bluuebunny/crossref_metadata_2025 huggingface dataset:
Baculovirus-mediated overexpression of transport proteins
Authors: Litherland* Gary J , Baldwintꝉ Stephen A
Published: November 2023

A major limitation to our understanding of the molecular mechanisms of membrane transport processes is the lack of detailed structural information on transport proteins, such as might be obtained using X-ray crystallography and other biophysical techniques. One reason for the paucity of structural studies on transporters using such techniques has been the difficulty in producing sufficient quantities (10-100 mg) of pure transport protein. The natural abundance of most transporters is low, and purification difficult. While many prokaryotic transporters have been successfully expressed at high levels in Escherichia coli (see Chapter 6), the use of bacteria to overexpress complex, often glycosylated or otherwise modified membrane proteins from eukaryotic sources has rarely been successful.
Choosing and characterizing antibodies
Authors: Forde* A , Coley† J
Published: November 2023

Choosing an antibody or antibodies is usually the most important preliminary to developing any immunoassay. The assay developer may have just raised antibodies for the particular application, or candidate antibodies may have been obtained from one of the many commercial and noncommercial organizations that produce antibodies. In either case, the best must be selected, or they all must be rejected and the search continued. An immunoassay is defined primarily by its analyte molecule, which can range in size from an intact cell such as a microorganism, to a natural antigen (which could be almost any size above about 2 kDa), to a specific antibody and to a peptide or small organic molecule.
Labels and endpoints
Authors: Abdul-Ahad* Wajdi , Brett† Martin
Published: November 2023

The sophistication of current immunoassay methodology allows the assay developer a wide choice of labelling substances and assay endpoints, so that it is not usually difficult to find a combination that well suits the specification required for a new assay. This chapter will deal primarily with. the three major kinds of labels and endpoints that are used when immunoassays for research purposes are being developed, i.e. radioisotopic (1), enzymatic (2) and fluorescent (3). Information on the preparation of ligand labels containing biotin and fluorescein (4) is also given.
Solid-phase reagents
Authors: Matson Robert S
Published: November 2023

This chapter is concerned with the preparation and use of solid-phase reagents in immunoassay. The merits in using a solid-phase, as well as the various approaches to preparing solid-phase reagents will be addressed. Information on the kinds of supports available will be given. Finally, detailed protocols for various formats (microtitre plates, membranes, beads, tubes) will be provided, along with sources of commercial solid-phase reagents.
Analysis of signal transduction pathways using protein—serine/threonine phosphatase inhibitors
Authors: Grahame Hardie D
Published: November 2023

A widely used approach for studying protein phosphorylation is the pharmacological one, i.e. to study the effects of cell-permeable inhibitors or activators of the enzymes which catalyse changes in phosphorylation. In Chapter 2 the use of protein kinase activators and inhibitors is discussed. Over the past 10 years, a number of potent, cell-permeable protein phosphatase inhibitors have also become available, and their impact has been considerable. Their applications may be summarized as follows: (a) Improving the specificity of protein phosphatase assays in cell-free extracts. This approach is discussed in Chapter 7, and the present chapter will concentrate instead on their uses in intact cell studies.(b) Provision of initial evidence that a physiological system is regulated by protein phosphorylation. This is particularly valuable in the case of a system where the major components are not well characterized, in which case more direct methods for the study of protein phosphorylation cannot be utilized.(c) In cases where the role of protein phosphorylation is already established, the inhibitors may in some cases be used to identify the protein phosphatase(s) active against particular substrates in intact cells.
Assay and purification of protein (serine/threonine) phosphatases
Authors: Mackintosh Carol , Moorhead Greg
Published: November 2023

The eukaryotic protein phosphatases comprise several families of enzymes that catalyse the dephosphorylation of intracellular phosphoproteins, thereby controlling diverse cellular functions in all eukaryotic cells. The activities of these enzymes are highly regulated in response to extracellular stimuli, and in many cases regulation is mediated by interactions of the catalytic subunits with diverse targetting and regulatory subunits and domains. The protein phosphatases responsible for dephosphorylation of serine and threonine (and histidine) residues in the cytoplasmic and nuclear compartments of eukaryotic cells are derived from two distinct gene families, PPP and PPM, that have been classified both by phylogenetic analysis of their sequences, and by their three-dimensional structures (reviewed in refs 1 and 2).
Analysis of protein kinase specificity using oriented peptide libraries
Authors: Songyang Zhou , Cantley Lewis C
Published: November 2023

This chapter focuses on the use of oriented peptide libraries to study the specificities of protein kinases. This approach not only predicts an optimal sequence from a single experiment, with no prior knowledge of in vivo phosphorylation sites required, but it also provides information about the relative importance of each position for selectivity, and about which amino acids are tolerated. In the following sections we will explain peptide library design strategies, detail the experimental techniques for peptide synthesis and selection, and discuss how the data obtained are interpreted.
Phosphatidylinositol 3-kinase signalling: a tale of two kinase activities
Authors: Coffer Paul J
Published: November 2023

Over the last 25 years considerable emphasis has been placed on the phosphorylation of proteins on tyrosine and serine/threonine residues as a mechanism of regulating cellular function. The importance of protein phosphorylation is highlighted in the other chapters of this book. However, there is another class of kinases that has recently taken centre stage, those kinases that phosphorylate phosphoinositide (Pl) lipids. In the original concept of membrane structure, it was thought that the membrane proteins were solely responsible for regulating membrane functions. More recently however, membrane lipids have been found to play not only a structural role, by isolating the intracellular environment from the extracellular, but also a critical role in the generation of second messengers. For example, the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate(Ptdlns(4,5)P2) to diacylglycerol(DAG) and inositol(l,4,5)P3 results in the release of Ca2+ from intracellular stores and the activation of protein kinase C (PKC). While the significance of Ptdlns(4,5)P2 hydrolysis has long been appreciated, the role of Pl-lipid phosphorylation is only now beginning to be fully understood. The reason for the relatively slow progress in this field of research is that, unlike protein kinases that were first cloned in the 1980s, the first PI-lipid kinase, phosphatidylinositol 3-kinase (Pl3K), was only cloned in 1992. This chapter will focus on recent discoveries concerning the structure and regulation of PI3K and its role in cellular functioning.
Genetic approaches to protein kinase function in lower eukaryotes
Authors: Plyte Simon E
Published: November 2023

The study of protein kinase function in higher eukaryotes such as Mammalia has been generally restricted to biochemical analysis of purified proteins, due to the limitations of genetic approaches in these species. Most of these enzymes were first identified biochemically and their genes isolated subsequent to peptide sequence analysis. However, the promiscuous nature of protein kinases in phosphorylating proteins in vitro has complicated study of their function in cells. In contrast, the tractability of lower eukaryotes to genetic analysis has resulted in a distinct approach in which genes involved in a given process are initially identified via their phenotypic effects. These genetic strategies, of course, do not specifically target protein kinase genes but this family of proteins has surfaced at a surprising frequency during analysis of regulatory genes, which is a testimony to their adaptability and ubiquity as biological tools. Once identified, the relative ease of genetic manipulation in lower eukaryotes enables the investigation of kinase function in vivo. Thus, mutational analysis can be used to determine whether a gene product is required in a particular pathway. Additionally, genetic epistatic experiments, between two genes in a particular pathway, may be applied to determine the order of action of those gene products. During the last decade, functional homologues of many mammalian protein kinases have been identified in lower eukaryotes, allowing a degree of extrapolation in the workings of the proteins in different species. In fact many of these kinases seem to function in an identical manner in lower eukaryotes and participate in evolutionary conserved kinase cascades, vindicating the use of lower eukaryotes as a model system in which to study protein kinase function.
Specificity in JAK-STAT signalling
Authors: Halupa Adrienne , Barber Dwayne L
Published: November 2023

Since the original identification of Janus kinases by Wilks et al. (l, 2) and the isolation of STATl and STAT2 by the Darnell laboratory, an entire new field of discovery has emerged and come to dominate research in cytokine signalling during the last decade. This wealth of information now provides us with some clues as to the function of these novel tyrosine kinases and transcription factors. To date, four Janus kinases (Fig. 1) and seven STAT (Fig. 2) transcription factors have been identified in mammalian species. With the exception of TYK2 and STAT2, gene-targeting experiments have been reported for all members of the JAK-STAT family. Drosophila expresses a Janus kinase homologue, called hopscotch, indicating that the JAK-STAT pathway is conserved throughout evolution. The STAT transcription factors are more widespread, having been isolated in Caenorhabditis elegans, Drosophila spp., and Dictyostelium spp.
Differential display analysis of alteration in gene expression
Authors: Cho Yong-Jing , Liang Peng
Published: November 2023

Temporal and spatial expression of the 100000 different genes in the genome of a mammal is a highly regulated process which determines the normal develop ment of an organism. The alterations in this process are therefore often the cause of developmental and pathological abnormalities such as cancer. Two-dimensional protein gel electrophoresis was developed for the purpose of comparative studies. However, the limitation of this method became evident mostly due to its sensitivity problem. as at most only up to 2000 of 10-15000 different types of protein expressed in any mammalian cell can be detected. Therefore, the majority of the genes, especially those expressed at lower levels, may not be represented. One other major pitfall of the method is the difficulty of being able to retrieve enough protein spots identified for future molecular characterizations, although there have been many recent technical advances in these areas (see Chapters 9 and 10 for further discussion of these issues).
Identification of differentially expressed genes by suppression subtractive hybridization
Authors: Desai Sejal , Hill Jason , Trelogan Stephanie , Diatchenko Luda , Siebert Paul D
Published: November 2023

SSH features several improvements over other cDNA subtraction methods that have been described in the literature (1-5). SSH eliminates any intermediate steps for physical separation of single-stranded (ss) and double-stranded (ds) cDNAs, requires only one round of subtractive hybridization, and can achieve greater than a 1000-fold enrichment for differentially expressed cDNAs (6-8). To overcome the problem of differences in mRNA abundance, SSH incorporates a hybridization step that normalizes (equalizes) sequence abundance by standard hybridization kinetics during the course of subtraction. This dramatically in creases the probability of obtaining low-abundance differentially expressed cDNAs and simplifies analysis of the subtracted library.
Gene expression analysis by cDNA microarrays
Authors: Mousses Spyro , Bittner Michael L , Chen Yidong , Dougherty Ed R , Baxevanis Andreas , Meltzer Paul S , Trent Jeffrey M
Published: November 2023

The Human Genome Project (1) has spurred the emergence of ‘genome-wide’ approaches to study gene function, regulation and interaction termed functional genomics. Comprehensive analysis of differential gene expression is becoming a cornerstone of functional genomics. To this end, several new technologies have been developed for high-throughput differential gene expression analysis (2-8). The deposition of cDNAs in an array format to filters or glass is a hybridization based analytical tool at the forefront of this field. The parallel gene expression of arrayed cDNAs on glass and subsequent analysis by means of two-colour fluor escence was pioneered in the laboratory of Dr P.O. Brown at Stanford. The first proof of principle paper showed gene expression analysis for an array with 45 Arabidopsis genes (2). This approach has since grown rapidly to high density microarrays with tens of thousands of genes. Today, many laboratories around the world have established cDNA microarrays for a range of applications and found it to be very useful for simultaneously profiling mRNA levels for thousands of genes.
Analysis of gene expression by two-dimensional gel electrophoresis
Authors: Tolkovsky A M
Published: November 2023

Two-dimensional (2-D) polyacrylamide gel electrophoresis using isoelectric focus ing (IEF) in the first dimension separates proteins by charge and size. Although simple in concept, the technique has been under-used for three main reasons: (i) the diffirulty in obtaining highly reproducible patterns of isoelectric focusing in the first (pH) dimension using tube gels and soluble carrier ampholytes; (ii) the lack of good analytical tools for analysis and assignation of differential patterns of gene expression; and (iii) inability to obtain sequencing information from other than the most abundant proteins. In the last few years, however, the practical and analytical tools have changed to such an extent that analysis of the proteome by 2-D gels can (and should) now be considered an accessible technique.
Bacteriology of the respiratory tract
Authors: O’Hara Steve , Lowes J Andrew , Smith Grace
Published: November 2023

The isolation and detection of potential pathogens of the respiratory tract in disease is often complicated by the presence of commensal flora in the upper tract. Unless specialized specimen collection techniques are used most respiratory tract specimens sent to a diagnostic laboratory are contaminated with commensal flora. Reporting of all isolates present with their antibiotic sensitivities is inappropriate and may be misleading. Microscopy and cultural findings must be interpreted taking into account the quality of the specimen, the expected commensal flora of a given site through which the specimen was collected, the relative amounts of potential pathogens, and the clinical history of the patient including antimicrobial treatment.
Bacteriology of superficial and deep tissue infection
Authors: Baker Nicola , Bushella Ann , Hawkey Peter M
Published: November 2023

The range and variety of clinical samples seen and microbes grown from these infections is enormous. The approach will be to outline general methods, concentrating on the major pathogens, and then to examine specific techniques appropriate to different sites of infection, with cross-reference to other chapters. To get the most useful results from these samples it is important to know the site (anatomical) and nature (primacy/secondary, acute/chronic) of the infection. If the sample is of high priority (pus, tissue, and some fluids) then the laboratories must liaise with the clinicians, and expend maximum effo1t to obtain a micro biological diagnosis. If the sample is of lower priority (most ‘wound swabs’ fall into this category) then the laboratory may legitimately process the sample in a routine manner. The samples that deserve extra effort are not always immediately apparent, and it is part of the role of the clinical microbiologist to pick them out. Finally, intelligent and informed interpretation of culture results and appropriate clinical liaison will make all the difference as far as the clinician (and patient) is concerned, between useful and useless laboratory reports.
Bacteriology of intestinal disease
Authors: Pedler Stephen , Graham Clive
Published: November 2023

There is a formidable list of bacteria which are known or suspected to cause gastrointestinal infection in human beings. This list is not unchanging; organ isms are sometimes added to it, while the status of other organisms varies with the passage of time. In the first edition of this book, for example, examination for enteropathogenic E. coli was recommended for an specimens from children aged three years or under, while current practice in many laboratories is not to examine specimens for these organisms routinely. Conversely, the criteria for examining specimens for Verocytotoxic E. coli have been expanded so that many more specimens are now routinely examined for this organism. Since the first edition antibiotic treatment has been increasingly recognized as useful in certain gastrointestinal (GI) infections, and with that the problem of antibiotic resistance in pathogenic bacteria has increased in importance.
Laboratory investigation of health care-associated infection
Authors: Hawkey Peter M , Kerr Kevin G
Published: November 2023

Whilst most hospital microbiology laboratories are very busy processing the large numbers of specimens detailed in the previous chapters, they will also be required periodically to investigate and direct the control of episodes of cross-infection. Although this chapter will concentrate on hospital-acquired infection (nosocomial infection), it is increasingly recognized that the infections previously regarded as typically nosocomial in origin, such as those caused by methicillin-resistant Staphylococcus aureus, may emerge, and be transmitted, in the community. Indeed the UK Department of Health Report Getting ahead of the curve (1) suggests that terms such as nosocomial infection are misleading and that ‘health care-associated’ infection is a more appropriate term.
Epidemiological methods in the investigation of acute bacterial infections
Authors: Palmer Stephen R
Published: November 2023

Epidemiology is the study of the occurrence and causes of disease in populations. In contrast to the clinical situation when an individual patient is the focus of concern, the epidemiological approach relates the disease in the individual to the occurrence or risk of disease in others within the relevant population. This means taking into account not only the microbiological, but all personal. environmental. and social factors which influence the occurrence and presentation of the disease. A well-defined methodology exists (1). which demands specialized skills, but every clinical microbiologist will need to be familiar with certain basic concepts and methods (2) and to take an epidemiological perspective from time to time. Examples include the investigation of sources and modes of transmission in outbreaks, applying public health measures to the control of outbreaks. preventing the spread of sporadic cases of certain infections (such as diphtheria, typhoid, meningococcal meningitis), and devising preventive strategies, for instance for nosocomial legionnaires’ disease.
Case study 3: Folding of influenza haemagglutinin in the living cell
Authors: Ellgaard Lars , Helenius Ari
Published: November 2023

Almost all the available information on protein folding has been obtained by analysing the refolding of denatured proteins in vitro in the presence or absence of added molecular chaperones. As described elsewhere in this book, studies using this approach are providing an ever more detailed picture of the biophysical principles underlying assisted and non-assisted folding of polypeptide chains.
Functions of carbohydrates revealed by transgenic technology
Authors: Stanley Pamela
Published: November 2023

Mammalian glycans are assembled as covalent attachments to protein or lipid by the sequential action of glycosyltransferases and processing glycosidases (see Chapter 1). The addition or removal of each sugar is a precise reaction catalyzed by an enzyme specific for the sugar and the linkage configuration. Therefore it is possible to block glycan synthesis, or to add an extraneous sugar to preformed glycan units, by, respectively, the inactivation or the induction of a glycosylation enzyme. This has been termed glycosylation engineering and has been used to define the functions of particular sugar residues or sugar determinants associated with glycoconjugates. Initially, this approach was implemented using somatic cell genetics. Both loss-of-function and gain-of-function glycosylation mutants have been isolated from cultured cells (1-4) and used in glycosylation engineering (5). Once glycosyltransferase genes were cloned, gain-of-function transfectants could be created to identify functions for sugars (6) and antisense constructs could be used to eliminate a glycosylating activity (7, 8). In the last few years these approaches have been applied to the mouse in order to study the consequences of overexpressing, misexpressing or eliminating a glycosylation enzyme in a complex mammalian organism. Mutant mice provide experimental models for known human diseases. They also may identify new bases for disease, as well as new steps in the synthesis and catabolism of mammalian glycans. This chapter will discuss the properties of transgenic and knockout mice with altered glycosylation, identify difficulties of interpretation at the mechanistic level, and discuss future directions and methods for obtaining mouse glycosylation mutants.
Roles of sphingolipids and glycosphingolipids in biological membranes
Authors: Hirabayashi Yoshio , Ichikawa Shinichi
Published: November 2023

In 1884, Thudichum showed that the sphingolipids, galactosylceramide and sphingomyelin, were the major lipid components of human brain. Since then, the chemical structures of these lipids have been extensively studied and many different species of sphingolipid have been identified. In mammalian cells, there are two major classes of sphingolipid: glycosphingolipids (GSLs) and sphingomyelin, both of which contain hydrophobic ceramide. GSLs, in which oligosaccharides are the hydrophilic component, are generally minor components of cell membranes in animals and plants, while sphingomyelin, in which phosphocholine is the hydrophilic component, is more abundant in membranes. The ceramide portion of these sphingolipids is embedded in the outer leaflet of the plasma membrane and the sugar moiety or phosphocholine group projects into the extracellular space. A characteristic feature of GSLs is their structural diversity, in both the ceramide and the oligosaccharide chain. GSLs with sugar chains containing sialic acid are termed gangliosides.
Viral transformation of lymphocytes
Authors: Meinl Edgar , Fickenscher Helmut
Published: November 2023

The main limitations of molecular and biochemical studies of human T lymphocytes are twofold. First, the phenotype of T-lymphoblastic tumour cell lines like Jurkat (1) is significantly altered in comparison to primary cells with respect to signal transduction (2) and gene regulation. Second, primary T-cell cultures are inevitably limited in their lifespan. It is often laborious and frequently impossible to grow primary T lymphocytes to large numbers, and it requires considerable effort to amplify antigen-specific T cells (as described in Chapter 10). The use of irradiated feeder cells may cause difficulties in the interpretation of results derived from T cells expanded in this way. Immortalization of human T cells should be the ideal way to solve such problems. T-cell transformation should ideally lead to permanent growth independent of restimulation procedures, whilst conserving the essential features of the primary, parental T cells. Various approaches for immortalizing human T cells have been tried. The technique of T-cell fusion hybridomas is successful for rodent cells, but is much more difficult to achieve with human T cells where it is hampered by the genomic instability of the clones (as discussed in Chapter 4).
Murine T-cell culture
Authors: Mills Kingston h. G
Published: November 2023

The development of techniques for propagating and cloning T cells has had a dramatic impact on the field of immunology. Indeed, the study of cellular immunology, and many aspects of molecular immunology, are now almost totally dependent on cell-culture techniques. T cells cultured from blood, spleen, lymph nodes, or other lymphoid organs have been successfully used to establish that CD4+ and CD8+ T-cell responses recognize antigen in association with major histocompatibility complex (MHC) class I or class II gene products (1, 2) and that functionally distinct T-cell subtypes can be discriminated on the basis of cytokine secretion (3). The ability to clone antigen-specific T cells has also allowed the definitive identification and functional analysis of distinct T-cell subpopulations, and has provided the ideal tools for studies on the mechanisms of antigen processing, presentation, and T-cell receptor interaction with peptide-MHC complexes. Furthermore, the study of T-cell subtypes in vitro has provided valuable information for manipulating the induction and function of these cells in vivo, and this has important implications for future developments in immunotherapy and vaccines (4).
Quality assurance in the clinical laboratory
Authors: Gratama Jan W , Barnettt David
Published: November 2023

Each clinical diagnostic laboratory must have a strategy to assure the quality of its products. The focus in this chapter is on flow cytometric immunophenotyping assays; cases in point are:

for quantitative assays:

(a) enumeration of lymphocyte subsets, and

(b) enumeration of CD34+ haematopoietic progenitor cells (HPC); and for qualitative assays:

(c) leukaemia/lymphoma immunophenotyping, and

(d) screening for the HLA-B27 antigen. Quality assurance strategy includes:
Analysis of cell proliferation using the bromodeoxyuridine/Hoechst ethidium bromide method
Authors: Ormerod Michael G , Poott Martin
Published: November 2023

The fluorescence of bis-benzimidazole dyes (Hoechst 33258 and Hoechst 33342) bound to DNA is quenched by the thymidine analogue, 5-bromodeoxyuridine (BrdUrd). Consequently, continuous labelling with BrdUrd and subsequent staining of DNA with Hoechst 33258 allows the discrimination of chromatids accord ing to the number ofreplications they underwent during the observation period. The potential of this technique for analysing the progression of cells through their cycle has been established in several laboratories (see, for example, refs 1 and 2 and references therein). Cell-cycle kinetics are analysed using continuous BrdUrd labelling and bivariate Hoechst 33258/ethidium bromide (EB) flow cytometry. By the quenching of fluorescence from the Hoechst dye, cells are separated according to how many phases of DNA replication they have traversed, whereas the fluorescence of the non-quenched EB resolves the cell cycle into the G1, S, and G2 compartment. In this chapter, we refer to Hoechst/EB but propidium iodide (PI) can also be used in place of EB. Both dyes have been used to prepare the data shown in the figures.
Genetic analysis of ascomycete fungi
Authors: Harris Steven D
Published: November 2023

Filamentous fungi, in particular Aspergillus nidulans and Neurospora crassa, have historically played an important, yet under-appreciated, role as model eukaryotic organisms (1, 2). In this context, the primary attribute of these fungi is the ease with which they can be manipulated using both classical and molecular genetic techniques. Because they are haploid, the generation and analysis of mutants is a relatively straightforward process in A. nidulans and N. crassa. A variety of approaches can be used to generate mutations; ranging from traditional chemical mutagenesis to more recent transformation-based strategies. Similarly, an unlimited array of screening protocols can be used to select the desired mutant. Once obtained, a mutant must be subject to rigorous characterization to determine if the relevant phenotype is caused by one or more mutations, and if the mutation is recessive or dominant. The protocols employed to answer these questions in the filamentous fungi have remained essentially unchanged over the past forty years. The purpose of this review is to summarize the approaches used for genetic analysis in the ascomycete fungi, with particular emphasis on A. nidulans, N. crassa, and the plant pathogen Magnaporthe grisea. The first section will cover strategies used to identify and isolate mutants, whereas the second section will summarize the protocols employed for the analysis of a mutant.
Meiotic studies in humans
Authors: Hulten M A , Barlow A L , Tease C
Published: November 2023

The investigation of meiotic chromosomes presents a special technical challenge. First, the material is difficult to obtain for ethical and practical reasons. Second, meiotic chromosomes undergo a highly complex pattern of behaviour and it is essential to have a clear understanding of this process before undertaking analysis of scarce human material.

Investigation of meiosis may be undertaken either for basic research purposes or as an aid to a clinical diagnosis. Study of germ cell chromosome behaviour is of particular relevance to reproductive medicine. Errors in the cell division and maturation processes that generate human eggs and spermatozoa often result in impaired fertility. Through analysis of germ cells, it may be possible to determine the underlying causes of infertility and to obtain information of considerable value to subsequent counselling of patients. The increasing use and importance of in vitro fertilization and intracytoplasmic sperm injection as approaches to overcome infertility enhance the desirability of data from meiotic cells.
Basic techniques for the preparation and analysis of chromosomes from bone marrow and leukaemic blood
Authors: Czepulkowski B
Published: November 2023

This chapter is the first of five covering all aspects of the cytogenetic study of the leukacmias and related disorders. It serves to provide basic and general infom1ation on the culture, staining and analysis of the tissues normally used for such studies, namely bone marrow and peripheral blood. Protocols for the use of other sample types such as lymph node and solid tumour tissue, and further techniques specific to particular disease types are provided in subsequent chapters.
Cytogenetics in acute lymphoblastic leukaemia
Authors: Czepulkowski B , Gibbons B
Published: November 2023

In the past, cytogrnctic analysis of ALL has been lmnpcrcd by the difficulty in obtaining good quality chromo omes for c1.naly i . Poor spreading, fuzzy chromosomes and indistinct bands me, mt that only about 50% of cases analysed were found to have an abnormal clone, or because of metc1phases that could not be analysed, a number of case were cla,sified as failures. Improvements in cytogcnetic techniques have increased the success rate and abnormality rates observed in ALL. and also given an insight into the importance of cytogenetic abnorm. ilities in the pathophysiology c1nd prognosis of haemato logical malignancies, including ALL ,\bnormalities were detected in only about halt of all ALL patients in initial banding studies (1) but now most centres report chromosomal abnormalities in 60-85% of ALL cases (2-6). In cases with a normal katyotype or in failed cases, interphase FISII provides an additional technique by which chromosome abnormalities of prognostic importance can be identified. Such cases are now routinely screened with panels of probe to detect cases of high hyperdiploidy.
Chaperones in signal transduction
Authors: Smith David F
Published: November 2023

Hsp90, typically the most abundant chaperone in the cytoplasmic compartment of cells, has gained a reputation for its functional interactions with a wide variety of signalling proteins (for recent reviews see 1, 2–:4). Examples include the steroid receptors and several transcription factors unrelated to steroid receptors, a variety of tyrosine and serine/ threonine kinases, and other regulatory enzymes such as telomerase (5) and nitric oxide synthase (6–:8). In vitro refolding assays with model substrates have shown that Hsp90 on its own has the general ability to stabilize proteins in a folding competent state (9–:11), but there is limited evidence to support a major role for Hsp90 in general nascent chain folding. Still, unstable or alternative folding appears to be a common feature among the signalling proteins that are native targets for Hsp90. Another striking feature of native Hsp90 complexes is the in-variable presence of some combination of partner co-chaperones that bind Hsp90.
Molecular chaperone systems in the endoplasmic reticulum
Authors: Pelletier Marc F , Bergeron John J M , Thomas David Y
Published: November 2023

The endoplasmic reticulum (ER) is an organelle defined microscopically as a network of saccular and tubular membranes in continuity with the nuclear envelope. This definition can be further refined into a distinction between the rough endoplasmic reticulum to which ribosomes are attached, and the smooth endoplasmic reticulum which consists of a network of ribosome-free tubules and is the site of exit of secretory cargo vesicles. The ER can also be defined functionally as the site of lipid metabolism, carbohydrate metabolism, drug detoxification, and the entry point for proteins into the secretory pathway. Proteins that are secreted from cells, and proteins which are destined for cellular membranes, are synthesized on the ribosomes of the rough endoplasmic reticulum and translocated into the lumen of the ER, where they are glycosylated, become folded, disulfide bonds are formed, and some multiprotein complexes are assembled. Before proteins are transported out of the ER they are subjected to a ‘quality control’ process which assesses their state of folding and then, if they are correctly folded, allows them to exit the ER by packaging into transport vesicles. Proteins that are not folded correctly can have several fates. They are usually retained in the ER for further cycles of the quality control process or they are retrotranslocated out of the ER and degraded in the cytosol by the proteasomal system, or they can accumulate in the ER as aggregates.
The function of chaperones and proteases in protein quality control and intracellular protein degradation
Authors: Maurizi Michael R
Published: November 2023

During the last few years we have witnessed a major revolution in our understanding of the importance and the mechanism of intracellular protein degradation. We have come to appreciate that highly selective protein degradation occurs in all organisms and is a component of many essential regulatory pathways. We have also learned that protein degradation plays an important part in maintaining protein quality control within the cell. Most excitingly, we now have a grasp of the basic mechanism of ATP-dependent protein degradation and have begun to develop an understanding of how cells protect their proteins from inadvertent destruction while assuring that appropriate proteins are efficiently targeted for degradation. Studies of the structure and mechanism of the ATP-dependent proteases responsible for the majority of intracellular protein degradation have revealed unanticipated parallels between the proteolytic machinery of cells and the molecular chaperone systems responsible for the folding of cellular proteins and the assembly or disassembly of macromolecular complexes. This progress has led to a unified model of protein degradation and protein quality control, in which protein surveillance and structural remodelling mediated by chaperone systems and the chaperone components of ATPdependent proteases serve as the keystone.
Patterning the Xenopus Embryo
Authors: Smith J C , White R
Published: November 2023

The problem we wish to address concerns the mechanism by which the vertebrate early gastrula forms from the fertilized egg, with the right genes activated in the right places and with cells poised to undergo the movements of gastrulation. We will concentrate on the amphibian embryo, where the detailed molecular events of early embryogenesis are probably better understood than in any other vertebrate. This understanding is due to what might be regarded as the ‘traditional’ advantages of the amphibian embryo: its large size, the ease of grafting, the ability to culture cells in a simple salt solution, and the ease with which molecules can be overexpressed by RNA or DNA injection. These advantages have recently been supplemented by the introduction of transgenesis in Xenopus (by Amaya and Kroll (1, 2)), allowing, in principle, genes to be expressed in virtually any tissue of interest.
Anteroposterior Regionalization of the Vertebrate Nervous System
Authors: Graham Anthony , Mason Ivor
Published: November 2023

The complex vertebrate nervous system is constructed in a stepwise fashion during development. Specification of the neural territory at the dorsal surface of the embryo occurs early in development: at the gastrula stage (see Chapter 5). The subdivision of the neural primordium into the four broad territories, which prefigure the gross anatomy of the adult brain (forebrain, midbrain, hindbrain, and spinal cord) occurs both concomitant with and following neural induction. As embryogenesis proceeds, these domains are further partitioned into smaller units, which define the placement of neuronal populations and pioneering of the first axonal pathways. Thereby, the ground plan of the brain’s neuronal architecture is established. Thus these early patterning events are critical to the development of the adult nervous system, as they provide the scaffolding upon which the elaborate cytoarchitecture of the brain and spinal cord is realized. In this chapter we discuss the current view of how the nervous system becomes patterned along its long axis (anteroposterior axis). Such a review is timely as we are now beginning to understand both the developmental processes and the molecular effectors that underlie the anteroposterior regionalization of the nervous system.
Axon guidance in the Developing Vertebrate Nervous System
Authors: Guthrie Sarah
Published: November 2023

During development, neurons originate in the inner, proliferative ventricular zone of the neural tube and then move away to form the outer mantle zone. Both progenitor cells and neurons may migrate radially or tangentially with respect to the developing neuroepithelium. Neuronal migrations occur over a protracted period and take place in a complex landscape containing many cells which have already differentiated and extended axons (1). As a general rule, large projection neurons are generated first, and smaller interneurons and glial cells are produced later. Neuronal migration is an active process, which results in neurons taking up their correct positions within laminar or nuclear structures (2). Axonogenesis often begins during this migration so that the processes of migration, differentiation, and axonogenesis are intimately linked. Thus the processes of migration and axon extension are regulated coordinately and are likely to depend on the same, or similar, receptor-ligand systems.
Evolutionary Aspects of Vertebrate Patterning
Authors: Shimeld Sebastian M
Published: November 2023

In the later half of the nineteenth century and early decades of the twentieth century, embryology and evolutionary biology where intricately linked, as the work of scientists such as Haeckel, von Baer, and Garstang testifies. However, during the early twentieth century evolutionary embryology essentially reached a technical boundary and, since the New Synthesis, when Mendelian genetics were unified with Darwinian natural selection, evolutionary biology has been largely quantitative. Principal themes have been the study of variation in natural populations, the effect of selection, and the establishment of phylogenies of living and extinct animals using molecular and morphological systematics. These studies essentially focus on diversity. Conversely, the model system approach of modern developmental biology has emphasized conservation, i.e. the similarity of developmental mechanisms between taxa. Modern evolutionary developmental biology attempts to reunite these two areas, using modern molecular and developmental techniques to investigate questions that were often first posed over 100 years ago.
Live Now, Pay Later
Authors: Hamilton W D
Published: November 2023

IT seems that Stephen Daedalus was either a poet too much or too little: he did not reply and presently watched as his friend Cranly chased ‘gipsy’ Temple away. For my part I sympathized with Temple most among all Joyce’s students in A Portrait of the Artist as a Young Man; he seemed more scientific than the rest. From the time I first read it I suspected his idea could well be profound. Later, combining some thoughts from R. A. Fisher, P. B. Medawar, and G. C. Williams along with new ones of my own, I became convinced of it as the paper in this chapter shows.
Citation analysis and mapping of genetics research in Iran
Authors: Sedghi Shahram , Ghaffari Heshajin Somayeh
Published: November 2023

Purpose

Genetics, a discipline of biology, is one of the most recent and rapidly advancing disciplines in science. This study aims to present a bibliometric analysis of the genetics research output of Iranian authors, map the intellectual structure of these studies and investigate the development path of this literature and the interrelationships among the main topics.

Design/methodology/approach

This study searched the Web of Science database for documentation of Iranian-published genetics research published up to 2020. Further, this study used HistCite software to profile and analyze the most cited articles and references and to draw their historiographies.

Findings

A database search revealed 21,329 documents that created the study population. The highest cited publications based on the Global Citation Score (GCS) and Local Citation Score (LCS) achieved scores of 602 and 47, respectively. The publication growth rate study demonstrated consistent expansion over time. The scientific maps based on LCS and GCS had five and four clusters, respectively. Furthermore, journal articles emerged as the predominant type of publication.

Practical implications

The significance of this study is in its contribution to understanding the genetics research position in Iran, informing policymakers and researchers, helping scientific collaboration and its impact on public attitudes and quality of life. The results of the present study, with benefits for various groups of communities, such as policymakers, academic groups and public society, can bridge the gap between theoretical research and practical implications.

Social implications

The results of this study, by helping future advancement in health care, medical genetics and disease prevention, may have a direct and indirect positive influence on the quality of life. Furthermore, it may lead to more informed discussions on health care and biotechnology as well as influencing public attitudes and perceptions.

Originality/value

Ultimately, this study concludes that despite the proliferation of publications in terms of quantity and complexity, especially in areas such as disease diagnosis, prevention and treatment, there remains a need for more attention to other facets of genetics such as biology and biotechnology. Iranian publications are most related to population genetics, human genetics, molecular genetics, medical genetics, genomics, developmental genetics and evolutionary genetics out of 10 branches of genetics. This study reveals patterns in scientific outputs and authorship collaborations and plays an alternative and innovative role in revealing Iranian research trends in genetics.
Measuring fiscal stress via fiscal vulnerability index: a case of Turkish economy
Authors: Yildirim Yusuf
Published: November 2023

Purpose

This paper aims to develop a compound measure, which is fiscal vulnerability index, provides early warning signals of fiscal sustainability problems for Türkiye's economy.

Design/methodology/approach

The index is constructed using twelve distinct fiscal indicators and applying the portfolio method, which considers the time-varying cross-correlation structure between the subindices.

Findings

Dynamics of the fiscal vulnerability index indicate that it accurately predicts to the well-known fiscal crisis occurring in Türkiye's recent history. As a result, such a compound measure should be used in the early identification of fiscal vulnerability in Türkiye.

Originality/value

The main contribution of this paper, relative to existing papers, is that a fiscal vulnerability index was constructed by employing the most contemporaneous method and evaluating its performance in terms of capturing historical stress periods.
Research on the production, application and management of virtual reality in the National Palace Museum
Authors: Wu Shao-Chun , Lin James Quo-Ping
Published: November 2023

Purpose

Virtual reality (VR) can be used as an alternative mean for viewing collections at home when it is not possible to visit museums due to COVID-19. This study took the development process of VR at Taiwan's National Palace Museum (NPM) as a case to discuss the characteristics of VR developed there in different periods and how NPM transforms the contents of its collections into VR.

Design/methodology/approach

This study used a case study to analyze the development process of VR at NPM from 2014 to 2019 and summarized the characteristics of the development and application of VR.

Findings

The authors find that the history of VR application in NPM is a process from exploring the technology to gradually getting familiar with the potential of its application. Its development can be divided into the exploration and experiment stage from 2014 to 2015, the single collection interpretation stage in 2016 and the multipurpose application stage from 2017 to 2019. It is suggested that museums should adopt a long-term strategy to introduce VR, make plans carefully and pay attention to the limitations of VR application.

Research limitations/implications

The results of this study are suitable for art and history museums.

Originality/value

Many research studies on the application of VR in museums mostly focused on the benefits and technologies of adopting VR in museums as well as specific museum VR projects. There is still scant literature on the development process of museum VR from the perspective of museum organizations.