can thus be used as a favourable case for the establishment of techniques for the production of anti-tumour Mabs.
Synthetic Peptides in Epitope Mapping
Authors: Rodda Stuart J , Tribbick, Gordon , Maeji N Joe
Published: November 2023

Synthetic peptides can be used as a tool for probing the nature of antibodydefined (B cell) epitopes or T cell receptor (TCR)-defined (T cell) epitopes. Linear synthetic peptides homologous with the sequence of a protein antigen are suitable for mapping the ‘linear’ (continuous) type of B cell epitope, and all T cell epitopes. The power of the synthetic peptide approach to epitope mapping has been greatly expanded by the development of techniques for the parallel synthesis of hundreds to thousands of peptides, and by improvements in the chemistry of peptide synthesis which make the synthesis of peptides beyond 20mers in length feasible on a mass scale.
Liposomes
Authors: Claassen Eric , Iwaarden Freek Van
Published: November 2023

Liposomes can be used in a number of different ways to influence the homeostasis of a given organism. Typical examples include their use as vaccine, adjuvant, drug delivery system, immunomodulator, imaging agent, or as a cosmetic. Liposomes in their most basic form consist of spontaneously formed phospholipid bilayers enveloping an aqueous phase. When preparing liposomes from naturally occurring (phospho)lipids, in the correct concentrations and without adding foreign substances to the preparation, one can assume that the ensuing vesicles are non-immunogenic (i.e. not a target for antibodies or specific T cells) and biodegradable. However, this type of vesicle can still be a target for immune attack by the innate branch of the immune system embodied in most cases by macrophages which will take up almost any particulate matter not bearing the proper MHC molecules. As reviewed recently (1) the innate immune response which is very swift and efficient in removing non-self does not receive enough attention in the design of liposomes. Consequently, ‘natural’ liposomes can be used to influence macrophages in several ways. First, by the inclusion of a toxic substance the macrophages can be killed or temporarily suppressed. The latter can also be achieved by overloading the macrophages with large doses of ‘empty natural’ liposomes. Secondly, by the inclusion of immunomodulators, both in the aqueous or lipid phase, one can stimulate the macrophages. The efficiency of elimination, stimulation, or suppression is critically dependent on factors such as: liposome size, charge, stability, content, and membrane composition. Minor variations in these factors may have major consequences for the eventual outcome.
Immunochemical assays for complement components
Authors: Wurzner R , Mollnes, T E , Morgan B P
Published: November 2023

The complement system consists of more than 25 different plasma proteins. Most components are circulating in the blood in an inactive form. Classical or alternative pathway activation of the complement system results in the generation of multiple complement proteolytic cleavage products, recruited from these inactive precursor molecules in a sequentially proceeding cascade (1). This leads to an alteration of their antigenic pattern and thus to the consumption of the native molecule. Concomitantly, complement activation initiates formation of multimolecular complexes which function as convertases within the cascade or express other functions such as the membranolytic effects of the membrane attack complex, the terminal complement complex (TCC) generated on biological membranes. Such activation-dependent changes can be revealed by the appearance of neoepitopes, and by the disappearance of native-restricted epitopes (2), and are usually assessed by means of monoclonal antibodies in enzyme immunoassays (abbreviated as EIAs or ELISAs). The present chapter focuses on sensitive and specific quantitation of complement proteins, fragments, and complexes to reliably assess complement activation in vivo. Figure 1 gives an overview of the complement system and indicates the proteins on which neoepitopes have been found upon activation.
Expressing cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris
Authors: Tuite Mick F , Clare, Jeff J , Romanos Mike A
Published: November 2023

When considering whether or not to express a heterologous gene in yeast, one has not only to decide which strategy should be used—vector, strain, promoter, etc.—but also which yeast. The term ‘yeast’, although widely regarded to be synonymous with the yeast Saccharomyces cerevisiae, is in fact an umbrella term covering approximately 500 different yeast species. The reason for this laxity in terminology is perhaps simple laziness, but there are ever increasing numbers of researchers turning to S. cerevisiae as a model organism for their studies on gene structure, function, and regulation. The completion and release of the entire genome sequence of S. cerevisiae will ensure that such interest is maintained.
Role of DNA methylation in mammalian development
Authors: LI E
Published: November 2023

Methylation of genomic DNA at the CS position of cytosine residues is the predominant form of DNA modification in plants and vertebrates. DNA methylation is a developmentally regulated process that acts in concert with other regulatory mechanisms to control gene expression. Evidence suggests that DNA methylation is involved in a diverse range of biological processes which include regulation of tissue specific gene expression, cell differentiation, genomic imprinting, X chromosome inactivation, regulation of chromatin structure, DNA replication, viral latency, carcinogenesis, and ageing. The importance of DNA methylation has been increasingly recognized as many recent studies have provided direct evidence for its role in cancer (1, 2). While numerous studies have focused on the role of DNA methylation in gene regulation, little is known about how DNA methylation itself is regulated during development. One major obstacle in studying the function of DNA methylation in eukaryotic organisms in the past had been the lack of a genetic model system. Recently, DNA methylation-deficient mutants have been successfully generated in several model organisms including mice, Arabidopsis thaliana, and Neurospora crassa (3-5).
Function and epigenetic modification of the imprinted H19 gene
Authors: Bartolomei Marisa S
Published: November 2023

The H19 gene encodes one of the most abundant RNA polymerase II transcripts in the mouse embryo. This gene was originally identified as a fetal liver-specific cDNA whose postnatal repression paralleled that of the u-fetoprotein gene (1). H19 has been identified in other screens including those for genes that are activated during the differentiation of C3H10T1 / 2 cells (referred to as myoH) (2), as well as during the differentiation of embryonic stem cells (3). The H19 gene was also isolated as a developmentally regulated gene in rabbit vascular smooth muscle cells (4) and as a gene associated with tumour suppression in syrian hamster embryo cells (5). While these studies have led to a more detailed understanding of the expression and nature of the gene product, it was not until H19 was found to be imprinted, or expressed exclusively from the maternally-inherited allele, that the function of the gene could be elucidated (6, 7). It is the purpose of this review to summarize what is known about the function and imprinting of the H19 gene as well as to assess the merits of using H19 as a general model to study imprinting.
The imprinted insulin-like growth factor 2 gene
Authors: Feil Robert , Moore Thomas F , Oswald Joachim , Walter Jorn , Sun Fanglin , Reik Wolf
Published: November 2023

The insulin-like growth factors IGFl and IGF2 are mitogenic polypeptides which are structurally and functionally homologous to the hormone insulin. Both single chain peptides play important roles in pathways regulating growth and development (1, 2). Their action is mediated by specific interactions with cell surface receptors and is further modified by at least six different IGF-binding proteins. Two IGF receptors have been identified. The type-1 receptor has a higher affinity for IGFl than for IGF2 and is related to the insulin receptor. The type-2 receptor, which is also a receptor for mannose-6-phosphate, binds IGF2 with high affinity, but is structurally distinct from the type-1 and insulin receptors. IGFl and IGF2 mediate their effects on growth and development through the type-1 receptor (3).
Genomic imprinting as a developmental process disturbed in cancer
Authors: Feinberg Andrew P
Published: November 2023

One of the most exciting recent discoveries in the study of genomic imprinting is direct evidence of its role in human cancer. Several indirect lines of evidence had suggested such a role. First was the observation that hydatidiform moles and ovarian teratomas arise from androgenetic (46 chromosomes, all of paternal origin) and parthogenetic embryos (46 chromosomes, all of maternal origin), respectively (1, 2). Thus, even if a normal number of chromosomes is present, neoplasia results from an imbalance between maternal and paternal chromosomes. Furthermore, an excess of the paternal genome leads to a more malignant growth than an excess of the maternal genome, suggesting that maternal and paternal chromosomes have differential effects on growth homeostasis.
Heterokaryons reveal that differentiation requires continuous regulation
Authors: Blau Helen M
Published: November 2023

As vertebrates develop, totipotent cells in the early embryo give rise to cell types specialized for function in tissues. In Waddington’s epigenetic landscape, differentiated cells enter grooves, or valleys (Waddington 1940), a depiction which suggests that their fate is not easily changed. Although this appears to be the case, the mechanism by which this stability is maintained and propagated to differentiated cell progeny remains unknown. In this review I present evidence which suggests that the differentiated state requires continuous regulation. The implication of this type of control mechanism is that at any given time the balance of positive and negative regulators present in a cell is critical. To sustain threshold concentrations of key regulators and ensure stability, feedback mechanisms seem essential. Yet, to date, relatively few feedback loops with a role in differentiation have been elucidated. A genetic complementation approach appears to hold promise of identifying novel regulators capable of tipping the balance, acting in a feedback loop, and reinforcing the decision either to grow or to differentiate.
Nuclear protein sorting in heterokaryons and homokaryons
Authors: Ringertz Nils R
Published: November 2023

Our interest in nuclear protein sorting began 25 years ago and was triggered by Professor Harris’ report that it was possible to reactivate RNA and DNA synthesis in inactive chick erythrocyte (CE) nuclei by Sendai virus-induced fusion of chicken red cells with human HeLa cells (Harris 1965, 1967). This finding suggested that human tumour cells contain factors capable of activating chick genes and raised a number of interesting questions about the nature of cytoplasmic signals regulating transcription and replication. In collaboration with Professor Harris we carried out a quantitative cytochemical study of chromatin changes during nuclear reactivation in CE x HeLa heterokaryons. Individual nuclei in fixed heterokaryons were analysed using microspectrophotometry, microfluorimetry, and microinterferometry (Bolund et al. 1969). During early stages of reactivation the chick chromatin underwent drastic biophysical changes resulting in an increased ability to bind intercalating fluorochromes to DNA, altered thermal stability of the DNA, etc. These changes were followed by a marked dispersion of the condensed erythrocyte chromatin. Model experiments with isolated CE nuclei suggested that the biophysical changes, which we named ‘chromatin activation’ (Ringertz 1969), were triggered by ionic signals causing conformational changes in the deoxyribonucleoprotein complexes. The drastic increase in nuclear size noted by Harris (1967) and illustrated in Fig. 3.1, was paralleled by a dramatic increase in nuclear dry mass and protein content. An interesting aspect was that the nuclei grew in size and weight before there was any significant increase in chick RNA and chick proteins. Using species-specific antibodies to nuclear proteins (Ringertz et al. 1971) we subsequently found that the chick nuclei grow because they selectively take up and concentrate human nuclear proteins contributed by the HeLa fusion partner. This led us to examine which human proteins entered the chick nuclear compartment and which chick proteins, if any, were lost from the inactive CE chromatin. We also tested if different cytoplasmic environments, i.e. different fusion partners, resulted in activation of the same or of different genes.
Some insights into the replication of damaged DNA in mammalian cells
Authors: Johnson R T , Downes C S , Godfrey D B , Hatton D H , Ryan, A J , Squires S
Published: November 2023

In a volume devoted to the legacy of cell fusion, this article on cellular responses to DNA damage contains only a single example of the use of the technique, though the information gained thereby has transformed our view of the mutant cell whose phenotype is described at some length below. In the wider sense cell fusion continues to play an invaluable role in the analysis of DNA repair problems, most notably in complementation analysis using heterokaryons (Giannelli et al. 1982) or permanent hybrid cells (Johnson et al. 1989). In this way the number of genes involved in a repair pathway can be identified, by separating a set of repair-defective mutant cells into different complementation groups.
The role of the nucleolus in the transfer of information from nucleus to cytoplasm
Authors: Hatanaka Masakazu
Published: November 2023

When the nucleus of a hen erythrocyte is introduced into the cytoplasm of a human or mouse cell in culture, it resumes the synthesis of RNA (Harris 1965, 1967). The reactivated erythrocyte nucleus undergoes great enlargement, but it does not, for at least 2 or 3 days, develop nucleoli. During this period, the heterokaryon, although it may contain active erythrocyte nuclei, does not synthesize any hen-specific surface antigens. But when, later, the erythrocyte nuclei do develop nucleoli, hen-specific antigens reappear on the surface of the heterokaryon and progressively accumulate. These and other nucleolar inactivation experiments suggested that the nucleolus may play a decisive role in the transfer of information from the nucleus to the cytoplasm (Harris et al. 1969; Sidebottom and Harris 1969; Deak et al. 1972; Harris 1972).
Transcription by immobile RNA polymerases
Authors: Cook P R
Published: November 2023

An unstated assumption in current models for transcriptions is that a polymerase tracks along the template as it synthesizes RNA. However, experiments using ‘nucleoids’ derived by lysing cells in a non-ionic detergent and 2 M NaCl originally suggested that RNA was synthesized by a polymerase attached to an underlying nucleoskeleton. These experiments were subject to the criticism that the associations seen resulted from the artefactual aggregation of nascent RNA. Further experiments using more physiological conditions have confirmed the existence of an intermediate-filament-like nucleoskeleton and have shown that active polymerases resist electroelution from nuclei, presumably because they are attached to the skeleton. Whether immobilization affects polymerase activity has also been tested directly by attaching to plastic beads a pure enzyme that is widely used for transcription in vitro-the RNA polymerase of bacteriophage T7. Although initiation is inhibited, immobilization has no effect on elongation. It is suggested that genes become active by binding to an attached polymerase and then transcripts are generated as the template moves past the fixed enzyme.
Lateral mobility of membrane proteins-a journey from heterokaryons to laser tweezers
Authors: Edidin Michael
Published: November 2023

Soon after virus-induced heterokaryons were described (Harris and Watkins 1965) it was observed by several laboratories that species-specific surface markers of the parent cells were randomly distributed over the heterokaryon surface (Watkins and Grace 1967; Harris et al. 1969; Gordon and Cohn 1970). All of these observations were made hours after the fusion of the parent cells, and were made with fairly low spatial resolution. Larry Frye, then a graduate student in my laboratory, decided to refine the experiments by resolving the time required for randomization of surface markers from parent cells. He also improved the spatial resolution of the distribution by using fluorescent antibodies to the class I MHC (major histocompatibility complex) molecules of the mouse cell parent and antibodies to undefined, but membrane-integral, antigens of the human cell parent of the heterokaryons. With this system, Frye was able to show that randomization of surface marker antigens began within minutes of fusing mouse and human cells with virus (Frye and Edidin 1970). Controls, including fixation of newly formed heterokaryons, showed clearly that redistribution was due to diffusion and not to cell metabolism (Edidin and Wei 1977). Together with studies of lipid motion in the bilayer, these experiments set us on the road to viewing cell membranes, especially cell surface membranes, as ordered fluids whose components are free to move in the plane of the membrane. The experiments that showed diffusional motions of lipids and proteins are summarized in an early review on the subject (Edidin 1974); the model embodying them, together with data on the solubility of membrane proteins is the well-known ‘fluid mosaic’ model of Singer and Nicolson (1972).
Radiation hybrid mapping: an idea whose time has finally arrived
Authors: Cox David R
Published: November 2023

In 1975, Stephen Goss and Henry Harris published a short paper in the journal Nature that promised to revolutionize the field of human gene mapping. Entitled ‘New method for mapping genes in human chromosomes’, this paper described a systematic approach for determining the linear order of human genes and estimating the distances between them. Previous workers had used rodent-human somatic cell hybrids to map genes to specific human chromosomes or to specific chromosomal segments in those regions of the genome where human translocation chromosomes were available. However, such maps were very crude in comparison with the genetic maps available for many other organisms. By exposing diploid human cells to a lethal dose of ionizing radiation followed by fusion to rodent cells to obtain viable hybrid clones segregating human chromosomal fragments, Goss and Harris were able to measure the frequency with which pairs of human genes were cotransferred after irradiation, thereby creating maps of much higher resolution than was possible previously.
Genes in control of cell proliferation and tumorigenesis in Drosophila
Authors: Mechler Bernard M
Published: November 2023

Cancer is generally considered as a failure in the normal progression of differentiation. As a result, the cancer cells escape the mechanisms controlling normal growth and proliferate. When the overgrowth of neoplastic cells reaches a critical threshold, complex syndromes arise and, ultimately, lead to the death of the organism. A genetic basis for cancer is now well established and includes the discovery of protooncogenes, whose activation or altered expression is associated with the cancerous state and tumour suppressor genes which appear to be lost or functionally inactivated. About 25 years ago, pioneering studies in the field of Drosophila, mouse somatic cell, and human genetics revealed that neoplasia may result from recessive mutations in regulatory genes controlling cell growth and differentiation (Gateff and Schneiderman 1967, 1969; Harris et al. 1969; Knudson 1971).
Somatic cell genetics and the search for colon cancer genes
Authors: Thomas H J W , Frischauf A -M , Solomon E
Published: November 2023

As this volume is in honour of Professor Sir Henry Harris, it seems appropriate, under the general topic of genetics of colon cancer, to focus specifically on the role of somatic cell genetics in the isolation of genes responsible for this disease. Two rather different types of somatic cell genetics have been used in the identification of the genes mutated in colon cancer. The first type might be called tumour somatic cell genetics and has developed from the fact that loss of genetic material from tumour cells can be detected either microscopically, as whole or partial chromo­some loss or by loss of heterozygosity (LOH) (for reviews see Goddard and Solomon, (1993) and Lasko et al., (1991)). This genetic loss in tumours has come to be recognized as the hallmark of tumour suppressor genes.
The use of flow-sorted chromosomes in genome mapping
Authors: Ross Markt. , Langford cordelia f.
Published: November 2023

Flow-sorted chromosomes are an invaluable resource for genome mapping. Sorted material can be cloned efficiently in E. coli to produce chromosomespecific libraries with moderate insert sizes which arc suitable for contig building, gene identification, and genomic sequencing. Alternatively, smallinsert libraries, which provide a source of chromosome-specific landmarks. can be generated either directly or following amplification of the sorted DNA by the polymerase chain reaction. This chapter gives a brief introduction to now-sorting itself. but the principal emphasis is on the use of flow-sorted material for the construction of libraries and as a PCR template for the chromosomal assignment of markers.
Long-range restriction mapping of genomic DNA
Authors: Tümmler and Burkhard
Published: November 2023

Long-range restriction maps are important tools to determine the arrangement and distance of gene markers, and a prerequisite before committing oneself to costly and time-consuming cloning and sequencing projects in order to identify gene organization and rearrangements. For the construction of a low-resolution physical map of a genome or chromosomal region, the intact DNA is digested with a restriction endonuclease that cleaves infrequently and the fragments are separated by pulsed-field gel electrophoresis (PFGE) (1, 2). Restriction fragment patterns of small genomes, such as those of prokaryotes or lower eukaryotes, can be visualized directly by gel staining. With larger genomes, the complex mixture of fragments is blotted onto membranes after electrophoresis, and hybridized with suitable probes to detect fragments of interest. The order of probes and of fragments is established from the combinatorial analysis of partial and complete digests with one or more restriction enzymes. In the simplest case, two probes will hybridize to the same restriction fragment, thus revealing physical linkage between them. However, independent confirmation must always be sought to exclude hybridization to distinct but co-migrating bands. This may be accomplished by, for example, hybridization of the two probes in question to the fragments produced by partial digestion with the same enzyme as was used in the complete digest: both probes should detect the same bands (and with the same relative intensities) in this more complex mixture of fragments. Combined usage of several different enzymes should then allow construction of a macrorestriction map around both loci, which may be refined by double digestion analysis.
RFLP analysis using heterologous probes
Authors: Aquadro Charles F , Noon William A , Begun, David J , Danforth Bryan N
Published: November 2023

DNA sequence variation is ideally assayed by direct determination of nucleotide sequences representing independent copies of a gene region or other homologous segment of the genome sampled from a natural population. We will refer to such multiple copies of single regions as 'alleles'. An estimate of the level and distribution of sequence polymorphism segregating in a population requires multiple alleles to be sampled and sequenced. The sequences must also be determined with great accuracy, since a 1% error rate in deter mination of the sequence would generate an apparent level of nucleotide polymorphism higher than that actually seen for many species. The large sampling variances of most of the descriptive statistics of variation require the investigator to sample perhaps 10 or more alleles in order to obtain statistic ally reasonable estimates. The development of the polymerase chain reaction (PCR) and rapid sequencing methodologies has made such efforts practical for many genes and organisms.
Avidin-biotin immobilization systems
Authors: Wilchek Meir , Bayer Edward A
Published: November 2023

Over the past two decades, the avid in–biotin system has evolved to be an extremely useful tool in the fields of biology, biochemistry, and biotechnology. The extraordinary affinity (Kd ∼ 10−15 M) of avid in (or its bacterial relative, streptavidin) for the vitamin biotin forms the basis for using this system. This approach has found broad application, mainly for localization studies, medical diagnostics, and recombinant genetics. The principles and scope of avidin–biotin technology have been extensively described and catalogued, and the reader is referred to the series of reviews which are available in the literature (1–5) as well as the book which has recently appeared (6).
Preparative gel electrophoresis
Authors: Lee Kelvin H , Harrington Michael G
Published: November 2023

Protein analysis has long served an important role in the biological and biomedical sciences. The most commonly used methods for separating protein mixtures are chromatography and gel electrophoresis. The essence of these two methods are the same because, in both, a sample mixture is separated into components by various molecular interactions while passing in a ‘liquid phase’ though a ‘solid phase’ matrix. The difference lies in the force used to drive the liquid phase through the solid phase: pressure in chromatography and an electric field in electrophoresis. Among its many applications, analytical protein electrophoresis can be used to determine biochemical characteristics such as the isoelectric point and mass of a protein, to catalogue protein content in complex mixtures with two-dimensional electrophoresis, and to identify the relative quantity of a particular protein in a mixture.
Polymer solution mediated size separation of proteins by capillary SDS electrophoresis
Authors: Guttman András , Shieh, Paul , Karger Barry L
Published: November 2023

Researchers in the late 1960s found that the molecular weight of protein molecules can be easily derived from their electrophoretic mobility, measured by SDS-PAGE (1). The methodology was later significantly improved (2) and progressed as a standard molecular weight determination method for proteins in slab or rod gel electrophoresis methods (3, 4). The gel acts both as a molecular sieve, enabling separations based on the size (hydrodynamic volume) of the SDS: protein molecules, and as an anti-convective medium, minimizing the conductive transport and reducing diffusion of the analyse molecules during the electrophoretic separation. Classical slab and rod gel electrophoresis is still performed manually, requiring gel casting, manual sampling, and laborious staining/destaining procedures for detection of the separated proteins.
Conventional isoelectric focusing in gel slabs, in capillaries, and immobilized pH gradients
Authors: Righetti Pier Giorgio , Bossi, Alessandra , Gelfi Cecilia
Published: November 2023

This chapter both updates our chapter in the previous edition of this book (1) and covers the most recent aspects of the technique, including capillary isoelectric focusing (cIEF), the rising star in the field of IEF methodologies. We also draw the reader’s attention to two textbooks on conventional IEF (2) and on IPGs (3). For capillary IEF (cIEF), we also recommend two recent reviews (4, 5). Fractionations which rely on differential rates of migration of sample molecules, for example along the axis of a chromatographic column or along the electric field lines in electrophoresis, generally lead to concentration bands or zones which are essentially always out of equilibrium. The narrower the band or zone the steeper the concentration gradients and the greater the tendency of these gradients to dissipate spontaneously. This dissipative trans port is thermodynamically driven; it relates to the tendency of entropy to break down all gradients, to maximize dilution and, during this process, to thoroughly mix all components (6). Most frequently, entropy exerts its effects via diffusion which causes molecules to move down concentration gradients and so produces band broadening and component intermixing.
Two-dimensional gel electrophoresis
Authors: Hanash Samir M
Published: November 2023

Two-dimensional gel electrophoresis (2DE) has been and remains a unique and powerful approach to resolve proteins and polypeptides in complex protein mixtures. However, in the past this approach has caused as much frustration as satisfaction to investigators because of its complexity, the difficulty of achieving good resolution and reproducibility, and the perceived existence of alternative molecular approaches, primarily at the nucleic acid level, to uncover gene products or gene expression patterns of interest. Thus, 2DE as a methodology has had a limited appeal to investigators. This situation has gradually been reversed in the past few years, due to a number of important developments, some technological and others related to a renewed emphasis on ‘going back to the proteins’ for a greater understanding of important bio logical processes, such as signal transduction, growth and differentiation, and cellular responses to a variety of agents and conditions. The renaissance in 2DE can also be linked to the greater availability of ready-made products for this approach, to an enhanced resolution over a wider pH range from the most acidic to the most basic, to the increased feasibility of identifying proteins separated by 2DE, and to the burgeoning availability of relevant resources on the Internet. A description of approaches and resources for the two-dimensional separation of proteins is provided in this chapter. The procedures described are all in use in the author’s laboratory and have been developed or adapted from published monographs.
Sequence analysis of gel-resolved proteins
Authors: Simpson Richard J , Reid Gavin E
Published: November 2023

It is well recognized that polyacrylamide gel electrophoresis, particularly 2DE (1), is one of the most powerful techniques for rapidly analysing complex protein mixtures. Indeed, when the human genome is fully sequenced, predicted to occur by the turn of the century (2, 3), the attention of biologists will move away from de novo protein sequencing and towards establishing the function of mature proteins in terms of their post-transitional modifications, spatial and temporal characteristics. In this post-genome era, 2DE, undoubtedly, will play a key role in strategies aimed at analysing the full protein complement expressed by a cell or tissue type. Moreover, electrophoretic techniques will continue to play an important role in dissecting biological processes such as development, differentiation, and signal transduction which involve the co ordinated expression of proteins that interact with one another in a regulated fashion.
Engineered growth factors and receptors, and their applications
Authors: Mckay Ian A , Bustin Atephen A , Brown Kenneth D
Published: November 2023

This chapter serves as an introduction to the rest of the book. In it you will find described the various types of growth factors and receptors, and their potential applications. This is followed by a discussion of systems for getting expression of recombinant growth factors and receptors, with protocols describing specific examples of in vitro and mammalian cell expression. Other examples will be found in Chapters 3, 4, 5, 7, and 9 in this volume.

In the first edition of Growth factors: a practical approach (1), published in 1993, were listed approximately 130 different known growth factors. By the time of the first reprint in 1995 this number had grown to over 170. At the time of going to press about 200 are known (see Appendix 1). Therefore it is apparent that new growth factors are still being discovered.
Structure-activity relationships of chemokines
Authors: Clark-Lewis Ian , Anderson Jennifer , Owen Philip , Vo Luan , Gong Jiang-Hong
Published: November 2023

This chapter focuses on structure-activity relationships (SAR) of protein growth and activation factors citing examples from our work on chemokines (1, 2). The emphasis is on general principles and approaches to SAR of secreted and soluble proteins, the primary function of which is to bind specific integral membrane receptors leading to a distinct cellular response. The study of SAR requires changing the covalent structure of a pro tein and determining the effects of the changes in terms of function and three-dimensional (3D) structure. The structure could be changed by either chemical modification or by de novo synthesis. Compared with chemical modification, the synthesis approach has the advantages that a large repertoire of changes can be introduced into any position and that the covalent structure is defined. Thus, the de novo synthesis approach will be the subject of this chapter.
Generation and selection of RNA ligands that inhibit the interaction of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) with its receptors
Authors: Green Louis S , Janjic Nebojsa
Published: November 2023

Storage and transmission of genetic information by nucleic acids is critically dependent on their ability to interact with proteins. The formation of specific protein:nucleic acid complexes is a function of precisely defined three dimensional structures of both binding partners. Like proteins, nucleic acids are capable of folding into a wide variety of three-dimensional structures. The ability of nucleic acids to form secondary and tertiary structures such as Watson-Crick base pairing, Hoogstein, and other non-canonical base pairing, pseudoknots, and G-quartets is determined by their sequence. Sequence randomized nucleic acid libraries therefore contain a collection of binding specificities.
Stability, Storage, and Sterilization of Liposomes
Authors: Zuidam Nicolaas Jan , Winden Ewoud van , Vrueh Remco de , Crommelin Daan J A
Published: November 2023

During the preparation process or upon storage liposomes may undergo chemical degradation or physical changes. In this chapter, the types of degradation, the consequences, and measures of prevention or minimization are discussed. Methods to establish the degree of degradation are presented in Chapter 2. Most of the relevant data on liposome stability published in the public domain was collected with bilayers based on phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine often in combination with cholesterol. Therefore, our conclusions are particularly relevant for liposomes composed of those lipids. A number of review articles/chapters dealing with liposome stability issues have been published by our group (1-5).
Vesicular Phospholipid Gels
Authors: Brandl M , Massing U
Published: November 2023

This chapter discusses some of the common limitations with small liposomes as drug carriers and how vesicular phospholipid gels can be used to circumvent them. The purpose of an intravenously injected liposomal drug carrier usually is to circulate in the bloodstream and carry the drug to the desired target organ or tissue. Under ideal circumstances the pharmacokinetics and biodistribution of the carrier-associated drug is merely dependent on the characteristics of the liposomes, not on the drug itself. Size and surface characteristics of the liposomes primarily determine the degree to which they are eliminated from the bloodstream by macrophages and end up in the RES organs spleen and liver (1, 2). Big liposomes (>200 nm) are rapidly eliminated, thus they are inappropriate for i.v. drug delivery. Smaller liposomes are more likely to escape the RES and may thus circulate long enough to reach targets within or nearby the vasculary bed (2). Smaller liposomes furthermore are more likely to extravasate.
Liposome-based DNA Vaccines: Procedures for Entrapment
Authors: Gregoriadis Gregory , McCormack Brenda , Perriea Yvonne , Bacon Andrew , Caparros-Wanderley, Wilson , Zadi Brahim
Published: November 2023

Numerous workers (1-3) have shown that intramuscular injection of naked plasmid DNA elicits humoural and cell-mediated immune responses against the encoded antigen. It appears {2, 3) that promotion of immunity is the result of DNA uptake by muscle cells which leads to the expression and extracellular release of the antigen to be subsequently taken up by antigen-presenting cells (APC). It is also believed (3) that some of the injected DNA is taken up directly by APC. There are two main disadvantages of immunization with naked DNA. First, DNA is taken up by only a minor fraction of muscle cells which, at any rate, are not professional APC. Secondly, naked DNA is exposed to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA.
Detection of mutations by hybridization with sequencespecific oligonucleotide probes
Authors: Saiki Randall K , Erlich Henry A
Published: November 2023

Genetic analysis by hybridization with sequence-specific oligonucleotide probes (SSOP) is one of the oldest methods commonly used to examine nucleic acids for the presence of known sequence variants (1, 2). The procedure takes advantage of the fact that under the appropriate reaction conditions, a short oligonucleotide probe will hybridize to its target only when it is perfectly matched; a single base pair mismatch is often sufficiently destabilizing to prevent a stable probertarget duplex from forming. By testing a specimen with a pair of probes—one complementary to the normal sequence and one to the mutant—the genotype at that position can be determined. Using a panel of probes, an entire series of mutations or polymorphisms can be surveyed.
Functional assay of the p53 tumour suppressor gene
Authors: Frebourg Thierry , Flaman Jean-Michel , Estreicher, Anne , Iggo Richard
Published: November 2023

Screening methods generally used to detect mutations are commonly based on structural analysis of genes. These methods are often labour-intensive and fail to distinguish inactivating mutations from polymorphisms or functionally silent mutations. The alternative is to use biological assays which test the function of the gene product. This chapter describes a functional assay which detects mutations in human p53 cDNA expressed in the yeast Saccharomyces cerevisiae. Somatic p53 mutations are the most common genetic defect known to occur in human tumours (for a review see ref. 1) and more than 2000 different mutations have been described. Germline p53 mutations predispose to the development of multiple tumours and are responsible for the majority of cases of the familial Li-Fraumeni cancer syndrome.
Analysis of antigen processing and presentation
Authors: Kaye P M
Published: November 2023

This chapter seeks to identify the key issues relating to the study of macrophages as antigen-presenting cells (APC) and presents protocols for the major assays used to measure antigen processing and presentation. In addition, it discusses some of the theoretical and practical issues related to the processing and presentation of intracellular pathogens for which macrophages are the major host cell. The methods presented are, for the most part, those used in the author’s laboratory or by colleagues. Whilst these provide working examples, it is stressed that the nature of individual host-pathogen interactions may necessitate alterations on a case-by-case basis. Potential areas for such modifications are highlighted, as appropriate.
Pattern recognition: classification of chromosomes
Authors: Graham Jim
Published: November 2023

The topic of pattern recognition is often associated with image analysis. Indeed several textbooks combine the topics (1, 2) and the titles are often used interchangeably. While the two topics intersect, they are distinct. Pattern recognition concerns classification: assigning labels to things on the basis of their properties. This is clearly important in image analysis, and Chapter 3 would not have been complete without some discussion of classification. In this chapter I will deal with classification methods and associated issues, which could be applied more widely ( e.g. in signal analysis, speech analysis, medical diagnosis, etc.). However, in keeping with the context of the book it will be illustrated by examples from image analysis. There are many textbooks which deal with the topic of pattern recognition in great depth, from the classic work of Duda and Hart (1 ), through several worthy contributions (2-5) to the recent comprehensive expositions of Ripley (6) and Bishop (7), which relate the topic to neural networks. The purpose of this chapter is to present a ‘user’s guide’, for which I will draw on all of this substantial body of work. I will make little attempt at mathematical or statistical justification and direct myself to those topics which I believe most ‘users’ (as distinct from pattern recognition practitioners) would need to be aware of. Where appropriate I will guide readers with an appetite for a greater depth of exposition to the appropriate sources. I have tried to keep mathematical and statistical argument to a minimum, but have found it necessary in defining some basic terms to make use of some vector-matrix notation. Where this has occurred I have tried to explain what it means. I do not think that a reader who is familiar with the basic concepts of probability, mean, and covariance will find their understanding impaired.
Projective stereology in biological croscopy
Authors: Carothers Andrew D
Published: November 2023

Stereology can be defined as the three-dimensional interpretation of flat images, such as sections and projections, by criteria of geometric probability. Although the definition encompasses both sections and projections, the two approaches differ in both theoretical underpinning and practical application. Most standard theory is concerned almost exclusively with sectioned material. No doubt this is a result of the early historical development of the subject, which was largely driven by geological applications where the main requirement was to determine patterns of inclusions within solid rocks. Sectioning is necessary wherever there is a need to determine the internal composition of large, opaque objects, such as body organs. There is little point in attempting to reproduce here the many useful results from this body of theory. For these, the reader is referred to standard texts (see for example refs 1-3). In many other applications of biological microscopy, however, it is more relevant to consider plane projections since sectioning is invasive and may be technically difficult, and since objects of interest, such as subcellular elements, whole cells, and tissues, are often transparent or nearly so. Another, and perhaps the earliest, application of the stereology of projections was the inference by the ancient Greeks that the earth was spherical from the shape of its shadow on the moon during lunar eclipses.