bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of beer proteins which affect foam stability
Authors: Mohan S B , O’Shaughnessy C , Shuttleworth G , Lyddiatt A
Published: November 2023

The presence of a stable head of foam is a major factor in assessing beer quality. Characteristics of components which concentrate in the liquid film of foam bubbles, produced by normal beer dispense, were therefore investigated. Three foam components, depicting drainage over 30 min, prepared from bitters, lagers and stouts were further fractionated by gel permeation on Superose 12. Immunoblotting of the isolated components, using an antiserum raised against foam produced from a commercially available lager, showed that similar proteins might be involved in the foam stability of the beers tested. The data obtained for their molecular characteristics and foaming potential are described.
Antibodies as tools for the study of the structure and function of channel proteins
Authors: Anne Stephenson F
Published: November 2023

The exquisite specificity of antibody molecules combined with the ease with which they can be labelled makes them valuable in the study of the structure and function of proteins. Historically, the generation of an antibody against a defined protein required the purification to homogeneity of high concentrations of the antigen followed by animal immunization and the production of polyclonal antibodies. The specificity of these antibodies was determined by the major antigenic regions of the immunogen. In general, channel proteins are not easily isolated in the amounts required for polyclonal antibody production. However, the advent of monoclonal antibody technology has circumvented the need for a pure antigen. There was a requirement instead for a rapid, selective screening method for the production of antibodies against those proteins expressed at low abundance. The specificity of these antibodies was again dictated, for the most part, by the antigenic regions of the protein of interest. Today, the availability of cDNAs encoding channel polypeptides means that the natural proteins themselves are no longer required for antibody production, since recombinant or synthetic antigens can be employed instead. In many cases, the specificities of the antibodies thus produced are predetermined, which means that they are even more powerful research tools.
Septal neurons
Authors: Svendsen Clive
Published: November 2023

Interest in the septum stems from numerous studies showing that cholinergic neurons in this, and other regions of the basal forebrain appear to degenerate in Alzheimer’s disease (AD). These express both low and high affinity receptors for nerve growth factor (NGF), and project to allo and neocortical regions which show pathological changes in AD. As in vivo studies have shown that NGF can reduce cholinergic cell death in the septum induced by axotomy of the rat fimbria/fornix (1), the prospect of using this factor to prevent these cells dying in AD has been suggested (2,3) and is now undergoing preliminary clinical trials.
Schwann cell culture
Authors: Morgan Louise
Published: November 2023

Schwann cells support neuronal function in the intact nerve by supplying the myelin sheath around larger axons. Schwann cells are likely to also regulate the ionic environment of axons, provide neurotrophic support, and regulate the periaxonal space. During development and after nerve injury, Schwann cells provide increased neurotrophic support for neurons both as a source of soluble neurotrophic factors and by providing a growth promoting substrate for axonal regrowth by synthesis of permissive molecules both on the cell surface and secreted into the extracellular matrix (1).

Cultures of Schwann cells facilitate analysis of both normal Schwann cell function and of disease where the primary effect is on Schwann cell function. Diseases in this category include neurofibromatosis, associated with loss of Schwann cell growth control, the hereditary peripheral neuropathy Charcot Marie-Tooth disease, and the immune-mediated polyneuropathy Guillan Barre syndrome, both of which are demyelinating diseases, and leprosy which is associated with loss of function by both non-myelin and myelin-forming Schwann cells (2).
Integrase
Authors: Craigie R , Hickman A B , Engelman A
Published: November 2023

The linear DNA copy of the HIV genome made by reverse transcription must be integrated into a chromosome of the infected cell in order to be stably maintained and efficiently transcribed. DNA integration is therefore a necessary step in the viral replication cycle. HIV DNA is inserted into the host genome by a specialized DNA recombination reaction in which the viral integrase protein is the key player. The mechanism of this reaction is similar to that used by many transposable elements to move from one location to another in DNA, but is dissimilar to any reaction known to be involved in the normal functioning of the host cell. The specialized nature of the HIV DNA integration reaction suggests that it may be a good target in the search for specific inhibitors that block viral replication without disrupting normal cellular processes.
HIV envelope glycoprotein/CD4 interactions: studies using recombinant vaccinia virus vectors
Authors: Berger E A , Nussbaum O , Broder Mdc.C
Published: November 2023

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immune deficiency syndrome (AIDS), the mechanism of HIV entry into target cells has been a major focus of scientific investigation (see ref. 1 for review). It is difficult to overstate the importance of the fortuitous fact that HIV ‘chose’ as its receptor the cell surface marker CD4. This glycoprotein, present on a subset of T cells as well as on monocyte/ macrophages, was already under intense scrutiny for its role in T cell function. The isolation and sequencing of the cDNA encoding human CD4 (2), the availability of a battery of anti-CD4 monoclonal antibodies (3), and the molecular cloning of numerous diverse HIV isolates (4) allowed rapid progress to be made in elucidating structural features of the binding interaction between CD4 and gp120, the external envelope glycoprotein (Env) subunit.
Cellular assays for antiviral drugs
Authors: Mahmood N
Published: November 2023

The detailed understanding of the growth of HIV, reflected by the chapters in this book has enabled chemists to produce compounds targeted at specific sites in the HIV replicative cycle. HIV infects human T lymphocytes, monocytes/macrophages, and nerve cells both in AIDS patients and in vitro. Compounds have been shown to exhibit differences in antiviral activity and cytotoxicity depending on the cell type studied. It is therefore necessary to evaluate prospective anti-HIV drugs in multiple cell lines of different origins.
Microbore and packed capillary HPLC
Authors: Oliver Ronald W A , King Brian W
Published: November 2023

At the present time most analysts using HPLC routinely employ columns of support material packed in stainless-steel tubing varying in length from 10 to 25 cm and with a bore between 3.9 and 8.00 mm. The most commonly used columns are the so-called analytical columns which have a bore of 4.6 mm. However, it has been demonstrated by many workers (1-4) that it is possible to achieve similar separations using columns of the same length but reduced bore. Clearly, a reduced-bore column requires a smaller quantity of support material to fill it, and the amount of sample which can be loaded on top of it is also smaller because of its reduced area compared with that of the analytical column. In addition, because the reduced columns are eluted at lower flow rates, smaller volumes of eluent will be consumed. Because of these clearly advantageous effects we believe that many analysts are interested in learning how to convert their analytical HPLC system into a reduced-bore system, and therefore the primary aim of this chapter is to explain how this may be done. In order to achieve this aim, the effect of reducing the size of the column bore on the operating parameters, and hence design of each of the other components of a HPLC system, will be discussed in turn in the following theoretical section. Before doing so, it should be noted in passing that when such column miniaturization was first mooted a number of practical difficulties were encountered, thus it proved difficult to pack stable, chromatographically efficient, reduced-bore columns suitable for routine use. In addition, the necessary low flow-rate pumps, low-volume injectors, and low volume, UV-detector flowthrough cells were not commercially available so that the separation efficiencies of the early "homemade’ systems were often poor. In spite of these difficulties, interest in reduced-bore columns continued primarily because of their ability to effect the analysis of much smaller quantities of sample such as that present in, say, unique historical specimens (5) or lysates of a single cell (6).
Size-exclusion, high-performance liquid chromatography of proteins
Authors: Welling Gjalt W , Welling-Wester Sytske
Published: November 2023

Since the introduction of Sephadex in 1959 (1) size-exclusion chromatography (also termed gel filtration, gel permeation, or molecular sieving) has become exceedingly popular in the field of protein purification. The high-performance mode of this type of chromatography became operational in 1980 after the development of uniform rigid particles where the uniform pores were sufficiently large to be entered by protein molecules.

Separation by size-exclusion (SE)-HPLC depends on differences in the size, more precisely the hydrodynamic volume, of the proteins in a sample. The larger molecules do not enter the pores of the column particles and are excluded. They elute in the void volume of the column ( V0). The pores of a column particle are differentially accessible to smaller particles, depending on their size. This volume of the column is called V;. The total accessible volume (V1) is the sum of the volume outside the particles (V0) and the volume accessible inside the particles: Vt= Vo+ Vi.
HPLC of oligosaccharides and glycopeptides
Authors: Michalski Jean-Claude , Alonso Catherine
Published: November 2023

Protein glycosylation is a major modification which occurs after translation. Carbohydrate chains (glycans) may be found linked to proteins in different ways with respect to the kind of amino acids that are modified. Commonly, carbohydrate chains may be found 0-linked to serine or threonine residues, or N-linked to proteins at Asn-X-Ser (Thr) sequons (1, 2). Glycans present a great chemical diversity which results in the ability of monosaccharides to combine with each other in a variety of ways differing in sequence, anomery(o: or 13) position of linkages, branching points, and chain length. Additive substitution by sulfate. phosphate, acetyl, or methyl groups of the different monosaccharides considerably increase the structural diversity. There are four major classes of N-linked glycans: oligomannose type (high-mannose type), so-called N-acety1lactosaminic type (complex type), polylactosaminic type and hybrid type (3) (Figure I). O-glycans present a greater diversity and are generally subdivided into different classes according to common cores (4) (Figure 2).
Equipment and automation; Appendix on dose-response curve fitting
Authors: Howes Iain , Edwards Raymond
Published: November 2023

The basic aspects of automation have been reviewed together with various degrees of automation and some practical applications. It has been impractical to consider all aspects of automation. Automation refers to tasks carried out by mechanical or electrical devices (essentially machines) assisting in the execution of part or all of the analytical technique.
Production of human monoclonal antibodies from B lymphocytes
Authors: Melamed M D , Sutherland J
Published: November 2023

The discovery in 1975 by Kohler and Milstein (1) that murine antibodies of pre-defined specificity could be produced in monoclonal culture unleashed an explosion of activity in almost all fields of biomedical research. However, the transfer of this technology to the production of human monoclonal antibodies has proved to be a slow and difficult process. The essential event in the creation of a monoclonal antibody by the Kohler-Milstein approach is the fusion of a spleen B cell of appropriate specificity from an immunized mouse with a myeloma or plasmacytoma cell adapted for indefinite growth in culture, to create productive hybridomas.
Immunotoxins
Authors: Derbyshire Elaine J , Gottstein, Claudia , Thorpe Philip E
Published: November 2023

Immunotoxins (ITs) are cell type-specific cytotoxic agents consisting of an antibody linked to a protein toxin. The antibody component is responsible for directing the toxin selectively to the target cell and the toxin component is responsible for killing the cell (for reviews on ITs see refs 1—5). Because of their potency and selectivity, ITs have several clinical applications. First, ITs have been employed ex vivo to kill residual tumour cells in autologous bone marrow grafts, or T cells in allogenic bone marrow grafts for the prevention of graft-versus-host disease (6,7). Secondly, they have been used systematically to treat leukaemias, lymphomas, and solid tumours (8). Thirdly, they have been used to treat solid tumours by intracavity administration (8). They have also been evaluated for the treatment of autoimmune diseases like psoriasis (9).
Confocal Laser Scanning Microscopy
Authors: Johnson G D
Published: November 2023

The development of confocal laser scanning microscopy (CLSM) has considerably enhanced the study of biological specimens stained by immunofluorescence (IF) which continues to be the preferred method of immunolabelling in a wide range of applications. These include rapid diagnosis of infection, autoantibody screening, and cell surface (FACS) analysis of leukaemic cells, in addition to the applications in the research field where the unique properties of the method are highly valued. The increasing availability of potent alternative fluorochromes having characteristic spectral features (1) enables different structures to be recognized and analysed for co-localization at appropriate excitation/emission wavelengths-a property not available by other methods of immunostaining, e.g. immunoenzyme staining where minor staining may be masked by coincident strong reactivity.
Chemical cross-linking and protein function
Authors: Kluger Ronald
Published: November 2023

The functional properties of most proteins are a function of their primary structure. which determines their tertiary and quaternary structures (t). Chemical modification of site within the protein will often produce changes in the protein· function. Relating changes in function due to modification was one of the earliest approaches to elucidating the basis of protein function (2). The effective use of chemical reagents to alter proteins required parallel development in organic chemistry and p role in chemistry. Reagents for particular amino acid residues (sec Chapter 10) and affinity labels (see Chapter 6) appeared to combine versatility and specificity (2).
Convergent themes in X chromosome inactivation and autosomal imprinting
Authors: Brockdorff Neil
Published: November 2023

X chromosome inactivation (X inactivation) is the genetic silencing of a single X chromosome in XX female mammals, providing the mechanism whereby X-linked gene dosage is equalized relative to XY males (reviewed in ref. 1). X inactivation occurs early in female embryogenesis and there is normally an equal probability that either the maternally-derived (Xm), or paternally-derived (XP) X chromosome will be inactivated. The stochastic nature of X inactivation gives rise to the classical variegated phenotypes observed in females heterozygous for X-linked coat colour mutations, for example the tortoiseshell cat. The pattern of variegation reflects the fact that the initiation of X inactivation occurs in early embryogenesis, and that the inactive state is clonally inherited in subsequent cell divisions. In certain instances X inactivation is non-random. Thus, for example, XP is preferentially inactivated in marsupial species, and also in restricted extra-embryonic lineages in mouse.
Multilocus and single-locus DNA fingerprinting
Authors: Bruford Michael W , hanotte olivier , Brookfield, john F Y , burke Terry
Published: November 2023

Over the last ten years DNA fingerprinting has been and continues to be applied in many diverse areas of biological science, and has now become a standard technique for rapidly screening genetic variation in animals, plants, and other eukaryotes. This chapter outlines the techniques of both multilocus and single-locus DNA fingerprinting using minisatellite sequences and out lines the various uses of these techniques in population genetics, evolutionary studies, and behavioural ecology.
Kinetic analysis of the interaction between an analyte in solution and an immobilized protein
Authors: Karlsson R , Löfås S
Published: November 2023

Proteins function by interacting with other biomolecules. On a molecular basis the interaction can be characterized in terms of the affinity and the kinetics of the interaction. The affinity is often easy to measure (1) and describes the strength of the interaction. Kinetic analysis is more informative than affinity analysis and gives additional data regarding the rate of complex formation and dissociation. When kinetic analysis is possible, the binding assay is facilitated and fewer experiments can be performed. This is illustrated in Figure 1. Here data obtained using a traditional solid phase assay (i.e. an ELISA) and a biosensor-based kinetic assay is compared. When a single concentration of analyte is used a traditional assay gives one data point that in itself is almost meaningless. In contrast a biosensor assay can provide direct and continuous information on how the analyte binds to and dissociates from the immobilized ligand. In theory a single binding curve can be used to determine both the association rate constant, ka, and the dissociation rate constant, kd.. The affinity constant KA can then be calculated from the ratio between ka and kd.
Peptide mapping
Authors: Andrews Anthony T
Published: November 2023

Current versions of one-dimensional techniques such as polyacrylamide gel electrophoresis (PAGE), polyacrylamide gel electrophoresis in the presence of detergents such as sodium dodecyl sulfate (SDS-PAGE), immuno electrophoresis (IE), and isoelectric focusing in polyacrylamide gels (IEF) have very high resolution, are reproducible, reliable, rapid, and relatively inexpensive and simple to set-up and use. Although slower and more complex two-dimensional separations give unparalleled resolution that no other analytical method can match, the number of samples which can be examined in a given time is much reduced. In spite of these capabilities, however, there are situations where neither one-nor two-dimenstional methods can give an unequivocal answer. Most of these devolve into a question of ‘relatedness’ between two or more proteins and this is where peptide mapping can make a major contribution. When working with intact proteins, identifying an un known or following changes in protein components typically involves comparison with known standard proteins.
Physical Methods Of Study: Differential Scanning Calorimetry
Authors: Taylor Kevin M G , Craig Duncan Q M
Published: November 2023

Thermal methods, and in particular differential scanning calorimetry (DSC), are well established within the liposome field, and are most frequently used to measure the phase behaviour of the phospholipid bilayers, from which information on phospholipid conformation, bilayer fluidity, drug-liposome interactions, and rate of drug release may be derived. In addition, knowledge of the transition behaviour of phospholipids is essential for the rational development of manufacturing protocols. There is also a growing interest in the use of thermal methods for the examination of freeze-dried liposomes, particularly in terms of characterizing the glass transitional behaviour of these systems.
Fluorescence Methods in Liposome Research
Authors: Düzgünes Nejat , Bagatolli Luis A , Meers Paul , Oh Yu-Kyuong , Straubinger Robert M
Published: November 2023

Liposomes of certain compositions can undergo fusion in the presence of the appropriate ionic conditions and pH, or fusion-inducing proteins or peptides (1-4). Thus the molecular requirements for membrane fusion can be explored systematically, using assays that monitor either the intermixing of membrane components or the coalescence of internal aqueous contents of liposomes. In this section we describe widely used fluorescence assays that monitor the kinetics of the fusion process.
Single-strand conformation polymorphism analysis
Authors: Hayashi Kenshi , Kukita Youji , Inazuka, Masakazu , Tahira Tomoko
Published: November 2023

SSCP (single-strand conformation polymorphism) is widely used for mutation detection because of its simplicity and versatility. In this method, a segment to be searched for a mutation is amplified by PCR from genomic DNA or cDNA, denatured, and separated by electrophoresis in media having sieving properties without denaturant (1). Mutation usually causes a mobility shift in electrophoresis, and so, is detected. Conformational change of the single stranded mutant DNA is believed to be the reason for the mobility shift.
The theory and practice of in situ hybridization
Authors: Wilkinson David G
Published: November 2023

The in situ detection of nucleic acid sequences, whether of genes on chromosomes or viruses, or of mRNA in tissues, provides a direct visualization of the spatial location of specific sequences that is crucial for elucidation of the organization and function of genes. As a consequence, methods of in situ hybridization have become important techniques in a number of fields, including diagnosis of chromosomal rearrangements, detection of viral infections, and analysis of gene function during embryonic development. In situ hybridization takes advantage of the specific annealing of complementary nucleic acid molecules, which can be DNA and/or RNA, through hydrogen bonds formed between bases attached to the sugar-phosphate backbone: adenine (A) anneals with thymine (T, in DNA) or uracil (U, in RNA), and cytosine (C) anneals with guanine (G). This base pairing underlies the formation of a double-stranded complex, in which one· strand has the opposite orientation to the other with respect to the sugar-phosphate backbone (Figure JA); the sequence is read from 5’ to 3’ (this refers to the positions of the sugar at which the phosphate residues are attached). Any nucleic acid sequence can therefore be specifically detected by use of a probe that is the ‘antisense’ reverse complementary sequence. An important practical asset is the speed with which such probes can be generated by the cloning of DNA sequences, amplification by the polymerase chain reaction (PCR), or the synthesis of oligonucleotides.
Oligonucleotide probes for in situhybridization
Authors: Rattray Marcus , Michael Gregory J
Published: November 2023

Oligonucleotide probes are convenient for determining the sites of gene expression using in situ hybridization. Since the probes are synthetic, their use does not require much specialized equipment for molecular biology, the growth of bacteria, or the purification of nucleic acids. Oligonucleotide probes can be designed to form complementary base pairs with a target sequence, that can be RNA or DNA. In this chapter we consider binding of oligo¬ nucleotide probes to RNA targets only. The probe is labelled so that its presence on tissue sections can be detected. Probe is applied to the tissue under conditions that hybridization takes place, and then the sections are washed to remove non-specific binding. After these steps have taken place, the presence of the probe can be detected by autoradiography (for radio¬ labelled probes) or by histochemistry (for non-radiolabelled probes). It is possible to use oligonucleotide probes to measure the level of gene expression in individual cells or in tissues using appropriate image analysis techniques.
Electron microscope in situ hybridization: the non-isotopic post-embedding procedure
Authors: Puvion-Dutilleul Francine
Published: November 2023

Although the advent of in situ hybridization for light microscopy goes back almost three decades (1, 2) and its extention to the electron microscope (EM) level occurred shortly after, it is only recently that this technique revolution¬ ized our knowledge about the spatial distribution of defined nucleic acid molecules in the cell. Binder et al. (3) broke new ground in this field by con¬ comitantly using for the first time hydrosoluble resin-embedded material, non-isotopic probes, and immunogold labelling of hybrids. This post embedding non-radioactive procedure was immediately adopted, and became widely used after some procedural improvements which rendered it a method applicable to a wide variety of biological systems.
Microscopy of chromosomes
Authors: Sumner A T , Leitch A R
Published: November 2023

Chromosomes have been studied for something like 120 years, and for most of this period they were visualized using uniform staining with a variety of dyes. This was adequate for studies of the gross structure and behaviour of chromosomes, but identification of individual chromosomes was difficult, being based only on chromosome morphology and size, and specific chromosome substructures could not be studied. Just over 25 years ago, two new techniques emerged that revolutionized the study of chromosomes: (a) Chromosome banding, a series of staining techniques that produced patterns of longitudinal differentiation on chromosomes, and allowed identification of specific chromosomes and chromosomal substructures. (b) In situ hybridization, which labels specific DNA sequences in chromosomes.
Nanovid microscopy
Authors: Lee Greta M
Published: November 2023

Nanovid microscopy, nanometre-sized particles viewed with video-enhanced microscopy (1-3) is used to directly observe the lateral mobility of gold labelled membrane proteins and lipids, to observe the precise location and mobility of gold labelled pericellular and extracellular matrix macromolecules, and to follow endocytosis of gold labelled molecules. The technique involves the use of 30--40 nm colloidal gold particles conjugated to antibodies, streptavidin, lectins, or other ligands. The association of the antibody to the gold is non-covalent but extremely stable. Since a single colloidal gold particle is typically conjugated to many antibody molecules (4), an individual gold particle may bind to a small group of like membrane molecules rather than to a single molecule (5). The gold tag is visualized using video contrast enhancement to detect the light scattering of the gold particle. The gold can be viewed with bright-field, DIC, or epipolarization optics. Examples of bright-field and DIC images are shown in Figure 1.
Embryonic feeder cells
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

To maintain their pluripotency, ES cells are usually cultured either on plastic plates (optionally can be gelatinized) in the presence of leukemia¬ inhibitory factor (LIF) or on a layer of mitotically inactive feeder cells derived from embryonic fibroblasts. These embryonic feeder, or EF, cells are prepared from day 13-14 embryos., We culture our ES cells on mitomycin C-treated primary EF cells and in the presence of LIF.

Preparation of EF cells obviously needs to be done in advance of any ES cell work. EF cells must be G418-resistant (or resistant to any other selection drug you wish to use in isolating homologous recombinants) and, therefore, must be prepared from mice harboring the neo gene. We use a 129-derived strain which harbors pSV2neo (135).
Chapter 16
Authors: picking ES clone
Published: November 2023

The number of clones that must be recovered will depend on a number of factors such as the type of selection (e.g. selection for homologous recombinants and against random integrants), number of colonies obtained after selection, method of screening for homologous recombinants (Southern vs PCR), knowledge about the targeting frequency for the locus of interest, etc. For obvious reasons, however, the more clones isolated the higher the probability of recovering a homologous recombinant. In situations in which we have targeted the same locus in multiple instances (e.g. the immunoglobulin heavy chain locus) we have generally observed a similar frequency of homologous recombination events and therefore know approximately how many colonies must be isolated to obtain a homologous recombinant. As a very general number, at least 250-300 colonies should be isolated for examination of a homologous recombination event.
Embryo aggregation
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

For embryo (morula) aggregation less equipment is needed than for blastocyst injection: required are a stereomicroscope (∼40x magnification) with transmission light for setting up aggregates and a CO2 incubator for culture so that researchers setting up a new lab may choose aggregation instead of blastocyst injection for the production of chimeric mice. For embryo aggregation eight-cell stage embryos up- to morulas should be isolated from the oviduct of superovulated or normally mated females at day 2.5 p.c. After removal of the zona pellucida the embryos are placed individually, together with a small clump of about 10 − 20 ES cells, into small ‘aggregation wells’ of a plastic dish for aggregation (aggregation plate). After overnight culture the majority of these aggregates develop into compacted morulas or early blastocysts which are then transferred into the uteri of pseudopregnant females in the same way as microinjected blastocysts (see Chapter 27). Our protocol is adapted from the one described by Nagy and Rossant (170) except that only one embryo is used per aggregate and that media contain a low concentration of phenol red.
Arf
Authors: Roth Michael G
Published: November 2023

The six mammalian ADP-ribosylation factors form a distinct family within the Ras GTPase superfamily (1). The amino-acid sequences of these 20 kDa guanine nucleotide-binding proteins are at least 70% identical in plants, protozoa, and animals, attesting to their importance in all eukaryotic cells. Two genes encode redundant Arf proteins in S. cerevisiae that are essential for vegetative growth and secretory function (2). A third gene, ARF3, encoding a protein 52% identical to Arf1p, cannot compensate for the loss of ARF1 and ARF2 (1). In mammalian cells, the major function of Arfs is to regulate a variety of membrane transport processes, although Arf proteins also activate phospholipase D enzymes (3, 4) and one member of the family, Arf6, may be involved in regulating the actin cytoskeleton (5). Sarl, a small GTPase that is 37% identical to Arf1, functions in the assembly of the COPII coated vesicles that are responsible for membrane transport from the endoplasmic reticulum to the Golgi complex (6). The molecular details of Sar1 function are better understood than those of Arf proteins (7) and Sar1 has therefore been used as a model for understanding Arfs, particularly Arf1. However, this is likely to be misleading, since the regulation of Arfl is clearly quite different from that of Sarl, and Arfl appears to have a greater number of activities than Sarl.
Proline isomerization and its catalysis in protein folding
Authors: Balbach Jochen , Schmid Franz X
Published: November 2023

In proteins the peptide bonds are usually in the trans conformation, which is favoured energetically over cis in both folded and unfolded proteins. However, an appreciable proportion of the peptide bonds preceding proline (prolyl bonds),* and occasionally those preceding other amino acids, arc in the strained cis conformation. These cis peptide bonds create a problem for protein folding. The non-native trans forms predominate at equilibrium in the unfolded or nascent protein molecule and, since cis⇌trans isomerization is an intrinsically slow reaction — because rotation about a partial double bond is required — the rate of folding to the native state is governed by the rate of isomerization. Non-native prolyl isomers do not block the early stages of folding of a protein chain, but they markedly retard the overall folding process. This is clearly seen in the case of small, single-domain proteins. Many of them refold within a few milliseconds when they contain native prolyl isomers, but when non-native isomers are present (in particular trans isomers of bonds that are cis in the native state) folding is retarded from the millisecond to the minute time range.
Measurement of cytoplasmic and nuclear antigens
Authors: Larsen J0rgen K
Published: November 2023

1 Introduction

Flow cytometry has become the technique of choice for the quantification of a variety of intracellular molecules involved in the regulatory pathways of cellular proliferation, differentiation, and death, as well as in signal transduction and cytokine production. This development is based on recent improvements of the immunochemical methods for detecting target antigens within the cytoplasm and nucleus, supported by the increased assortment of commercially available specific antibodies and fluorochrome conjugates, new signal amplification methods, and improved instrumentation of multiparameter data acquisition and analysis.
Analysis of DNA-measurement of cell kinetics by the bromodeoxyuridine/ anti bromodeoxyuridine method
Authors: Wilson George D
Published: November 2023

Cell kinetics is primarily concerned with the measurement of time parameters in the growth of biological systems. This definition sets it apart from other techniques involved in the study of cellular proliferation. Most methods involve a ‘snapshot’ assessment of cells in a particular phase of the cell cycle (e.g. mitotic index, tritiated thymidine labelling index or S phase fraction from DNA histograms) or cells actively involved in a proliferation cycle (e.g. Ki-67, PCNA staining-see Chapter 9). Whilst static parameters of cell proliferation can be informative, they give no indication of the rates of cell-cycle progression and are subject to ambiguity as will be discussed later in this chapter.
Nucleic acid isolation and analysis
Authors: Talbot Nicholas J
Published: November 2023

The ability to carry out molecular genetic analysis of filamentous fungi is very heavily dependent on being able to extract and purify large amounts of DNA and RNA from fungal cells. Many procedures have been devised in order to purify nucleic acids from eukaryotic cells and a large number of commercially available kits have been formulated for this purpose. Most procedures devised, however, have been optimized for use in cells of model organisms such as yeast, Arabidopsis, Drosophila, and human cells. Adapting these procedures for use in filamentous fungi produces highly variable results and some species have proved recalcitrant to molecular analysis largely due to workers being unable to purify high-quality nucleic acid samples.
DNA-mediated transformation of fungi
Authors: Bowyer Paul
Published: November 2023

The advent of techniques for manipulating and isolating genes from plant pathogenic fungi has lead to a rapid increase in our understanding of how these organisms cause disease (1). An important factor in these advances has been the ability to re-introduce homologous or heterologous genes into these organisms. Transformation systems for model fungi such as yeast, Neurospora crassa, and Aspergillus nidulans, have existed since the 1970s and these techniques are being adapted for use in a growing array of pathogens. This chapter concentrates on techniques developed for pathogenic fungal species, but the techniques have often been found to be broadly applicable among fungal species. The vast majority of transformation protocols for filamentous fungi are based on permeabilizing cell membranes with polyethylene glycol (PEG), or electroporation.
Investigations of Oomycete cell biology
Authors: Hardham Adrienne R
Published: November 2023

The Oomycetes are a distinct group of organisms which, like many true fungi (Basidiomycetes, Ascomycetes, and Zygomycetes), form filamentous hyphae during vegetative (somatic) growth. They are also heterotrophs with absorptive nutrition. Many characteristics of Oomycete organisms, however, indicate that they are not true fungi but are most closely related phylogenetically to the Chromophyte algae and other flagellate protists which possess tripartite flagellar hairs (1–3).
The cytogenetics of pregnancy
Authors: Boyle T , Griffin D
Published: November 2023

Prenatal cytogenetic diagnosis is made possible by the in vitro culture of human cells and tissues. Amniocentesis is the removal of amniotic fluid usually performed between 15 and 17 weeks’ gestation, although sampling has been carried out as early as 12 weeks (1). Chorionic villus sampling allows for first trimester analysis with transcervical or transabdominal aspiration of villi performed at 10-11 weeks’ gestation. Solid tissue specimens may be examined at various stages of gestation and encompass both fetal and extra-embryonic tissues. The techniques supplied are also applicable to adult skin biopsy.

At the time of writing, the cytogenetic study of pre-implantation embryos is a promising new field and, together with advances in the study of sperm and oocyte chromosomes, may provide further help to couples with infertility problems or pregnancies at high risk of genetic disease.
Microscopy and image analysis systems
Authors: Levy E R , Maher E J
Published: November 2023

The microscope is one of the most important tools for a cytogeneticist. It is essential that the microscope provides contrast and resolution of the sample, along with suitable magnification, and to achieve these, three criteria must be met:

the lens system must reveal the required detail, i.e. provide adequate resolution

the image must have sufficient contrast to allow the eye to distinguish what has been resolved

the magnification must be sufficient so that the image of separate points resolved by the microscope fall upon separate cones of the retina.

The aim of this chapter is to provide enough information for the beginner to be able to understand the workings of both light and fluorescence compound microscopes, and to be able to set up and maintain them.
Micromanipulation by laser microbeam and optical tweezers
Authors: Greulich Karl Otto
Published: November 2023

In several chapters at the beginning of this book, techniques were described using light for observation of, and making measurements on cells. This chapter will describe techniques, using light not only for observation but also for manipulation. Here the fact that light is a carrier of energy (heat) and a carrier of momentum (force) is exploited. With the heat effect one can perforate plant cells, cut subcellular structures such as cytoplasmic strands or chromosomes, and can fuse plant protoplasts in a controlled manner. With the force effect it is possible to hold or move cells and subcellular structures as if light were a pair of tweezers, which in contrast to conventional tweezers can also work in the interior of closed objects. Books on laser microbeams and optical tweezers (1) or solely on optical tweezers (2) and a number of reviews (for example refs 3-7) are available in the literature.
Electron microscopy
Authors: Hawes Chris , Satiat-Jeunemaitre Béatrice
Published: November 2023

Despite the recent resurgence in the use of light microscopy (LM) due to the popularity of the confocal microscope coupled with the wide availability of image processing techniques (see Chapters 1 and 2), the unparalleled resolution offered by the electron microscope will maintain its importance as a basic tool in cell biology. Recent developments in preparative protocols, such as ultra-rapid freeze-fixation and freeze-substitution have permitted the preservation of cellular ultrastructure with great fidelity. Whilst the use of immunogold label ling techniques permit the location of antigens within cells at a resolution not possible by LM or confocal based immunocytochemical techniques.
In vitroDNA recombination
Authors: Miyazaki Kentaro , Arnold Frances H
Published: November 2023

This chapter illustrates in vitro DNA recombination methods frequently used in directed evolution. Four different methods are presented with full experimental detail: DNA shuffling (Stemmer method), random priming recombination, Staggered extension process (StEP), and in vitro heteroduplex formation in vivo DNA repair (heteroduplex recombination). One problem in recombination experiments is unwanted nonrecombinant parent sequences and misrecombined sequences. Protocols to reduce these backgrounds are included.
Phage selection strategies for improved afinity and speciicity of proteins and peptides
Authors: Dennis Mark S , Lowman Henry B
Published: November 2023

Filamentous phage display of peptide or protein variants has been widely used for rapid selection of protein variants that bind with improved affinity and improved specificity to a target molecule (for reviews, see 1, 2). The key feature of such selection schemes is that the genotype of a particular variant is linked through packaging inside a virion particle to the phenotype of a displayed protein or peptide that has been fused to one of the coat proteins, such as gene III protein (pIII), of the phage. Phage particles can be selected by binding to an affinity matrix, propagated in Escherichia coli , and identified by DNA sequencing ( Figure 1 ).
Affinity maturation of phage antibodies
Authors: Nielsen Ulrik B , Marks James D
Published: November 2023

Phage display can be used to increase the affinity of antibodies more than a 1000-fold (1, 2). The starting point is typically a specific antibody isolated from a phage antibody library (see Chapter 13). To accomplish affinity maturation (increased affinity) the sequence of the antibody is diversified, the mutant gene repertoire is displayed on filamentous phage, and higher affinity binders are selected on antigen. While the process is reasonably straightforward, the investigator should be reasonably certain that increasing the affinity of their particular antibody will lead to the desired biologic effect prior to undertaking in vitro affinity maturation. Higher affinity is especially important when trying to neutralize a circulating toxin or growth factor. In this case, the antibody dis- tributes in the same compartment as the antigen, allowing the antigen-antibody interaction to proceed to equilibrium. The higher the affinity, the greater the amount of antigen bound. In contrast, affinity is only one factor determining the amount of antibody fragment that will accumulate in a tumor in vivo (3). Factors such as antibody fragment size, pharmacokinetics, and valency may have a more important impact (4).
Human chromosome mapping of single copy genes
Authors: Beatty Barbara G , Scherer Stephen W
Published: November 2023

One of the most common applications of FISH is the localization of single copy genes to a specific chromosomal band on metaphase chromosomes (1-3). Identifying the sub chromosomal position of a gene in both normal and disease states can provide valuable information regarding its biological and/or clinical significance. In some situations, mapping a newly identified gene to regions of chromosomal deletion or amplification, to translocation breakpoints, or to a region near a gene known to be involved in a particular disease process (4-6) can supply essential clues which may lead to important clinical applications (see Chapter 9).
FISH on three-dimensionally preserved nuclei
Authors: Solovei I , Walter J , Cremer M , Habermann F , Schermelleh L , Cremer T
Published: November 2023

This chapter focuses on the fluorescence in situ hybridization (FISH) of DNA probes to three-dimensionally preserved cells, so-called 3D FISH. This technique allows three-dimensional visualization of specific DNA and RNA targets within the nucleus at all stages of the cell cycle. It provides information about the arrangement of chromosome territories and the organization of sub chromosomal domains, about the pattern of chromatin density within a chromosome territory, about positions of individual genes and RNA transcripts read from them. Accumulation of such data is necessary for understanding relationships between the spatial organization of the genome and its functioning in the interphase nucleus.
FISH in clinical cytogenetics
Authors: Squire Jeremy A , Marrano P , Kolomietz E
Published: November 2023

In recent years the possibilities for visualizing several chromosomal targets simultaneously has meant that FISH analysis has an increasing role to play in the study of patient samples. The clinical cytogenetics service laboratory no longer focuses on the morphological analysis of chromosomal aberrations alone, but now has to provide comprehensive molecular cytogenetic analysis of diverse gene sequences consistently involved in certain classes of cancer and a variety of human genetic diseases (1).
The Effect of Accelerated Continuous Theta Burst Stimulation on Weight Loss in Overweight Individuals With Schizophrenia: A Double-Blind, Randomized, Sham-Controlled Clinical Trial
Authors: Kang Dongyu , Zhang Yi , Wu Guowei , Song Chuhan , Peng Xinjie , Long Yujun , Yu Guo , Tang Hui , Gui Yawei , Wang Quan , Yuan Tifei , Wu Renrong
Published: November 2023

Background and Hypothesis

Obesity is a common comorbidity in individuals with schizophrenia and is associated with poor clinical outcomes. At present, there are limited effective approaches for addressing this issue. We conducted a double-blind, randomized, sham-controlled clinical trial to investigate the efficacy of noninvasive magnetic stimulation techniques in reducing obesity in individuals with schizophrenia.

Study Design

Forty overweight individuals with schizophrenia were recruited and randomly assigned to receive either the active or sham intervention. The active group received 50 accelerated continuous theta burst stimulation (cTBS) sessions over the left primary motor area (M1), while the sham group received sham stimulation. The primary outcomes were the change in body weight and body mass index (BMI), and the secondary outcomes were the psychiatric symptoms, eating behavior scales, metabolic measures, and electrophysiological to food picture stimuli.

Study Results

The study demonstrated a significant decrease in body weight and BMI after the intervention selectively in the active group (mean = −1.33 kg, P = .002), and this improvement remained at the 1-month follow-up (mean = −2.02 kg, P = .008). The score on the Barratt Impulsivity Scale (mean = −1.78, P = 0.036) decreased in the active group and mediated the effect of accelerated cTBS on body weight. In the food picture cue electroencephalograph task, the late positive potential component, which is related to motivated attention and emotional processing, decreased in frontal brain regions and increased in posterior regions after the active intervention.

Conclusions

The accelerated cTBS may offer a promising approach for treating obesity in individuals with schizophrenia. Further research with a larger sample size or individualized stimulation protocol should be promising.

Trial Registration

Clinical trial registered with clinicaltrials.gov (NCT05086133).