bluuebunny/crossref_metadata_2025_split huggingface dataset:
Culture and characterization of human endothelial cells
Authors: Hinsbergh Victor W M Van , Draijer Richard 
Published: November 2023

Abstract
Endothelial cells, the lining cells of all blood vessels, are involved in many physiological and pathophysiological processes, including haemostasis, vasoregulation, inflammation, angiogenesis, and the extravasation of fluid, macromolecules, hormones, and leucocytes. The recognition that endothelial cells are involved in all these processes went in parallel with the ability of investigators to study these cells in culture. Endothelial cell culture was first established in the early 1970s (1, 2). Nowadays, a whole repertoire of molecular and cellular approaches are available to study the activation and metabolic regulation of endothelial cells in vitro and in vivo. Although one has to realize that endothelial cells from different types of vascular beds have distinct features, much information regarding these cells can be obtained using cell culture methods. This chapter describes the culture and characterization of human umbilical vein and microvascular endothelial cells, and simple techniques to study the permeability of human endothelial cell mono-layers in vitro.
Working with Laboratory Fermenters
Authors: Collins Stephen 
Published: November 2023

Abstract
Shake flasks are commonly used to grow microbial cells in small quantities at relatively low cell densities (up to 5-10 g/litre). However, the experimenter has little control over the environment experienced by the microorganisms. Process variables such as pH or oxygen concentration are likely to change during the course of the experiment. Terms such as late log phase are commonly used to describe such cultures when there is, in fact, no data to support the assumption of constant growth rate implied in the term. Indeed. it is likely that changes in the environment will affect the physiology of the cells.
Separation of cells using free flow electrophoresis
Authors: Eggleton P 
Published: November 2023

Abstract
The principal physical characteristics that have been exploited for cell separation techniques are their size (volume), density, or surface electrical charge. If cells differ sufficiently in density, then density gradient sedimentation techniques should be used (see Chapter 2). Cells which differ in size or volume can be separated by countercurrent centrifugal elutriation (see Chapter 3). As all mammalian cells carry a net surface negative charge at physiological pH, free flow electrophoresis (FFE) is able to maximize subtle differences in the charge of cell surface molecules leading to the separation of cells from one another. As the electrophoretic separation normally occurs in a continuous flowing buffer which exceeds 1 gsedimentation, within certain limits, the size or density of the cell is not normally influential in the separation.
Evaluation of Antisense Effects
Authors: Nellen Wolfgang , Lichtenstein Conrad 
Published: November 2023

Abstract
Antisense-mediated gene silencing by expression of antiscnse RNA in transfected cells has become a complementing alternative to gene disruption or gene ‘knock-outs’ by homologous recombination. Though powerful and often successful. there are unknown mechanisms which may sometimes hamper the effectiveness of the experiment. As with experiments with antisense oligonucleotides used to transiently interfere with gene expression, there may also be unexpected side-effects leading to the wrong interpretation of the results (see Chapter 11).
Selecting, Preparing, and Handling Antisense Oligodeoxyribonucleotides
Authors: Toulmé Jean-Jacques , Cazenave Christian , Moreau Serge 
Published: November 2023

Abstract
Combined developments in DNA sequence analysis and in oligonucleotide (oligo) chemistry make the antisense oligo approach a very attractive one to regulate gene expression. Knowing the primary sequence of the gene one wishes to turn down allows the design of a complementary antisense oligo sequence which, upon binding to its cognate RNA, may selectively prevent the function of the target gene. Since this very simple idea was first proposed by a Russian team in the late 1960s (1), numerous examples of successful inhibition of gene expression by antisense oligos have been published in the scientific literature. This ranges from pioneering work in cell-free extracts and cultured cells (2, 3) to recent in vivo experiments (4, 5). Even therapeutic trials in human beings are under way for AIDS and cancer (6). See Chapter 10 for medical applications.
Non-Antisense Effects of Oligodeoxynucleotides
Authors: Stein C A , Krieg Arthur 
Published: November 2023

Abstract
A conceptual problem in the application of oligodeoxynucleotides (oligos) as antisense, anti-gene, or anti-protein agents is that there is no essential difference in the oligo synthesized for either of these three applications. The designation of an oligo as ‘antisense’, or ‘triplex’, or ‘aptamer’ is actually frequently based more on the intention of the investigator than on any scientifically determined mechanism of action. In principle, any oligo might have any of these activities, depending on the relative values of KJ for different oligo binding sites and on the intracellutar localization of the oligo. As an example, a 16 base oligo synthesized to be a triplex-forming oligo was shown to have fortuitous complementarity to an mRNA sequence at 12 non-contiguous bases; this was sufficient to hybridize and activate RNase H, causing degradation of that RNA (1). In addition, many ‘antisense’ oligos have been found to show non-sequence-specific effects; this may perhaps be due to their polyanionic backbones (reviewed in ref. 2).
Enzymology of the replication fork
Authors: Burgers Peter M J 
Published: November 2023

Abstract
The assembly of the replisome is an ordered process mediated by both protein DNA and protein-protein interactions. The initial steps in this process, origin recognition by the initiator proteins and localized melting of the origin into a replication bubble, will be discussed in Chapters 2 and 3 of this volume. This chapter focuses on the elongation process of DNA replication and the activities of the proteins at the fork. It combines our knowledge of individual replication proteins from mammalian systems and their application to SV40 DNA replication with genetic and biochemical studies of the analogous proteins in yeast. Comprehensive reviews detailing each of these fields have been published (1-10). With the exception of the putative DNA helicase that unwinds the DNA, all enzymes and factors that are predicted to function at a chromosomal replication fork from studies with model systems have been identified in these two eukaryotes, and most of their genes have been cloned. The eukaryotic replication fork contains both DNA polymerase a and DNA polymerase 8 and their accessory factors. Although not evident from viral replication studies, DNA polymerase £ may also participate at the chromosomal DNA replication fork. Finally, recent progress on the maturation of Okazaki fragments initiated by ribonuclease Hl and a 5’-3’ exonuclease will be discussed.
The initiation of DNA replication in the yeast Saccharomyces cerevisiae
Authors: Marahrens York , Stillman Bruce 
Published: November 2023

Abstract
The discovery that DNA consists of two complementary antiparallel strands associated by hydrogen bonds immediately led to the realization that the genetic material duplicates by using the preexisting strands as templates (1). DNA poly­merase was discovered shortly thereafter (2) and marked the onset of increasingly detailed enzymological studies of the template-driven reaction whose mechanism is now understood in great detail (Chapter 1). Insight into how DNA replication is initiated was gained much more slowly. Most progress has been made by studying the replication of the genomes of prokaryotes, bacteriophages and some eukaryote viruses, and the basic mechanism has been deduced for some of these organisms. How eukaryote cell chromosomes initiate DNA replication remains an unsolved problem.
Role of nuclear structure in DNA replication
Authors: Hozák Pavel , Jackson Dean A , Cook peter R 
Published: November 2023

Abstract
The idea that DNA polymerases track along the template as they duplicate the DNA pervades our thinking (Figs 1a-c) (1, 2; and this volume). We imagine that it must be the small polymerase that moves along the very much larger template and that each polymerase acts relatively independently of others. However, recent evidence suggests many active polymerases are concentrated together into large nuclear structures - ‘factories’ - and that replication occurs as a number of templates slide past the many enzymes fixed in the factory; the template moves whilst the enzyme remains stationary, rather than vice versa (Figs ld-f). Then, higher-order structure dictates how and when DNA is replicated. Discussion concentrates on eukaryotic enzymes, but as the catalytic domains of all polymerases are structurally related (3), prokaryotic enzymes probably work in the same way.
Affinity maturation of antibodies using phage display
Authors: Johnson Kevin s. , Hawkins robert e. 
Published: November 2023

Abstract
Protocols described in this section will deal with alteration of the properties of existing antibodies, formatted as scFv and displayed on bacteriophage. Although the title implies a focus on improving affinities, provisions are made for alteration in off-rate and specificity. Where the aim of the ‘matura tion’ process is a shift in specificity, this can be achieved by introducing bias into the selection process.
Human antibody repertoires in transgenic mice: manipulation and transfer of YACs
Authors: Davies Nicholas p. , Popov andrei v. , zou xiangang , bruggemann marianne 
Published: November 2023

Abstract
Therapeutic applications of an accessible human antibody repertoire are vir tually limitless. Considerable interest in the techniques for producing human antibodies are apparent from clinical results which highlight their benefits (1). The problems that occur with immunogenicity, when antibodies of a foreign species are being used for therapy, unambiguously point out that the affinity and authenticity of the antibody are critical (1-3 and references therein).
Measuring antibody affinity using biosensors
Authors: Hefta Laura J , Wu Anna M , Neumaier Michael , Shively John E 
Published: November 2023

Abstract
Antibodies are often engineered to improve their specificity and affinity for a given antigen. One of the first goals in evaluating newly engineered anti bodies is to measure their affinity constants. Recently, a new technology has become available that allows the measurement not only of antibody equilib rium constants, Ka (association) or Ka (dissociation), but also of their kinetic rate constants, k0n and koff, where Ka= k0fflk00 The biosensor-based technol ogy permits real-time mass measurements using either surface plasmon reso nance (SPR, BIAcore) or a resonant mirror (IAsys, Fisons). In general, a protein sample (ligand) is coupled to the sensor chip via a hydrogel (car boxymethyl dextran) and solutions containing different concentrations of a binding protein (ligate) are allowed to flow across the chip while monitoring binding with mass sensitive detection (association step). At the end of a bind ing experiment, it is possible to flow buffer across the chip and observe the dissociation step. This is usually done at saturating conditions, where rebind ing of the ligate is not appreciable. Appropriate analysis of the binding and dissociation steps allows the calculation of kinetic constants k00 and k0ff, and as mentioned above, Ka can be calculated from the ratio of the two kinetic constants.
Assembly of chromatin and nuclear structures in Xenopus egg extracts
Authors: Almouzni Genevieve 
Published: November 2023

Abstract
The structural organization of DNA within the eukaryotic nucleus is tightly defined to accommodate the functional properties of a given cell type. Nuclear functions such as replication and transcription operate in a highly regulated fashion using chromatin as a template. During every cell cycle, each nucleus has to replicate itself, requiring duplication not only of DNA but also of the individual chromosomes together with all their structural and regulatory features. Chromatin organization is known to influence gene expression ( 1 ) and its remodelling during replication and cell cycling can affect the transcription process. To study the relationship between the structural organization of DNA and macromolecular events such as replication and transcription, an adequate system is required, accurately reproducing both structural and functional features of the eukaryotic cell.
Epitope mapping of carbohydrate binding proteins using synthetic carbohydrates
Authors: Nilsson U J , Magnusson G 
Published: November 2023

Abstract
Specific recognition of carbohydrates by proteins is involved in a number of important biological phenomena. For example, carbohydrates may act as cell surface antigens, e.g. in the ABO, Lewis, P, and I blood group systems or as tumourspecific antigens; they control growth and tissue development in an embryo, and are involved in fertilization; they regulate nerve growth and neural cell adhesion; and they direct the recruitment of leukocytes to damaged tissue (1, 2). Furthermore, carbohydrates often act as recognition sites for pathogenic bacteria and viruses (3). As a consequence, detailed knowledge on a molecular level concerning the recognition mechanisms and driving forces would open up possibilities to specifically interfere with, or promote, biological phenomena stemming from carbohydrate-protein interactions (i.e. development of therapeutics that target carbohydrate-binding proteins) (4).
Transposons—gene tagging and mutagenesis
Authors: Grigliatti T A 
Published: November 2023

Abstract
The discovery of mobile genetic elements and their association with ‘spontaneous’ mutations in nature has led to novel ways of cloning genes, of germline transformation, and has added methods for creating certain types of mutations. To date, the P element is the most exploited transposable element in Drosophila, but there are many others, several of which have been characterized sufficiently to allow similar applications with perhaps only a little more effort. For example, the hobo element has been used for both transformation and gene tagging, the IS elements have been used for mutagenesis and gene tagging, and mariner has been used for transformation. These other mobile elements may have properties which, in the future, make them better for certain applications, such as target site specificity, efficiency of movement, or homologous target integration (gene replacement). But, this chapter focuses on some of the common applications of the P transposable element technology for creating useful mutations. Specifically it focuses on:
Looking at embryos
Authors: Wieschaus Eric , Nusslein-Volhard Christiane 
Published: November 2023

Abstract
In Drosophila, the pattern observed in mutant embryos often provides the first indication for the role of the wild-type gene in normal development. The loci which affect embryonic pattern, represent only a small fraction of the total genome and their phenotypes are often very discrete. With practice, new mutations in a given locus can be recognized strictly on abnormalities observed among the unhatched embryos from a mutagenized line. The easy recognizability of embryonic phenotypes greatly simplifies mutagenesis experiments and has proved very useful in subsequent analysis of the mutations obtained.
Immunolabelling of Drosophila
Authors: White Robert A H 
Published: November 2023

Abstract
Specific antibodies provide the easiest way, in general, of studying the distribution and subcellular localization of proteins. Such knowledge can give important clues to their role in development and cell biology. Using an antibody to detect the protein product of a specific gene is a convenient way of following gene expression patterns in development and allows the analysis of both transcriptional and post-transcriptional gene regulation. Antibodies are also widely used as markers for individual tissue and cell types enabling these to be followed during morphogenesis (see ref. 1). Multiple labelling can be used to correlate the expression patterns of different proteins. The use of immunolabelling is now so established that many research papers merely say ‘Antibody labelling was by standard methods’. However, it is clear that appropriate fixation, labelling, and detection can make a huge difference to the sensitivity and clarity of the result, and indeed whether the labelling actually gives a tolerably faithful representation of the in vivo protein distribution.
Eukaryotic DNA polymerases
Authors: Wang Teresa S -F , Conger krista L , Copeland William C , Arroyo Martha P 
Published: November 2023

Abstract
DNA polymerases are the fundamental enzymes that synthesize cellular DNA to produce two identical daughter chromosomes. Cellular DNA polymerases in conjunction with DNA repair enzymes play critical roles for the transmission and maintenance of error-free genetic information from one generation to the next. In the last decade, genetic studies in yeast, biochemical studies of in vitro replication of the simain virus 40 (SV40) origin-containing DNA, and isolation of genes and cDNAs of DNA polymerases from budding yeast, fission yeasts, and mammalian cells led to the identification of five distinct DNA template-dependent DNA polymerases, named polymerase α, β, γ, δ, and ε(1,2).
Saccharomyces cerevisiae as a model system to study DNA replication
Authors: Foiani Marco , Liberi Giordano , Piatti Simonetta , Plevani Paolo 
Published: November 2023

Abstract
The budding yeast Saccharomyces cerevisiae represents a powerful tool to study DNA replication and the cell cycle regulatory pathways controlling entry into, and progression through S phase. In fact, the availability of the complete sequence of the S. cerevisiae genome, the identification of several DNA replication genes, together with the possibility of combining molecular genetic tools with classical techniques of genetics, biochemistry, and cell biology, make this system particularly useful to improve our knowledge of the DNA replication process at the molecular level. Moreover, by considering that the structure and function of most of the replication proteins and the regulatory circuits connecting DNA replication to other cell cycle events have been highly conserved during evolution, the information obtained using the S. cerevisiae or the S. pombe systems can be generally extended to other eukaryotic cells.
Cell cycle checkpoints: safe passage through mitosis
Authors: Yu Kristina R , Duronio Robert J , Sullivan William 
Published: November 2023

Abstract
Mitosis results in the production of two daughter cells containing identical genetic complements. Achieving this requires a carefully orchestrated series of nuclear and cytoplasmic events leading to the accurate replication and segregation of the chromosomes. A powerful combination of molecular genetics, cell biology, and biochemistry have led to the isolation of key structural, enzymatic, and regulatory components governing these events. One of the most gratifying aspects of this research has been the realization that many of these components are highly conserved throughout the phyla. Therefore many of the lessons learned from one system will probably apply to others.
Meiosis: chromosome behavior and spindle dynamics
Authors: Karpen Gary H , Endow Sharyn A 
Published: November 2023

Abstract
Meiosis is an alternative pathway of development that leads to the production of haploid cells, usually spores or gametes, for sexual reproduction, rather than the identical or near identical diploid cells produced by mitosis. The recent discovery of analogous proteins that are involved in the regulation of both meiosis and mitosis makes it increasingly apparent that meiosis is closely related to mitosis, and can be regarded as a highly specialized mitotic division. Although meiosis differs from mitosis in several respects, many of these differences are variations of events that occur during mitosis. Some, however, are not. Probably the most radical departure from mitosis occurs during meiosis I and concerns the behavior of the meiotic chromosomes: homologous chromosomes in most organisms undergo pairing (initial alignment of homologs in zygotene), synapsis (intimate association of homologs at pachytene) and recombination (physical exchange) during meiotic prophase, which are not observed in mitotically dividing cells. These events are followed by two successive divisions without an intervening replication, instead of the single division that occurs in mitotic cells, resulting in reduction of the chromosomes to the haploid number (Fig. 1).
Changes occuring in polyphenol substances content during wort production
Authors: Srogl Jiri , Kosar Karel , Mikyska Alexandr , Bousová Pavla 
Published: November 2023

Abstract
This article deals with the release of polyphenol substances to the solution during the wort production. The hop polyphenols are being released to the wort easily. Addition of neither unmalted barley nor of husks during mashing will increase the content of polyphenols in the wort, not even in the mixture with a significant surplus of well modified malt. In the course of mashing the polyphenol substances are not being released, not even from the malt. Decisive factor influencing the content of malt polyphenols in the wort is the malting process.
Rate analysis of dimethyl sulfide volatilization during wort boiling
Authors: Mitani Yutaka , Ishida Fumito , Akiyama Hiroshi , Tamaki Teruo 
Published: November 2023

Abstract
The decrease in dimethyl sulfide (DMS) in boiling wort indicates how much volatile compounds have evaporated during this operation. DMS volatilization behaviour could be analysed as a consecutive process.
High gravity brewing and its negative effect on head retention
Authors: Bryce James H , Cooper Dan , Stewart Graham G 
Published: November 2023

Abstract
The aim was to establish why beer brewed using high gravity techniques has poor head formation and retention. Work focused on measuring hydrophobic polypeptides, identified as important foam stabilising molecules, throughout the brewing of lagers at high (20°P) and low (10°P) gravities. The efficiency of hydrophobic polypeptide extraction during high gravity mashing was significantly less than during the mashing of normal gravity wort. Furthermore, high gravity fermentations had a greater loss of hydrophobic polypeptides. Thus, the low gravity lager had up to 40 % more hydrophobic polypeptides. Modifications to high gravity brewing can now be recommended to maximise foam potential.
New molecular probes for assessing stress conditions in brewing yeast: biotechnological implications and applications
Authors: Lopez Victoria , GiJU Rosario , Navarro José V Carbonell & Alfonso 
Published: November 2023

Abstract
Modem day brewing stimulates technological innovations allowing more rapid processing and greater consistency and contml over the fermentation and the physiological condition of pitching yeast. However, we are still far from being able to predict and control yeast performance. Trying to choose the right probe to allow us to detect and monitor stress situations in yeast, we have targeted our attention to 20S RNA and 23S RNA which are single stranded yeast replicons whose copy number is greatly induced under stress conditions. The molecular characterization of brewing yeast strains in relation to the presence or absence of these yeast RNA viruses together with their potential applications in process control are described.