bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of TGF-β in bone growth and bone repair
Authors: Ballock R T , Roberts A B
Published: November 2023

Transforming growth factor beta (TGF-β) is a multifunctional regulator of cell growth and differentiation. Although nearly all cells synthesize and respond to TGF-β, the highest amounts of this peptide growth factor have been found in bone and cartilage (1). TGF-β has also been found to be an important regulator of extracellular matrix synthesis and degradation, suggesting a potentially criti cal function for this growth factor in the regulation of bone formation and repair.
Determination of growth factor receptor distribution by immunocytochemistry or autoradiography, and quantitation by ligand binding or FACS analysis
Authors: Linge Claire , Green Martin R
Published: November 2023

Antibody and ligand binding are the two most important tools used to specifically analyse and quantitate growth factor distribution on tissue sections and cells. However each tool and associated techniques, (e.g. immunohistology, autoradiography, ligand binding including Scatchard analysis, and fluorescence activated cell sorting [FACS] analysis) have their own strengths and weaknesses and should be selected only after a careful appreciation of the research problem in hand. As examples, some of the general applications and restrictions of antibodies and ligands in receptor research are listed below. The text assumes some general background knowledge of growth factor receptor properties.
HLA antigens in tissues
Authors: Rose Marlene L
Published: November 2023

Mhc antigens are the major target of the immune response following allograft rejection. It has been suggested that aberrant expression of autologous Mhc antigens is involved in the pathogenesis of some autoimmune diseases. Two facts about expression of Mhc antigens account for the current interest in understanding their precise distribution in organs. First, expression is not a constant feature of a cell, it can be up-regulated by cytokines and down regulated by substances that counteract or inhibit cytokine production. Second, quantitative changes in expression alter the magnitude of the immune response. It follows therefore that the precise distribution of Mhc antigens in organs will determine both the target and the strength of the immune response. This response will be manifest as an anti-allograft response should the organ be transplanted, or may be an autoimmune response following aberrant expression of autologous Mhc antigens. Furthermore, expression of Mhc antigens may be involved in the regulation of inflammatory responses, viral infections, and immune recognition of tumour cells.
B cell activation
Authors: Klaus Gerry G B
Published: November 2023

B lymphocytes are the precursors of antibody-forming cells (AFC), which express clonally distributed surface immunoglobulin (slg) receptors for antigen (generally slgM and slgD). B cells are generated in the bone marrow throughout adult life: this process involves an ordered sequence of gene rearrangements which gener ates a primary B cell repertoire of some 106-107 specificities. Binding of certain polymeric, cross-linking antigens (so-called T-independent (TI) antigens) to slg receptors is in itself sufficient to induce B cell activation and antibody formation. Generally, however, for T-dependent (TD) antigens (i.e. protein antigens) additional T cell-derived stimuli are required to elicit antibody formation. In either case, the clonal precursors bearing slg receptors specific for epitopes on the antigen become activated, enter the cell cycle, proliferate, and differentiate into high-rate AFC, which, ultimately become plasma cells. For an overview of the biology of B cells see ref.
Comparison of the localization of protein kinase C subspecies by in situ hybridization and immunocytochemical methods
Authors: Mcginty Jacqueline F , Couce Marta E , ways D Kirk
Published: November 2023

A variety of immunocytochemical protocols have been developed over the last 15 years which provide us with the ability to map the heterogeneom distributions of proteins and/or peptides of significance to neurotrammitter signalling. This major advance in histochemical technology has been joined recently by the advent of standardized in situ hybridization histochemical protocols that allow us to map the mRNAs that give rise to protein (e.g. receptors, enzymes) or peptide (transmitters, e.g. enkephalins) products. The cellular resolution and complementary RNA/protein information provided by both methods yield a unique combination for addressing complex neurochemical questions at a new level of detail. Furthermore, quantification by computerized image analysis is beginning to be applied to these histochemical techniques enabling them to become powerful quantitative, and not just qualitative, methodologies (see Chapter 4, this volume). Using these combined techniques, we are now able not only to map the distribution of peptide neurotransmitters, transmitter synthesizing and metabolizing enzymes, receptors, and, most recently. signal transduction molecules, but also to investigate the regulation of these molecules in response to physiological, pharmacological, and pathological challenges.
Immunofluorescent and immunogold methods to localize G proteins
Authors: Lewis J M
Published: November 2023

OTP-binding regulatory proteins (G proteins) underlie pathways of signal transduction that include inhibition of adenylyl cyclase, stimulation of K+ channels, and stimulation of phospholipase C. Members of the family of G proteins—G1, Gs, Go, Gz, or G1—may also regulate events such as fusion of endosomes, and there are probably other, a yet unknown receptors and effectors modulated by G proteins. While the participation of G proteins in the regulation of events at the cell surface has been well characterized, less is known about the intracellular control of such signal transduction pathways. One way of modulating G protein function, and thereby co-ordinating different paths of information transfer, involves spatial and temporal controls over G protein function in intact cells. Through these controls a cell can modulate the ability of its complement of different G protein to interact directly with particular receptors or effectors. Spatial and temporal parameters governing G protein function within intact cells possibly include the compartmentalization of different G protein subtypes with selected receptors and effectors within the plasma membrane. In addition, the redistribution of G proteins within cells may take place in response to specific agonists, and qualitative or quantitative changes in G protein subunits may occur throughout the cell cycle.
Molecular structure of anitnal lectins
Authors: Drickamer Kurt
Published: November 2023

Many effects of the carbohydrate portions of glycoconjugates result from the physical properties of the sugars themselves and from interaction of the saccharides with each other and with the proteins and lipids to which they are covalently attached. Thus, for example, the properties of proteoglycans in various biological matrices are dominated by the interactions of the constituent glycosaminoglycans with water (1). Other effects of carbohydrate conjugated to proteins can be more subtle and varied (2, 3). A striking example is modulation of activation of tissue type plasminogen activator by the oligosaccharide attached to it (4).
Antibody screening of bacterial expression systems
Authors: Luzio J Paul , Ellis juliet A , Thomas stephen , Banting George
Published: November 2023

Screening expression libraries with specific antibodies is a well-established method for the identification of cDNA clones (1, 2). It is often used when an accurate protein sequence is not available, thus precluding the design of appropriate oligonucleotide probes. It takes advantage of the high specificity and avidity of antibodies together with the availability of simple, robust, and relatively rapid methods of detection of antibody-antigen complexes.
Isolation of cDNA clones by direct expression in mammalian cells
Authors: Smith Craig A , Goodwin Raymond G , Maliszewski Charles R
Published: November 2023

The isolation of cDNAs encoding specific proteins historically required biochemical purification of the protein itself, followed by determination of a portion of the amino acid sequence. Using this information, degenerate oligonucleotides could then be synthesized and used as probes to obtain the desired cDNA by hybridization methods. Though this procedure continues to be successfully employed, the laborious and time-consuming nature of purifying sufficient protein from conditioned media or cell membranes, where expression levels can be quite low, spurred the development of alternative approaches based, in essence, upon purification of the cDNA itself. One of the first, termed hybrid selection, employed pooled cDNAs to hybridize mRNA which could then be translated either in vitro (1) or in Xenopus oocytes (2). The translated products could then be assayed appropriately to determine if the cDNA encoding the protein product of interest was present in this pool.
Gene targeting in mammalian cells
Authors: Johnson Randall , Killeen Nigel
Published: November 2023

A wide variety of techniques has been employed to inhibit the expression of specific genes selectively in mammalian cells. This is a useful experimental option because by depriving a cell of a given gene product, one can often obtain invaluable information about the function of the gene. As a highly specific means for achieving gene-ablation, the targeted mutagenesis of endogenous chromosomal genes by homologous recombination (gene targeting) has recently become a widely used technique (1-4). Embryo-derived stem (ES) cells have been used in the majority of such experiments, since these cells offer the researcher the opportunity to pass the mutation from tissue culture to the mouse germline and, therefore, they allow for the phenotypic analysis to be conducted in vivo (5, 6).
Immuno-electron microscopy
Authors: Clark Michael W
Published: November 2023

The budding yeast, Saccharomyces cerevisiae, well known as a model experimental system for the relative ease of its genetics and molecular biology, has disappointed many modern molecular cell biologists because of the limited cytological techniques that are available for this organism. As a result of the small size of a single S. cerevisiae haploid cell, 3–5 µm in diameter, most immunofluorescence observations provide relatively simple mapping information, such as whether the protein is in the nucleus or the cytoplasm, or whether or not it is in the vacuole. Most of the important cytological questions for yeast can be adequately answered only by the higher resolutions obtained with the electron microscope. The search for such efficient and effective cytological methods has thus far provided many usable techniques for mapping specific yeast constituents at this higher resolution. Various cell preparation methods have been reported which demonstrate the antibody mapping of specific yeast proteins with the electron microscope (1–3). These techniques are all variations upon the traditional electron microscopy (EM) methodology of sectioning fixed, resin-embedded samples. The diversity in these EM techniques arises from the different viewpoints taken by the investigators as they overcame problems specific to the interactions of a particular antibody with a particular protein. Thus far, each immuno-EM localization published for yeast has had the EM procedures ‘tailored’ for the specific protein and the antibody reagents utilized in the investigation. At this time, there is no single procedure for fixation and resin-embedding that works for all antibody-based EM examinations.
Industrial Saccharomyces yeasts
Authors: Kjelland-Brandt Morten C
Published: November 2023

Genetic and biochemical work with industrial Saccharomyces yeasts usually has close connection with their industrial applications, because basic biological phenomena are generally best studied in organisms that ‘behave well’ in certain respects. When Saccharomyces yeasts are chosen as model organisms, the highly developed genetics are usually one of the key virtues, and one therefore chooses members of a large group of closely related strains of Saccharomyces cerevisiae that behave regularly in crosses and carry convenient genetic markers (e.g. laboratory strains and genetic reference strains). These strains are ill suited for the traditional practical applications of Saccharomyces yeasts, such as bread-making or brewing, while industrial strains suffer from difficulties when it comes to crosses and various other genetic techniques. The present chapter describes some ways of dealing with these difficulties.
Streptomyces plasmid vectors
Authors: Wohlleben W , Muth G
Published: November 2023

Bacteria of the genus Streptomyces are Gram-positive, mycelium-forming soil bacteria that are generally non-pathogenic and which comprise a large number of different species. They are classified according to the following criteria: morphological (e.g. colour and shape of the mycelium and spores), physio logical (e.g. utilization of different nitrogen and carbon sources, production of primary and secondary metabolites), and genetic (e.g. rRNA sequences) (1, 2).
Lipid degradation via β3-oxidation in germinating barley
Authors: Holtman W L , Heistek J C , Kokkelink I , Douma A C
Published: November 2023

Lipids and their degradation and oxidation products can negatively affect beer quality and brewing process technology. Barley malt is a major source of lipids and lipid degrading enzymes in the brewing process.
Influence de I’ensemencement de micro-organismes au maltage sur le developpement de la flore et la qualite du malt
Authors: Boivin Patrick , Malanda M’baka
Published: November 2023

In the production of malt, natural microflora growing during the malting process plays an important role in the ultimate malt quality. A biological process (starter cultures) of increasing interest in food processing has been used to manage the development of microflora during the malting process.
Profil hormonal de l’orge et qualité brassicole
Authors: Boivin P , Kohl S , Label P , Doumas P
Published: November 2023

Endogenous plant cereals, including synthesis. These An HPLC separation different groups growth substances mediate physiological processes in seed dormancy, germination and hydrolytic enzyme processes are central to the production of good malt. and an enzyme linked immunosorbent assay (ELISA) with of polyclonal antibodies have been used for the determination of barley endogenous hormones: gibberellins, abscisic acid, auxin and cytokinins. Differences in main endogenous plant growth substances have been detected in barley varieties having different malting qualities.
The role of malt components in mash flocculation and wort filtration
Authors: Anderson I W
Published: November 2023

A wide range of “porosity” values are encountered when laboratory-scale mashes are assessed using a simple filtration test. This “porosity” can be greatly improved by adding a cationic polyelectrolyte (protein or polysaccharide) which flocculates anionic particles present in the mash. It has been shown that cationic proteins isolated from malt can flocculate mash particles in a laboratory assay, whereas addition of a cationic polysaccharide to the mash of a pilot-scale brew reduces lautering time.
High performance immobilized yeast reactor design for continuous beer fermentation
Authors: Winkel L Van De , Beveren P C Van , Borremans E , Goossens E , Masschelein C A
Published: November 2023

Primary fermentation with immobilized yeast has not yet been successful. Major drawbacks are gas-channelling, plugging, support-particle compression or abrasion in packed-bed, fluidized-bed and stirred reactors. These problems may be overcome using a sintered silicon carbide carrier in a multi-channel loop reactor design where the solid-liquid contact area and loading capacity are maximized. Complete attenuation was achieved in a continuous two-stage bioreactor with a hold-up vessel arranged in series.
Mesure de l’activite antioxygene de l’orge et du malt: approche multiple
Authors: Boivin P , Allain D , Clamagirant V , Maillard M N , Cuvelier M E , Berset C , Richard H , Nicolas J , Forget-Richard F
Published: November 2023

Malt contains natural antioxidants originated either from barley (mainly polyphenols) or from the malting process (mainly Maillard reaction products). These compounds could delay the staling of beer and thus avoiding the use of exogenous antioxidant compounds. Oxidation, responsible for the flavour deterioration of beer is both enzymatic and non enzymatic. Therefore, different methods have been developed for assaying the antioxidant properties of barley and malt: inhibition of purified oxidases from green malt (lipoxygenase land 2), accelerated lipid auto oxidation test (apolar medium) and an electrochemical method (polar medium).
Lipid oxidation during wort production
Authors: Kobayashi Naoyuki , Kaneda Hirotaka , Kano Yukinobu , Koshino Shouhei
Published: November 2023

It has been reported that lipid oxidation during wort production has a relationship with flavour staling of beer. To clarify lipid oxidation mechanism during wort production, fatty acid hydroperoxides were analyzed using a chemiluminescence-HPLC method. The production of the hydroperoxides during mashing increased with lipoxygenase activity in malt. Adding an inhibitor of lipoxygenase to mash just before mashing or lowering pH in mash from 5.5 to 5.0 significantly inhibited the production of the hydroperoxides.
Influence of insufficient wort aeration on unbalanced ester formation in brewing
Authors: Yoshimura T , Endoh A , Matsuzawa K , Kuki M
Published: November 2023

After successive use of brewer’s yeast in a 10 kl scale mini-brewery, enhancement of strong eatery flavour was noted, which was responsible to the higher formation of ethyl caproate (> 0.15 mg/1). It was elucidated to be deterioration of yeast vitality caused by insufficient wort aeration. Well-balanced ester flavour was restored by making adequate aeration, which could be judged by strenght of ethyl caproate by tasting of young beer, and exchanging deteriorated yeast for fresh one for next fermentation.
Improved CO2 purification system
Authors: Oeveren P W van
Published: November 2023

The most important impurities to be removed from fermentation gas are organic compounds as well as sulphur compounds like hydrogen sulphide and dimethyl sulphide. The commonly applied carbon dioxide purification plant has a water wash tower and an activated carbon filter in order to eliminate these impurities. The removal principle is based on physical processes, which makes the process sensitive to small disturbances like flow, pressure and temperature fluctuations.
Modélisation de la microfiltration tangentielle de biere sur membranes— comprehension du mecanisme de colmatage
Authors: Lenoël M , Blanpain P , Weishaupt R , Shingleton M
Published: November 2023

The classical laws of membrane filtration have been used to understand the filtration of beer and progressive blinding of the membrane. Experiments have been applied to laboratory filtration and to a pilot tangential¬ filtration unit. Filtration models confirm the transition from an internal blockage of the membrane to the formation of an external layer (dynamic secondary membrane). initial blinding The importance of the operating conditions of filtration (starting conditions, trans-membrane pressure) is confirmed. The results show the limits and the possibilities of the beer.
The measurement of the function of ion channels reconstituted into artificial membranes
Authors: Williams Alan J
Published: November 2023

As is clear from the first chapter in this book, the function of individual ion channels or populations of ion channels in the cell surface membrane can be monitored in tissues and cells using voltage-clamp techniques. This chapter describes a more biochemical approach to the study of ion channel function. The procedures outlined here are commonly referred to as ‘ion channel reconstitution’, and involve the incorporation of channel proteins into artificial phospholipid bilayers so permitting the characterization of channel function under voltage-clamp conditions. In practice, this term is applied both to procedures which involve true reconstitution (i.e. studies involving purified channel proteins), and to protocols in which intact isolated membrane vesicles, containing the channel protein of interest, are incorporated into an artificial membrane. For more information on (membrane) protein purification, see refs 1 and 2 in conjunction with Chapter 4.
Concepts of single-channel analysis: inferring function from fluctuations
Authors: Moss Guy W J , Moczydlowski Edward
Published: November 2023

The defining act of an ion channel protein is to allow an electrical current of ions to flow passively across a cell membrane. Thus, among the variety of approaches discussed in this text, perhaps the most basic measurement of channel function is its unitary current. As described in Chapters 1 and 2, single channel currents can be recorded with techniques that involve patch pipettes or artificial bilayers. Depending on the noise characteristics of the system, a patch-clamp amplifier allows one to measure unitary currents as small as ∼0.1 pA. Regardless of how the single-channel recording is obtained, the primary objective is to interpret or make sense of it in terms of channel function. For this formidable task, the record supplies only two basic types of information: current amplitudes and dwell times. These pieces of information are illustrated for a recording of a Ca2+ -activated K+ channel in Figure I. The first trace in Figure I shows that this particular channel fluctuates between a well-defined closed (down) and open (up) state. The unitary current, i, of the channel is measured as the difference between the closed and open levels. A dwell time in the open state is defined as the duration of an event in the open state between two consecutive closing events. Likewise, a dwell time in the closed state is that between two consecutive openings.
Ion channels and signal transduction systems in plant and animal cells
Authors: Mcfadzean I , Brownlee C
Published: November 2023

The advent of the patch-clamp technique in the late 1970s brought about a revolution in the study of ion channels. Whilst in the main this arose as a result of being able to resolve single channel currents, an equally important development was that it became possible to manipulate the intracellular environment of the cell, allowing the control of ion currents by cytoplasmic messengers to be studied in far greater detail than hitherto conceivable. The vast majority of such studies have been performed in animal cells, in which the transduction pathways coupling neurotransmitter receptor activation to changes in ion channel activity have been elucidated in great detail in a wide range of cell types.
Channel structure
Authors: Findlay John B C , Marsh Derek
Published: November 2023

It is now well appreciated that integral membrane proteins in general and ion channels in particular present formidable problems for the determination of their 3D structure. The first is that most proteins of this class are naturally present at low levels and can be isolated only with considerable difficulty. Thus far, expression systems have not really solved this problem in that, unlike water-soluble proteins, the amounts which can be produced and purified are in most cases still very low. Although there are prospects that heterologous expression may eventually provide the answer, progress at present is slow. The second major problem is that proteins with substantial intramembranous domains do not, with a few notable exceptions, readily form 3D crystals suitable for X-ray analysis using present-day methods. Much more hope lies with 2D systems where ordered arrays are more easily produced and electron microscopy, although more difficult, can be used to provide evidence at low to medium resolution.
Neural crest cells: strategies to generate lineage diversification in vitro
Authors: Duband Jean-Loup , Delannet Muriel , Monier Frederique
Published: November 2023

The neural crest is both a curiosity and a spectacular invention of vertebrates. It consists of a population of precursor cells endowed with striking migratory properties and possessing an extraordinary range of potentialities. The neural crest develops all along the embryonic axis, in the neural folds, at the bound ary between the neural plate and the superficial ectoderm. However, neural crest cells do not remain at their site of origin after appearance. At a precise stage of embryonic development, e.g. soon after neural tube closure in birds, they venture out of the neural epithelium and disperse within the embryo along defined migratory pathways. After migration, neural crest cells settle in various regions of the embryo to generate a wide spectrum of derivatives ranging from cells that are truly neural (i.e. neurons of the peripheral nervous system) to others that are essentially mesodermal (1,2) (Table 1).
An introduction to the growth cycle of human immunodeficiency virus
Authors: Karn J
Published: November 2023

The major biochemical steps in virus growth are shown in Figure 1. Soon after infection of the cells, the viral RNA genome is reverse transcribed into a DNA copy, transported to the nucleus, and integrated at random sites in the chromosome. Once integrated, the proviral genome is subject to transcriptional regulation by the host cell, as well as its own transcriptional control mechanisms. The later stages of the life cycle involve expression of the viral genes and eventual assembly and release of virus particles. The following informal summary is intended to provide an introduction to the gene products encoded by HIV and their roles in the viral life cycle.
HIV protease
Authors: Debouck C , Tomaszek T A , Ivanoff L A , Culp J
Published: November 2023

The retroviral protease, which plays an essential role in virion maturation (1), has been subjected to intensive studies and drug discovery efforts. The HIV-1 protease has a unique cleavage specificity and belongs to the family of aspartyl proteases. It is required for the proteolytic processing of the large Gag and Gag-Pol viral polyprotein precursors into the mature virion structural proteins (matrix, capsid, and nucleocapsid) as well as the virion enzymes (protease, reverse transcriptase, and integrase) (Figure I). When the HIV-1 protease is inactivated by mutation or by a specific inhibitor, non-infectious virions with immature morphology are formed and the life cycle is halted. It is the essential nature and the unique specificity of the HIV-1 protease that called for its consideration as a therapeutic target.
Tyrosine phosphorylation
Authors: Clark Edwin A , Brugge Joans.
Published: November 2023

1. A role for tyrosine phosphorylation in signal transduction One of the central questions in cell biology is how do cells communicate with each other and their environment, and how does an individual cell regulate itself? Characterization of the proteins that regulate extracellular and intracellular signal transduction has lead to an understanding of their role in biochemical pathways involved in cell growth, proliferation, and differentiation. One key regulatory event involves tyrosine phosphorylation of cellular proteins. The enzymes responsible for these phosphorylation events, protein tyrosine kinases (PTKs), fall into two general classes. First, the receptor tyro-sine kinases, which have both extracellular (ligand binding) and intracellular (tyrosine kinase) domains that allow them to directly couple external stimuli to intracellular signalling (1). Secondly, the non-receptor tyrosine kinases, which functionally couple with membrane receptors via various protein interaction domains or carry out functions downstream of membrane triggered events. Such coupling allows this class of signal transducers to relay signals from the external environment to the cytoplasm and nucleus (2).
In vitro and in vivo detection of free radical metabolites with electron spin resonance
Authors: Mason Ronald P
Published: November 2023

Electron spin (or paramagnetic) resonance (ESR or EPR) is a spectroscopic technique that detects the unpaired electron present in a free radical. As such, it is the only general approach that can provide direct evidence for the presence of a free radical. In addition, analysis of the ESR spectrum generally enables determination of the identity of the free radical.
Salicylic acid and phenylalanine as probes to detect hydroxyl radicals
Authors: Kaur Harparkash , Halliwell Barry
Published: November 2023

It is now widely accepted that reactive oxygen species (ROS) and reactive nitrogen species (RNS) (including free radicals) are involved in the pathogenesis of several inflammatory and degenerative disease states (for a recent review see ref. 1). In spite of their existence these species have not as yet been measured in clinical diagnosis. This is partly due to the lack of agreed formats and standardized methodology for measuring oxidative stress and/or levels of free radicals in humans.
F2-lsoprostanes: prostaglandin-like products of lipid peroxidation
Authors: Morrow Jason D , Roberts L Jackson
Published: November 2023

Free radicals are believed to be involved in a variety of disease processes (1). Much of the evidence for this, however, is indirect and circumstantial because of limitations in the techniques currently available to quantify free radicals or their products in biological systems. This is a particular problem when assessing oxidant injury in vivo in humans (2).
Measurement of products of free radical attack on nucleic acids
Authors: Maidt M Lindsay , Floyd Robert A
Published: November 2023

High performance liquid chromatography (HPLC) methods with electrochemical methods able to measure oxygen free radical adducts to DNA became available in the 1980s (1, 2). Due to their low presence in nature, where they are found at about one adduct per 100000 bases, they required the sensitivity afforded by HPLC coupled with electrochemical detection in order to detect and quantify them.
Solid-phase supports
Authors: Blincko Stuart , Rongsen Shen , Decun Shen , Howes Iain , Edwards Raymond
Published: November 2023

Optimum immunoassay performance is achieved when only the reaction of a specific antibody with its antigen is measured (1). Assuming the antibody preparation is of high affinity and high specificity then the key to achieving highly sensitive and highly specific assays is the reduction of non-specific signals. Such signals are caused by a number of effects. These include non specific interactions of the labelled immunoreagents, background signals from the sample and assay components and detector noise (e.g. optical and electrical noise). Furthermore the method adopted must demonstrate a high degree of precision to achieve maximum sensitivity (2).
Analysis of sequence-specific DNA-binding proteins
Authors: Rhodes Daniela , Fairall Louise
Published: November 2023

This chapter describes the use of two extremely sensitive electrophoretic assay systems, ‘band shift’ and ‘foot printing’, in the analysis of sequence specific DNA-binding proteins. In the last 15 years these techniques have permitted the identification and characterization of a large number of very scarce proteins involved in cellular processes such as transcription and replication. In both these assay systems the identification or analysis of a protein is based on studying the effects of protein binding on the DNA. Extremely small amounts of protein (in the region of 10-20to 10-15 mol) can be detected utilizing radioactively-labelled DNA fragments. The principal applications of band shift and foot printing assays are for obtaining information on the specific DNA binding site of a protein, including the affinity of the protein for the DNA, and for detecting and monitoring the purification of sequence-specific DNA-binding proteins from crude cell extracts.
Systetnatic approaches for the identification of imprinted genes
Authors: Feinberg Andrew P
Published: November 2023

Genomic imprinting has been shown to influence development, growth, and behaviour in mammals as a result of parent of origin-dependent expression of imprinted genes (1-3). Some human genetic diseases and cancers are also attributable to genomic imprinting (4, 5). These effects are apparently due to loss or gain of function of imprinted genes when one or more of these genes affect specific tissues and developmental stages. How many imprinted genes in total are there throughout the lifespan in mammals and what are their precise functions? How are imprinted genes regulated and what are the molecular mechanisms controlling their expression? Answers to many of these questions are still rudimentary. For further progress in the field, it is desirable to identify imprinted genes so that a comprehensive understanding of the mechanism and role of genetic imprinting in development and disease can be attempted.
lmprinting in the Prader-Willi/Angelman syndrome region on human chromosome 15
Authors: Horsthemke Bernhard
Published: November 2023

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are distinct neurogenetic disorders which are caused by a deficiency of paternal (PWS) or maternal (AS) contributions for chromosome 15. The affected genes are located in an imprinted chromosomal domain, which is controlled by an imprinting centre. Mutations of this centre block the paternal - maternal or the maternal - paternal imprint switch during gametogenesis. DNA methylation appears to be instrumental in regulating individual gene activity and can be employed for diagnostic purposes.
The p53 pathway, past and future
Authors: Lane David P
Published: November 2023

Mutation of the p53 tumour suppressor gene and the accumulation of the p53 protein are amongst the most common specific changes found in human cancer. An understanding of the precise biochemical and biological affects of these changes will provide new insights into the fundamental nature of the neoplastic process. New results show that the p53 protein is a critical part of a damage-induced cell cycle checkpoint control that maintains genetic stability. The p53 protein provides an attractive target for the development of new diagnostic and therapeutic agents that will hopefully reduce the impact of cancer on society.
Happy mapping
Authors: Dear Paul H
Published: November 2023

Most methods for genome mapping rely on some form of cloning to isolate a subfraction of the genome for analysis, whether into yeast or bacterial hosts (as in physical mapping, Chapters 10 and 11), into hybrid cells (as in radiation hybrid mapping, Chapter 4), or as offspring amongst which polymorphic markers segregate (as in linkage mapping, Chapters 1-3).
Fluorescence in situ hybridization
Authors: Leversha Margaret a.
Published: November 2023

In situ hybridization enables the visualization of a probe of interest within a cytological context. For the purposes of gene mapping, the probe is usually a particular segment of cloned DNA which is labelled to allow detection. The target is normally a preparation of chromosomal DNA on a microscope slide. Both target DNA and probe are denatured, and then annealed together to allow the labelled probe to hybridize to the complementary sequences in the target. Unbound probe is then washed away and the specific hybridization signals revealed. Non-isotopic probe labelling methods, with the advantages of improved speed and safety, have largely replaced the earlier isotopic procedures (1-3). An increasing range of fluorescent labelling or cytochemical detection systems enables simultaneous hybridization of multiple probes to a single cytological preparation (4).
Fixation of tissues for the polymerase chain reaction
Authors: Hopwood David
Published: November 2023

Cell and tissue fixation was originally devised to produce good morphology. The aims of fixation have been defined variously but a number of features are in common (1,2):to prevent autolysis and bacterial attack to preserve cell shape and volume during subsequent procedures to retain as life-like a condition as possible to facilitate subsequent processing and staining These aims are met in part and workable and reproducible morphological results achieved. Some substances, such as lipids, glycogen, and iron, are lost to different degrees during routine processing. Nonetheless, the microanatomy of the tissues is well enough preserved to allow a histopathological diagnosis to be established in most cases. The needs of hospital histopathology are the processing of large numbers of specimens received under a variety of conditions and from a multitude of anatomical sites. The system must be robust, simple, and reproducible. It is important to remember that it has been devised and has evolved to give good morphology rather than the optimum preservation of DNA and RNA.
In situ genetic analysis with selective ultraviolet radiation fractionation (SURF)
Authors: Ireland Adrian , Shibata Darryl
Published: November 2023

Genetic analysis of specific cells and tissues provides opportunities for better correlation of genotype with phenotype. Often the relative locations of cells are as important as or mere important than their cytological features. Therefore, in situ genetic analysis may yield insights difficult to obtain by other techniques.
Spectroscopic characterization of immobilized proteins
Authors: Bright Frank V
Published: November 2023

As a nation the United States spends billions of dollars annually on diagnostic assays. Unfortunately, most of these assays are performed in well-outfitted laboratories, and require skilled personnel, large amounts of costly reagents, and often demand long analysis times to quantify clinically important analytes. In order to overcome the disadvantages inherent with this approach, one must develop new sensing schemes that are reliable, inexpensive, fast, simple to construct and operate, redundant and self-checking, accurate, and precise, with adequate detection limits. Finally, the demand of a given sensing scheme to be used in point of care or field situations requires small, simple, and robust sensing and detection platforms.
Immobilization using electrogenerated polymers
Authors: Schuhmann Wolfgang
Published: November 2023

The reproducible immobilization of biological recognition elements, preserving their catalytic activity and biological selectivity on the surface of a suitable transducer, is a fundamental presupposition for the successful development of biosensors. As future applications will aim at miniaturized biosensors and multisensor arrays which integrate several different biological recognition elements, immobilization procedures have to be developed which allow one: (a) To pre-define the immobilization site (i.e. the miniaturized transducer surface) avoiding manual deposition procedures.
Analysis of protein: protein interactions by gel electrophoresis
Authors: Coghlan Vincent M
Published: November 2023

With the application of modern techniques for ultrastructural imaging and macromolecular characterization has come an appreciation for the high degree of structural organization found within cells. An emerging theme in cellular organization is that for many metabolic pathways, cells utilize large multienzyme complexes that are maintained through specific protein: protein interactions (1). Defining these interactions has become, and will remain, an essential part of the functional characterization of proteins.
Engineering of novel neurotrophins
Authors: Ilag Leopold L , Ibanez Carlos F
Published: November 2023

Neurotrophins constitute a class of growth factors that is essential for neuronal survival and differentiation in both the central and peripheral nervous systems. This class of proteins includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Neurotrophins influence the differentiation, survival, and regeneration of vertebrate neurones and although there are specific activities associated with each neurotrophin, overlapping functions also exist. Illustrating this point is the fact that all members of the family support, in varying degrees, the survival of embryonic neural crest-derived sensory neurones, NGF.specifically supports the survival of embryonic sympathetic neurones but not placode-derived sensory neurones, which are supported by BDNF and NT-3 (1).
Immunolocalization and RT-PCR for the detection and quantification of growth factor and receptor gene expressi•on
Authors: Myers Simon R , Navsaria Harshad A
Published: November 2023

In many research fields that impact directly on clinical medicine (e.g. wound healing) a new emphasis on the examination of human tissue has emerged facilitated by the availability of more sophisticated laboratory techniques. This immediately defines the limiting factor in choosing an assay as the volume of human tissue available. To investigate human growth factor and re ceptor gene expression in any depth, assessment of both protein and messenger RNA (mRNA) for ligand and receptor must be made, in terms both of quantity and of distribution, on small tissue samples. 6 mm punch biopsy wounds of skin for example can be sutured directly, or converted into a small ellipse. In the former case the sutures tend to pull out to leave a pale, circular scar, and in the latter a linear scar of< 1 cm length remains. A 6 mm punch biopsy of skin provides around 60 mg wet weight of tissue. For protocols that require repeated biopsy of an individual, 3 mm punch biopsies are preferable. These can be left to heal by secondary intention, leaving a minimal ‘pit’ scar. Note: researchers must meet all local and/or national ethical requirements before taking biopsies for experimental purposes from human subjects.
Nucleotide- and dye-ligand chromatography
Authors: Mattiasson Bo , Galaev Igor Yu.
Published: November 2023

A.s the level of competition between the manufacturers of rival biotechnological products increases, downstream processing costs, which can account for up to 80% of the overall production costs, will become more and more critical in determining product competitiveness (1). Since the late 1960s bio specific separation methods have come into focus and have been continuously improved. A fundamental advantage of affinity techniques is their predictive and rational character, since the ligand selected is designed to interact specifically with the protein to be purified. Among main important developments in affinity techniques is the use of more robust adsorbents and coupling chemistry that are not prone to leaking immunogenic degradation products. One such group of robust adsorbents is presented by matrices with nucleotide-ligands or dye-ligands, the latter exhibit a remarkable propensity to bind, sometimes specifically, to a plethora of proteins and enzymes.
Immunoaffinity chromatography
Authors: Cutler Paul
Published: November 2023

The principle of immunoaffinity or immunoadsorption chromatography is based on the highly specific interaction of an antigen with its antibody (1). Immunoaffinity chromatography is a specialized form of affinity chromatography and as such utilizes a ligand (either antigen or antibody) immobilized onto a solid support matrix in a manner which retains its binding capacity. Although the technique includes the separation of antibodies using immobilized antigens (2-4), it is more commonly performed for the identification, quantification, or purification of antigens (Figure 1). The crude extract is pumped through the column and the unbound material washed clear prior to elution of the retained antigen by alterations to the mobile phase conditions which weaken the antibody-antigen interaction.
Purification of DNA-binding proteins
Authors: Hanoa Hiroshi , Yamaguchi, Yuki , Wada Tadashi
Published: November 2023

Sequence-specific DNA-binding proteins are involved in many biological processes, such as DNA repair, recombination, replication, and transcription. To characterize these processes, it is quite important to purify the products that participate in them to homogeneity. The purified proteins can then be used to analyse their biochemical properties, to raise antibodies against them, and to isolate the genes encoding them.
Preparation of liposomes
Authors: Lasch J , Weissig V , Brandl M
Published: November 2023

Store organic solutions of phospholipids in a glass container layered with argon or nitrogen below -20 °c, preferably at -78 °C. The closure for the vial should be lined with Teflon. Alternatively, the vial can be sealed with Parafilm®. Never store organic solutions in polymer or plastic containers as this will leach impurities out of the container material (2). When transferring a portion of the material, remove the container from the freezer and allow it to reach room temperature before opening the bottle.
Long-Circulating Sterically Protected Liposomes
Authors: Klibanov Alexander L , Torchilin Vladimir P , Zalipsky Samuel
Published: November 2023

Soon after liposomes were proposed as vehicles for drug delivery and targeting, their major disadvantage was discovered: Typical liposome compositions rapidly exited the bloodstream and accumulated in the Kupffer cells in the liver, as well as in spleen macrophages (RES) (1). Various approaches to overcome this problem have been tested. Initially, the simple approach of blocking RES cells by administering an excess of ‘empty’ liposomes, a colloid dispersion or dextran sulfate was tested (2, 3). However, blocking RES is not the most appropriate approach for human therapy. On the other hand, to target liposomal pharmaceuticals to organs other than the liver and spleen, liposomes have been modified with specific targeting moieties, e.g. antibodies. However, immunoliposomes also do not exhibit prolonged circulation times and fail to accumulate sufficiently in the targets with limited blood supply and/or low antigen concentration (i.e. tissues and organs that require increased circulation times for liposome-to-target interaction). It is evident that a longer circulation time increases the probability that the liposomes will interact with the target tissue, yielding better targeting. Therefore, numerous attempts have been made to prolong the circulation time by varying liposome properties.
Gene expression databases
Authors: Davidson Duncan , Baldock Richard , Bard Jonathan , Kaufman Matthew , Richardson Joel E , Eppig, Janan T , Ringwald Martin
Published: November 2023

The rapid accumulation of large amounts of information from in situ hybridization experiments, together with the spatial and temporal complexity of this kind of data, presents serious difficulties for conventional publishing methods and for the individual researcher who wants to collate and analyse other people's results in relation to his own studies. The solution to these problems is to construct a database of gene expression and several are now being built; most apply to embryos, but in the future it is likely that they will be extended to include the adult.
Basic optical microscopy
Authors: Lacey A J
Published: November 2023

The perception of, or the sight of, an object whether in a microscope or in an everyday situation is the result of a complex process. It begins with light inter acting with the object. The modified light is collected by the lens of the eye and brought to a focus on the retina of the eye. The nerve responses in the retina are interpreted by the eye/brain system programmed to a greater or lesser extent by previous experience. The area of the specimen seen by the eye is the perception of the extension in x and y directions, while the experience of the of the z direction is the perception of depth.