bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assays for macrophage activation by cytokines
Authors: Doyle Anthony , Stein Michael , Keshav Satish , Gordon Siamon 
Published: November 2023

Abstract
The circulating monocyte may undergo a wide spectrum of functional changes during further differentiation. In the absence of inflammatory stimuli monocytes enter the tissues constitutively to become resident macrophages (1). These cells are relatively quiescent with respect to secretory or microbicidal activity and may have as yet uncharacterized trophic functions. In contrast, inflammatory recruitment causes monocytes to rapidly induce their secretory activity. These cells produce a large variety of enzymes, growth factors and cytokines when appropriately triggered. Lymphokines produced at the site of inflammation regulate the fate of elicited macrophages determining whether they are further stimulated to a heightened state of activation with maximal host-defence function, e.g. by interferon-γ (IFN-γ) or whether their secretory and defence activity is held in check (e.g. by IL-4 and IL-10).
Measurement of proliferative, cytolytic, and cytostatic activity of cytokines
Authors: Burke Frances , Rozengurt Enrique , Balkwill Frances R 
Published: November 2023

Abstract
Most cytokines have proliferative, cytostatic, and/or cytolytic activity which is often dependent on the context in which they are acting. IFN-α, β, and γ, TNF, Lymphotoxin, IL-1, IL-4, have all been shown to have proliferative, cytostatic and cytolytic activity depending on the environment (1). Synergistic interactions between cytokines, can often produce powerful antiproliferative effects (2). There is some experimental evidence to suggest that growth inhibitory cytokines such as IFN-a may act in vivo as autocrine or paracrine growth inhibitors (3). Some of the methods used to assess the above par ameters will be described in the following chapter.
Production of cytokine transgenic and knockout mice
Authors: Pasparakis Manolis , Kollias George 
Published: November 2023

Abstract
Transgenic mice have served for many years as fine tools in the dissection and understanding of complex biological phenomena. Studies on gene function in multifactorial and multicellular systems have most benefited, because they often require experimental settings which faithfully simulate the in vivo situation. Additional technological advances which become accessible to a growing number of investigators, offer the opportunity to inactivate or modify endogenous genes in the mouse and open up new possibilities for research. In general, perturbation of gene expression in transgenic systems often leads to measurable alterations in the physiology of the animal and important functional implications come to light.
Development of antibodies to cytokines
Authors: Poole S 
Published: November 2023

Abstract
Antibodies to cytokines can greatly facilitate research on many aspects of these mediators: including the development of specific and sensitive immuno-assays (see Chapter 22); the detection of cytokines in biological fluids, tissues, and cells using immunochemical and immunocytochemical techniques; immunoneutralization of biological activities, and delineation of external regions (epitopes) of the cytokines.
RIA, IRMA, and EUSA assays for cytokines
Authors: Meager A 
Published: November 2023

Abstract
Instructions for 125I-labelling with Enzymobeads (Bio-rad) and the Bolton Hunter reagent (Amersham) are supplied by the manufacturers. A further alternative is the N-bromo-succinimide (NBS) method of Reay (3) and this has been successfully applied to the radioiodination of interleukin-1β (3 (4).
Generally speaking, most current radioimmunoassays for cytokines employ a competitive inhibition assay method. In brief, this means that variable amounts of cytokine, as serial dilutions of a standard or in samples, are incubated with a fixed amount of diluted polyclonal anti-cytokine antiserum, followed by a further incubation period with a fixed quantity of 125I-labelled cytokine. Finally, antibody-cytokine complexes are removed from solution by addition of a second antibody (to the first species immunoglobulin) or other antibody-binding reagent, e.g. protein A. The amount of 125I-cytokine bound therefore decreases as the concentration of unlabelled cytokine increases.
Measuring parameters of growth
Authors: Baserga Renato 
Published: November 2023

Abstract
This chapter deals with the various parameters of growth and how they can be measured. A tissue can grow by: (i) increasing the number of cells; (ii) increasing the size of the cells; or (iii) increasing the amount of intercellular substance. Since the intercellular substance of a tissue is usually a secreted product of the cell, for example collagen, it can be considered as an extracellular extension of the cytoplasm. We can therefore consider an increase in intercellular substance as a variation of an increase in cell size and thereby reduce tissue growth to two mechanisms, growth in size and growth in the number of cells. This is true regardless of whether we are dealing with normal or abnormal growth, with the intact animal or with cells in culture.
Studying transport processes in absorptive epithelia
Authors: Artursson Per , Karlsson Johan , Ocklind Goran , Schipper Nicolaas 
Published: November 2023

Abstract
Transport processes in epithelia have traditionally been studied in a variety of organ and tissue preparations from different species. However, native epithelial tissues are complex and contain many different cell types. Monolayer cultures from established epithelial cell lines have the advantages of being structurally simple, homogeneous, and easy to manipulate. Transport studies in epithelial monolayers are, therefore, often easier to perform and interpret (1). The major disadvantage of epithelial cell cultures is that in most situations they are unlikely to serve as a complete substitute for the more complex normal epithelium. Moreover, epithelial cells in culture may express carriers and enzymes in a different or more variable manner than seen in vivo. Despite these disadvantages, recent studies in epithelial cell monolayers have brought new insights into the functions of a variety of transport mechanisms of endogenous as well as exogenous molecules such as drugs. The aim of this chapter is to present some of the protocols used in our laboratory to study passive and active drug transport processes across monolayers of the human intestinal epithelial cell line Caco-2 grown on permeable supports (Figure 1). However, before such studies are performed it is important to know how to culture and assess the integrity of the cell monolayers on permeable supports in a reproducible manner. Methods for such characterization are presented initially. The methods for studying integrity and transport processes are general and should, therefore, be applicable to solutes other than drugs and to epithelial cell cultures other than Caco-2.
Reconstruction of human skin epidermis in vitro
Authors: Lenoir-Viale Marie-Cécile 
Published: November 2023

Abstract
Since the pioneering work of Rheinwald and Green in 1975 (1, 2) for establishing keratinocytes in culture, numerous advances have been made in the reconstruction of human epidermis in vitro. The more recent techniques are based on the reconstruction of a substrate resembling dermis on which keratinocytes of different sources can be cultivated. After immersion for a few days, these cultures are raised to the air-liquid interface in order to mimic in vivo conditions.
Design and Optimization of Growth Media
Authors: Greasham Randolph L , Herber Wayne K 
Published: November 2023

Abstract
The primary objective in developing a microbial culture medium is to ensure that the required nutrients are present in appropriate forms and at non inhihitory concentrations. This objective can he quite challenging when considering the tremendous diversity of microorganisms present in nature, exhibiting diverse nutritional requirements ranging from simple chemicals, such as atmospheric carbon dioxide and a few inorganic salts, to complex chemicals, such as lipids, vitamins and nucleotides.
Measurement of Biomass
Authors: Ison Andrew P , Matthew Gavin B 
Published: November 2023

Abstract
The on-line measurement of some physical parameters in fermentation broths, such as pH and temperature, is relatively easy and commonplace (see Chapter 4). This has been made possible by the advanced technology that has now become standard. As yet, on-line monitoring of biomass in the fermenter has not become routine. However, there are many techniques available for biomass determination.. This chapter gives detailed protocols for those techniques likely to be used by microbiologists, biochemists. microbial physiologists. and bio chemical engineers. For those requiring substantial investment and specialist equipment, a strategy for the application of the technique is given. In these cases it is necessary to assume that the operator has gained some experience with the equipment and is familiar with the operating manuals of commercially available equipment. Most techniques will require some investment of time, dependent on the experience of the researcher, to personalize the technique for the specific application and to ensure its reproducibility.
Detection Of Sense: Antisense Duplexes By Structure-Specific Anti-Rna Antibodies
Authors: Lukacs Noemi 
Published: November 2023

Abstract
It is generally assumed that inhibition of gene expression by antisense RNA involves the formation of sense: antisense RNA hybrids (for review see refs 1-3). The formation of such hybrids is well established in prokaryotic systems and has also been shown experimentally in a few cukaryotic systems (4-6). In most eukaryotic organisms, however, attempts to detect sense:antisense RNA complexes have failed, although the biological effects of the antisense RNA were obvious. Since in several cases the simultaneous synthesis of both RNA strands has been confirmed by nuclear run-on transcription assays, and since reduced steady state levels of sense RNA have been detected, it has been proposed that sense:antisense hybrids are very rapidly degraded in vivo (7-9). In addition to the classical duplex formation degradation mechanism other antisense control mechanisms may also exist, and have been discussed (1-3, 7-9).
SV40 DNA replication
Authors: herendeen Daniel , Kelly thomas j. 
Published: November 2023

Abstract
Much of what is currently known about the mechanisms of DNA replication in eukaryotic cells has come from studying SV40 and related viruses. SV40 is a small DNA-containing virus that is indigenous to Asian macaques. It produces a lytic infection in permissive primate cells in culture and it is capable of transforming nonpermissive rodent cells to a tumorigenic state. The virus has served as a power-ful model system for understanding many aspects of the molecular biology of eukaryotic cells, including transcription, DNA replication, and the regulation of growth (1-4).
JJroducing antibodies in Escherichia coli: from PCR to fermentation
Authors: Pluckthun Andreas , Krebber Anke , Krebber Claus , Horn Uwe , Knupfer Uwe , Wenderoth Rolf , Nieba Lars , Proba Karl , Riesenberg Dieter 
Published: November 2023

Abstract
The expression of antibody fragments in Escherichia coli has become a widely used technique for several reasons; it is, in principle, an easily accessible methodology for anyone used to growing E. coli, and it can be advanta geously combined with phage-display technology which also makes use of E.coli (1-3).
Counting nucleosome cores on circular DNA using topoisomerase I
Authors: Clark David J 
Published: November 2023

Abstract
The nucleosome is the basic structural repeat of chromatin. It contains a nucleosome core, a single molecule of histone H 1, and a segment of linker DNA. The nucleosome core is composed of a central octamer of core histones (two molecules each of the four core histones: H2A, H2B, H3, and H4) and about 150 hp of DNA which is tightly wrapped around the octamer, forming nearly two complete turns. A nucleosome core protects one negative DNA supercoil from relaxation by topoisomerase I ( l ). This was shown by counting the average number of nucleosome cores reconstituted on a plasmid in vitro using the electron microscope and then comparing this with the average number of supercoils protected from relaxation in the same samples. This fact is now used routinely to determine the average number of nucleosome cores formed on a circular DNA: the DNA between nucleosome cores is relaxed using topoisomerase I, plasmid DNA is extracted, and the number of negative supercoils is measured in a gel containing chloroquine. The number of nucleosome cores is equal to the number of negative supercoils protected from relaxation.
Purification of RNA
Authors: Wilkinson Miles 
Published: November 2023

Abstract
The most important consideration in the preparation of RNA is to rapidly and efficiently inhibit the endogenous ribonucleases which are present in virtually all living cells. There are several classes of ribonucleases which have been well characterized in eukaryotic and prokaryotic organisms, including both endonucleases and exonucleases. Methods designed for the preparation of RNA depend on speed and/or efficiency of neutralization of ribonucleases for their success. Most methods stipulate the use of ribonuclease inhibitors such as RNasin, vanadyl ribonucleoside complexes, guanidinium hydrochloride, guanidinium isothiocyanate, heparin, and dextran sulphate, to name a few. Proteinase K is sometimes used to degrade ribonucleases, and organic solvents such as phenol and chloroform are used to remove ribonucleases by extraction procedures.
DNA sequencing
Authors: Brown T A 
Published: November 2023

Abstract
The rapid generation oflengthy, contiguous DNA sequences is one of the corner stones of molecular biology. Not every molecular biologist wishes to sequence an entire genome, but most will, at some stage in their careers, sequence a cloned fragment from a genomic or cDNA library or use sequencing to check the construction of a recombinant DNA molecule. This chapter describes the methodology for carrying out these tasks.
Combined B cell and T cell epitopes
Authors: Atabani Sowsan F 
Published: November 2023

Abstract
B and T cell epitopes can either be overlapping or contiguous within a single sequence. Indeed, numerous studies have determined that up to, and within, a synthetic peptide sequence as small as ten amino acids, the B and T cell recognition sites may be distinct (1). Surface immunoglobulins are capable of binding to both linear sequences of amino acid residues and conformational epitopes which represent non-adjacent amino acid sequences brought into close proximity within a native protein, whereas the T cell receptor recognizes linear processed peptide epitopes bound to major histocompatibility complexes on molecules. B cells are among antigen-presenting cells capable of presenting peptide epitopes to CD4+ T cells. T cells produce cytokines and other immunomodulators that provide help for antibody production and induction of memory B cells. A single B and T cell epitope comprises the minimal built-in subunit able to trigger T and B cell co-operation in vivo (2). Moreover, studies have shown that overlapping B and T cell epitopes within a synthetic peptide sequence does not impair its B cell immunogenicity (3). The ability of a free synthetic peptide representing a single amino acid sequence, which varies minimally between strains, to act as a functional immunogen able to induce both long-lasting cellular and antibody responses has major implications for the design of future vaccines. Indeed, there exists numerous examples of peptides which are immunogenic in the free, uncoupled state. These results suggest that a satisfactory single sequence can readily induce full protection of all vaccinated animals against a severe challenge with the virulent parent virus (4, 5). Therefore, the topographical relationship between T and B cell epitopes within a native protein sequence is of great interest. This chapter describes experimental methods by which to identify combined and overlapping B and T cell epitopes within the same amino acid sequence with the aid of synthetic peptides.
Generating monoclonal antibody probes and techniques for characterizing and localizing reactivity to antigenic determinants
Authors: Nelson Paul N 
Published: November 2023

Abstract
This chapter initially provides details of protocols useful for the generation of monoclonal antibody probes. Monoclonal antibodies (mAbs) in general, serve as powerful reagents in diagnostic applications and as tools for the investigation of macromolecules and cells (1, 2). However, it is the further scrutiny of their reactivity to desired target antigens, subsequently pursued in this chapter, which paves the way for the development of secure assay systems and reliable functional studies. The localization of a target epitope may be grossly identified though the combination of enzyme (or chemical) degradation studies, e.g. digestion of IgG to yield Fc and pFc’ fragments, and the analysis of mAb reactivity using techniques such as enzyme-linked immunosorbent assay, haemagglutination, and slot-blotting. Alternatively, in situ localization of cell surface or intracellular antigenic determinants may be achieved by immunocytochemical techniques.
Site-directed mutagenesis in epitope mapping
Authors: Perdue Samuel S 
Published: November 2023

Abstract
The utility of amino acid variants in epitope mapping originated with studies of naturally occurring evolutionary variants of hen egg white lysozyme (HEL) (1, 2) and myoglobin (3, 4). Seven avian lysozymes have been isolated that differ from HEL at 3–10 amino acids (human milk lysozyme differs at 52 positions). The epitopes for several anti-HEL antibodies have been crudely mapped to the lysozyme surface based on their differential recognition of these evolutionary variants (1, 2, 5). Similar studies were also performed using monoclonal antibodies specific for either human or sperm whale myoglobin and a panel of myoglobin variants from a number of vertebrates (3, 4).
Preparation of nucleic acids
Authors: Jowett T 
Published: November 2023

Abstract
This chapter describes protocols for:
(a) Purifying DNA for making libraries, pulsed-field gel electrophoresis (PFGE), Southern blot analysis, amplification.
(b) Purifying DNA and total RNA with and without organic extractions.
(c) Separately extracting both RNA and DNA from the same sample.
The methods described for preparing mRNA directly from whole animals utilize affinity chromatography in combination with spin columns or magnetic bead technology. These methods are suitable for work on a microscale to provide material for Northern blots or RT-PCR. Emphasis is placed on those methods which are easiest and quickest to perform as well as produce high purity of the final products.
Isolation and characterization of lagging strand processing activities
Authors: Bauer Glenn A , Melendy Thomas 
Published: November 2023

Abstract
As a result of the bidirectional DNA replication process, lagging strand synthesis results in the formation of Okazaki fragments, hybrid polynucleotide molecules consisting of a short 5’ RNA primer and a long DNA extension. Lagging strand processing involves the removal of the RNA moiety from these fragments and the completion of DNA replication to synthesize a continuous DNA daughter molecule on the lagging strand at the replication fork.
A moveable feast: the centromere-kinetochore complex in cell division
Authors: Sullivan Kevin F 
Published: November 2023

Abstract
The goal of cell division is to transmit the genome from one generation to the next. This occurs in two essential steps: the genome is first replicated in S phase and then during mitosis the two copies are separated and transported into separate cytoplasmic domains destined to become new cells. This transport function is specified primarily by the centromeres, specialized nucleoprotein domains present in a single copy on each chromosome (1). Centromeres direct assembly of the machinery for microtubule binding and motor activities, known as the kinetochore, at the surface of each sister chromatid, maintain cohesion between sister chromatids, and possess regulatory elements that integrate chromosome motility and spindle function with cell cycle control pathways. During mitosis the kinetochores interact with spindle microtubules first to bind the spindle in prometaphase, then to achieve the crucial bipolar orientation in metaphase and finally to drive poleward movement in anaphase. A parallel system of regulatory elements located within the centromere monitors kinetochore attachment and communicates with the spindle to control anaphase onset. On the other side of the anaphase switch, centromeres very probably function as targets for regulated proteolysis of the 'glue' that holds sister chromatids together.
Telomeres: structure, synthesis, and cell cycle regulation
Authors: Hughes Timothy R , Lundblad Victoria 
Published: November 2023

Abstract
Genomic integrity depends on a number of different processes affecting chromo some replication and stability. Telomeres are specialized structures found at the ends of linear chromosomes and are functionally defined based on their properties, which differ from those of ends resulting from chromosome breakage. Studies in Drosophila and maize from the 1930s and 1940s first demonstrated that broken chromosomes that lack telomeres are unstable and capable of fusing with other newly broken ends. These observations, showing that telomeres are required for proper linear chromo some function, have since been confirmed and extended in a number of experimental systems. Over the last two decades, advances in our molecular knowledge regarding features of telomeric DNA structure, the identification of a novel DNA polymerase responsible for replicating telomeres, and the characterization of numerous other proteins that interact with telomeres have greatly extended our understanding of this essential chromosomal element.
Les enseignements de 50 ans de relations entre sciences et technologies
Authors: Devreux A 
Published: November 2023

Abstract
Elie a été initiEe d’abord par des raisons économiques (main-d’oeuvre, énergie, …), parfois sanitaires (nitrosamines) ou environnementales et, plus rarement, par un apport scientifique inattendu. C’est le cas du trempage alterné, de l’utilisation de I’acide gibberellique et du PVPP et, plus récemment, des isohumulones réduits.
Amplified fragment length polymorphism: a new technique for barley varieties identification
Authors: Lauer I , Brignon P , Gendre (F) F 
Published: November 2023

Abstract
Plant fingerprinting is useful to identify varieties, to detect differences between species and to assess relatedness between them. AFLP technology is a novel DNA fingerprinting technique based on the selective amplification of restriction fragments from a total digest of genomic DNA. This technique is reliable, reproducible, rapid and allows us to visualise DNA polymorphisms without any prior knowledge of nucleotide sequence. Our results show that barley varieties are clearly distinguishable and moreover that one variety can be identified by its pattern.
Generation of fertile transgenic barley synthesizing thermostable 0-amylase
Authors: Kihara Makoto , Okada Yukio , Kuroda Hisao , Saeki Kazuo , Yoshigi Naohiro , Ito Kazutoshi 
Published: November 2023

Abstract
ß-Glucan solubilase releases ß-glucan and probably pentosans from cell walls making them more accessible to endo-B-glucanases and pentosanases respectively. An improved assay for measuring solubilase activity has been developed. Solubilase is present in raw barley and is relatively heat stable. Four fractions of solubilase activity have been located. Two of the fractions contain feruloyl esterase. Ester linkages involving ferulic acid, probably with the arabinoxylan component of the cell walls, may be disrupted by solubilase. The control of feruloyl esterase activity should therefore facilitate the efficient processing of B-glucan and pentosan.