bluuebunny/crossref_metadata_2025 huggingface dataset:
Technology for real time fluorescent ratio imaging in living cells using fluorescent probes for ions
Authors: Mason W T , Hoyland J , Davison I , Carew M A , Jonassen J , Zorec R , Lledo P M , Shankar G , Horton M
Published: November 2023

In this article, we shall discuss the means for applying fluorescent probes and computer-controlled image acquisition and analysis to previously intractable problems. Image processing is at the heart of virtually all of these new approaches. This article will focus mainly on how image acquisition and analysis can be used to study ionic gradients in living cells with optical probes. Some specific applications from our laboratory using this technology will be used to illustrate the potential for such techniques.
Chemical synthesis of oligosaccharides
Authors: Khan Shaheer H , Hindsgaul Ole
Published: November 2023

That oligosaccharides can function as ligands in biological recognition, usually through their binding to protein receptor sites, is now beyond dispute. The active structures have been identified in cases ranging in diversity from bacterial and viral adhesion to cell-cell recognition in the development of whole organisms. But the potential range of activities seems almost unlimited as noted in a recent, comprehensive and unusually open-minded review article entitled 'Biological function of oligosaccharides: all of the theories are correct’ (1).
The Contribution of the molten Globule model
Authors: Christensen Henriette , Pain Roger H
Published: November 2023

One of the conclusions that have been drawn from the 'thermodynamic hypothesis' of Anfinsen (1) is that amino acid sequence determines protein conformation. While ribonuclease has a very different structure from lysozyme there are NAD binding domains in different dehydrogenases in which the structures are very similar despite the lack of sequence homology (2). It is evident that the code relating sequence to conformation is highly degenerate as, by extension, must be the information that determines how the polypeptide chain folds. If we assume that similar 3D structures are the end-products of similar pathways, it seems reasonable to ask whether we should expect to find similarities, based on the physicochemical and structural properties of the amino acid side-chains, between the ways in which both the pathway and the final conformation are determined.
DNA radiolabelling and detection
Authors: Carvajal-Valdecasas Jaime
Published: November 2023

The hybridization process, whereby a target nucleotide sequence can be detected by the complementary binding of a labelled nucleic acid (DNA or RNA probe) is one of the most fundamental techniques used in molecular biology. Based on the procedure originally described by Southern (1), electro phoretic separation of DNA according to molecular weight, usually after endonuclease restriction, is followed by transfer and immobilization on a solid support such as a cellulose membrane. The target sequence is detected by hybridization (2) with a radiolabelled probe and subsequently visualized using autoradiography. Alternatively, non-radioactive probes may be used, coupled with an appropriate detection method. In this chapter, I describe the techniques and methods commonly used for the generation of radiolabelled probes to be used for the detection of DNA fragments by Southern blotting or RNA fragments by Northern blotting, respectively, for colony or plaque hybridization, and for in situ hybridization. All the main methods are covered except the use of PCR which is described in Chapter 6. The preparation of non-radioactive DNA probes is covered in Chapter 4.
Antibody probes
Authors: Banting George
Published: November 2023

The use of antibodies as tools in the fields of molecular and cell biology is widespread. They are used in the affinity purification of diverse molecules, in immunolocalization studies, as specific inhibitors of intermolecular inter actions, as a method of targeted delivery of conjugated molecules, and in a wide range of other applications. The aim of this chapter is to provide practical guidance regarding the production, characterization, and use of antibodies. For information not given here, the reader is referred to other sources, particularly the guide by Harlow and Lane (1). The focus of this chapter will be antibodies which recognize protein anti gens, although it is clear that antibodies to other molecules exist and are of undoubted importance and use. The structures recognized by antibodies are known as epitopes. An epitope can be defined as that region of a protein which is recognized by the antigen-binding site of an antibody. It may result from the local folding of a linear sequence of amino acids (a continuous epitope) or it may arise due to the spatial proximity of specific residues in the secondary or tertiary structure of the protein; residues which are far apart in the primary amino acid sequence (a discontinuous epitope). Thus, anti protein antibodies recognize specific epitopes; they do not recognize specific proteins. More than one protein may possess the same epitope, and this ‘epitope sharing’ may occur irrespective of linear sequence homology.
Screening recombinant DNA libraries by hybridization and amplification
Authors: Mason Philip J , Vulliamy Tom J
Published: November 2023

The methods described in this chapter are all designed to isolate and purify a particular recombinant DNA molecule from a complex mixture of such molecules. Whether one is screening a library containing an entire mammalian genome or subcloning from a few dozen DNA fragments, the techniques are essentially the same. In these days of large-scale genome projects, much gene isolation is performed using gridded libraries in cosmid or yeast artificial chromosome (YAC) vectors and these techniques are described in the following chapter. However, before detailed analysis of such clones can be made they must be subcloned into manageable pieces; moreover, there is not a genome project running for every organism. This chapter is concerned with the day-to-day use of library screening for the typical laboratory. The most common use of these techniques nowadays is probably in screening genomic or cDNA libraries in phage X using a radiolabelled DNA probe, so we will describe this procedure in detail. Modifications required when using different cloning vectors and probes will also be mentioned.
In situ hybridization of nucleic acid probes to cellular rna
Authors: dÉcimo Didier , Georges-labouesse Elisabeth , DollÉ Pascal
Published: November 2023

Hybridization of a nucleic acid probe to endogenous mRNA present in cytological or histological preparations is the most precise method for studying gene expression since it permits detection of the target mRNA virtually at the resolution of a single cell. For this reason, in situ hybridization (ISH) is widely used in various research areas, especially in the field of developmental biology, as well as for diagnostic purposes in pathology. Several types of nucleic acid probes and labelling methods have been described over the past years. At present, it appears that complementary RNA (cRNA) probes (riboprobes) labelled with a low-energy p emitter offer the best sensitivity and are thus most widely employed. We describe here a complete ISH protocol using S-labelled cRNA probes, applied to sections of paraffin- embedded tissue.
Directed mutagenesis and mutant analysis
Authors: White Roger , Butler Alison , Parker Malcolm
Published: November 2023

The ability to introduce specific mutations into a cloned DNA sequence provides a powerful technique for studying both gene regulation and the structural and functional properties of the encoded proteins. In particular, the construction of defined mutations allows analysis of specific properties and the organization of functional domains within a protein, and the determination of the role of specific promoter or enhancer sequences in gene regulation. The aim of this chapter is to describe the approaches and methodology involved in the design and construction of specific mutations and to explain how these techniques may be applied to the modification of any DNA sequence. The basic principles and methods of mutagenesis will be discussed, this will then be followed by a description of methods for identifying mutated sequences and a list of some of the applications of mutagenesis and methods of mutant analysis.
Human gene mutational analysis
Authors: Roberts Roland G
Published: November 2023

Mutational analysis of human genes is a key procedure in both human molecular biology and molecular genetic diagnosis. At the most fundamental level, it is rapidly becoming the rate-determining step in the identification of disease genes; recent advances in large-scale physical mapping, contig assembly and gene-hunting have meant that the route from meiotic linkage data to a set of candidate cDNAs has become a relatively short one. The ability to pare this set down to a single gene locus is then dependent on an efficient method for screening the candidate sequences for disease-associated mutations. After identification of a disease gene, much can be inferred about the function of parts of the gene product by ascertaining the nature and distribution of naturally occurring mutations which cause the disease phenotype. Characterization of such mutations also allows direct prenatal and carrier diagnosis in relatives of affected individuals.
Quantification of DNA and RNA by PCR
Authors: Kang Jie , Kühn Jochen E , Schäfer Peter , Immelmann Andreas , Henco Karsten
Published: November 2023

The ability to quantify low copy numbers of a specific nucleic acid target in limited amounts of sample is important in both basic and applied medical research. The combination of PCR and TGGE (temperature gradient gel electrophoresis) provides an exact and reliable system for such quantification (1).
In vitro expression of proteins from PCR products
Authors: Lesley Scott A
Published: November 2023

The primary advantage of in vitro translation of gene products over the in vivo expression of cloned genes is that in vitro translation allows the use of a defined template to direct total protein synthesis. There is a direct correlation between the sequence of the DNA added as a template for transcription and translation, and the protein produced without any background synthesis of endogenous proteins. This is a particular advantage when one would like to specifically radiolabel a protein of interest. The advent of PCR as a common tool for manipulating DNA sequences presents additional opportunities to accelerate such studies, by providing template for in vitro translations without the usual cloning and in vivo expression. By utilizing in vitro translation, the gene product can be amplified, translated, and assayed in a single day.
PCR-based approaches to human genome mapping
Authors: Morrison J F J , Markham A F
Published: November 2023

Genetic maps are constructed by determining how frequently two markers are inherited together. These maps allow the ordering of DNA markers and give estimates as to the distances between these markers. Genetic maps differ from physical maps in that they can be used to site loci which are associated with disease. Physical maps, in contrast, specify nucleotide distances between markers along a chromosome. PCR is used to generate both types of map through the generation of sequence-tagged sites (STS) (1). Sequence-tagged sites are used to identify DNA segments in both genetic and physical maps, and usually comprise 200-500 bp of DNA sequence unique to that segment, sufficient to allow a PCR reaction to be performed. Thus these STSs can conveniently be identified by specific PCR primers which can be used to screen genomic DNA contained in yeast artificial chromosomes (YACs) or cosmids.
Linear amplification for the in vivo study of ligand/DNA interactions
Authors: Saluz Hans Peter , Jost Jean-Pierre
Published: November 2023

Genomic footprinting is a tool to study specific ligand/DNA interactions in vivo (1). The DNA in the region where proteins bind shows altered reactivity towards footprinting reagents and therefore usually escapes modification and/ or cleavage. The cleavage products of the DNA are visualized by either electroblotting and indirect end-labelling (1, 2), linear amplification (3, 4), or via primer extension with labelled primers (5). Protected areas appear as a ‘window’ in the sequence ladder, when compared with treated protein-free control DNA.
Production of foreign proteins at high level
Authors: Gérard Loison
Published: November 2023

The yeast Saccharomyces cerevisiae is one of the most commonly used host cell systems for the production of foreign proteins for research and for industrial or medical use. Although the state of knowledge on yeast molecular biology is increasing day by day, a large part of the yeast biotechnologist’s work remains based on empirical approaches rather than on assertive predictions. Insertion of a foreign coding sequence into a standard multiple-copy expression vector for yeast is not always sufficient to ensure a high level of accumulation of the foreign protein. Various strategies have been tested by different groups to overcome limitations in protein production; mostly these depend upon empirical mutagenesis-screening. Reviewing all these cases is beyond the scope of this chapter. Interested readers should consult the recent and comprehensive review of Romanos et al. (1), which is entirely devoted to foreign gene expression in S. cerevisiae as well as in other yeasts. I will rather limit myself to emphasizing some basic pieces of advice and outlining some successful strategies we have experienced ourselves.
Cell-free translation of natural and synthetic mRNAs in yeast lysates
Authors: Tuite Mick F , Zhu, Delin , Hartley Aland.
Published: November 2023

All three phases of the translational process—initiation, elongation, and termination—can be faithfully reconstituted in the test tube with defined natural and synthetic mRNA templates. The establishment of such in vitro translation systems has underpinned not only studies into the mechanism of protein synthesis in both prokaryotes and eukaryotes, but has also provided a key experimental tool for the elucidation of the genetic code. While the fundamental details of how one prepares a cell-free translation system and uses it to translate mRNA templates have not really changed since Pelham and Jackson first described the mRNA-dependent reticulocyte cell-free lysate (1), progress has recently been made in refining these systems for the study of post-translational processing of secretory proteins (2, 3) and for generating quantitative yields of translation products (4). There have also been impressive developments in the area of in vitro transcription of cloned DNA templates (5) to generate both natural and engineered mRNAs for cell-free translation studies. Cell-free systems have been described from a relatively limited number of eukaryotic cells with most researchers still focusing on the rabbit reticulocyte and wheat germ systems primarily due to their commercial availability and reproducibility rather than any innate biological relevance.
Historical introduction
Authors: Gershoni J M
Published: November 2023

In 1975 Southern published his method for the identification of specific DNA fragments which had been resolved by gel electrophoresis via transfer of the band pattern to nitrocellulose membrane filters (1). This technique immediately gained recognition, proving to be a milestone in molecular biology, and has since been referred to as Southern blotting. It was not long thereafter that a new technique was developed in which the macromolecule to be analysed was RNA, also immobilized to a filter matrix; this technique was coined Northern blotting (2).
Protein blotting and immunoblotting using nitrocellulose membranes
Authors: De Maio Antonio
Published: November 2023

During the last decade protein blotting has emerged as a powerful technique for the study of proteins or glycoproteins immobilized on a membrane filter, without the necessity for painful protein purification procedures. The detection of antibody epitopes in total cell homogenates has been without any doubt the preferred application. However, the use of protein blotting has been expanded to other applications such as the detection of receptors through the binding of their respective ligands, physical chemical analysis of protein-protein interactions, and the study of glycoconjugates (see Chapters 12-14 of this text).
In situ hybridization approaches to the study of the nervous system
Authors: Seroogy K B , Herman J P
Published: November 2023

In situ hybridization methodology derives from a combination of techniques from the two very different disciplines of molecular biology and neuro anatomy. Over the past decade, in situ hybridization has emerged as a powerful means by which to examine not only the localization, but also the regulation, of cellular phenotype in the peripheral and central nervous systems. This method takes advantage of the ability of mRNA immobilized in tissue sections to form duplexes (i.e. hybridize) with complementary DNA or RNA sequences (referred to as ‘probes’). Visualization at the cellular level is accomplished by autoradiographic or immunohistochemical detection of tagged nucleic acids incorporated into the probe molecules. Compared to immunocytochemical techniques which detect post-translational products, in situ hybridization analysis of cellular mRNA provides information concerning pre-translational events, a level of understanding one step closer to the gene.

The value of in situ hybridization analysis of mRNA becomes particularly manifest when studying heterogeneously organized tissues. In the nervous system, it is quite common for functionally distinct cellular populations to produce the same neuroactive substances, often within microns of one another. Such cellular populations can synthesize different amounts of mRNAs and respond to very diverse regulatory input. Even cells that are morphologically and functionally similar and situated in the same nucleus can express different neurochemical phenotypes or respond quite differently to similar regulatory signals. In addition, laminar structures, such as the hippo campus, olfactory bulb, and cerebellum, possess different cell populations which interdigitate in such a fashion that accurate dissection is extremely difficult.
Protein phosphorylation and dephosphorylation in the nervous system
Authors: Dunkley Peter R , Jarvie Paula E , Sim alistair t. R
Published: November 2023

Phosphorylation of proteins plays a crucial role in controlling the functions of the nervous system. The brain has evolved to contain many unique phosphoproteins whose functions and characteristics have been reviewed (1-4). The neuronal phosphoproteins have phosphate covalently attached to them mainly at Ser and Thr residues. These phosphoproteins are associated with both protein kinase(s) and protein phosphatase(s) and this grouping will be referred to here as a protein phosphorylation system.
Receptor-linked phosphoinositide metabolism
Authors: Batty Ian H , Carter A Nigel , Challiss R A John , Hawthorne john n.
Published: November 2023

Activation of many cell surface receptors is linked to the metabolism of phosphorinositides and there has been an explosive growth of interest in the field over the last 15 years. Most of the phosphoinositide-linked receptors raise intracellular free Ca2+ concentration when activated. The receptors are linked by a G protein to phosphorinositide C (PIC), which hydrolyses phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to inositol 1,4,5- trisphosphate (Ins(l,4,5)P3) and diacylglycerol, both of which can act as second messengers. Ins(l,4,5)P3 causes release of Ca2+ from stores associated with the endoplasmic reticulum and diacylglycerol can activate protein kinase C (1,2). lns(l,3,4,5)P4 may also have a signalling function and many other higher inositol phosphates including InsP5 and InsP6 (phytic acid) have been identified in tissues. In addition a PtdIns 3-kinase has been described and the lipids it produces, particularly PtdIns(3,4,5)P3 may regulate growth (3).
Neurotransmitter transporters: purification, reconstitution, and assay of the brain GABA transporter
Authors: Keynan Shoshana
Published: November 2023

The process of chemical neurotransmission involves the regulated release of neurotransmitter from the presynaptic terminal into the synaptic cleft followed by binding and activation of specific receptors on the postsynaptic mem brane. Termination of the biological response and dilution of the neurotransmitter in the synaptic cleft are critically important events ensuring constantly high concentrations within the nerve and the prevention of excessive stimulation or delay of the response (1).
Plasmids from Gram-positive bacteria
Authors: Alonso Juan c. , Espinosa Manuel
Published: November 2023

Bacterial plasmids are self-replicating dispensable genetic elements that often specify resistance to antibiotics, heavy metals, bacteriophages, or encode the production of bacteriocins, amino acids, antibiotics, enzymes (e.g. proteases, lipases, catalases, restriction enzymes), etc. Plasmid biology is studied for several reasons. From a basic point of view, plasmids can be used as model systems to study the genetics and biochemistry of cellular processes, and from an applied point of view, they can be used to produce large amounts of industrially important compounds.
Light regulation of nuclear-encoded thylakoid proteins
Authors: Green Beverley R , Salter A Hugh
Published: November 2023

The thylakoid membrane system is the site where light energy is absorbed and converted into the chemical energy of reduced coenzymes and the trans-membrane electrochemical potential which drives ATP synthesis. Since green plants are obligate photoautotrophs, it should come as no surprise that the development and maintenance of the thylakoid membrane system is regulated by light at many different levels, both during development and in response to external factors after development. Chloroplast ultrastructure and development are discussed in several comprehensive reviews (1,2).
Import, sorting, and assembly of photosynthetic proteins in higher plant chloroplasts
Authors: Robinson Colin , Knott Tracy G
Published: November 2023

It has long been known that protein translocation on a massive and complex scale is required during chloroplast biogenesis, since the majority of chloroplast proteins are imported into the organelle after synthesis in the cytosol. Although the chloroplast is responsible for a wide variety of metabolic functions, the most prominent of these is, of course, photosynthesis, with the result that the most abundant chloroplast proteins are all involved in photosynthetic light capture or CO2 fixation. As a direct result, most studies on chloroplast protein transport have focused on the biogenesis of photosynthetic proteins, and considerable progress has been made in elucidating the mechanisms by which stromal and thylakoidal proteins are imported into chloroplasts and directed to their functional locations. Photosynthetic proteins have widely differing physical characteristics and are variously located in the stroma, thylakoid membrane, and thylakoid lumen; as a consequence they are imported and ‘sorted’ by a corresponding variety of mechanisms.
Peptide antigens and anti-peptide antibodies
Authors: Wisdom G Brian
Published: November 2023

Antibody molecules, especially of the immunoglobulin G (IgG) class, are extremely useful reagents for the identification, measurement, purification, and characterization of various molecules (1). This arises from the fact that antibodies can be induced to most structures of molecular weight greater than 200-300 Da (provided their size is increased by attachment to a carrier) and these antibodies usually show high avidity and specificity. In addition, IgG molecules are relatively stable and can be labelled and immobilized in various ways without serious detriment to their binding abilities. Antibodies to peptide antigens have become particularly useful tools.
Epitope predictions from the primary structure of proteins
Authors: Pellequer Jean-Luc , Westhof Eric , Regenmortel Marc H V Van
Published: November 2023

Rapid progress in recombinant DNA techniques has led to the sequencing of an ever-increasing number of genes for which the gene products are unknown. The most common method for detecting and localizing the putative gene product is to use antibodies raised against a synthetic peptide corresponding to an epitope of the putative protein, predicted on the basis of its primary structure. Prediction of antigenicity is thus used to indicate which peptide within the protein should be synthesized in order to obtain anti-peptide antibodies that will recognize, i.e. cross-react, with the intact protein.
Probe labelling methods
Authors: Hopman A H N , Speel E J M , Voorter C E M , Ramekers F C S
Published: November 2023

Several methods have been developed to detect specific nucleic acid sequences in various preparations, such as in situ localized DNA or RNA in intact cells or biochemically isolated DNA or RNA immobilized on membranes. In the latter case nucleic acids are either directly immobilized on filters (spot hybridization) or transferred to the filter after electrophoretic fractionation. The presence of specific nucleic acids is then detected on the membrane by means of hybridization with labelled nucleic acid probes. Techniques aimed at the localization of target DNA or RNA sequences within microscopic preparations, so called in situ hybridization, require several pre-treatment steps to allow the penetration of such probes into the matrix of the cell. Nucleic acid probes can be labelled either radioactively, followed by auto radio graphic detection, or non-radioactively, enabling direct detection by different forms of microscopy.
Cytogenetic analysis
Authors: Klinger K W
Published: November 2023

In situ hybridization was first demonstrated in 1969 by Gall and Pardue (1). With further refinement (2), isotopic in situ hybridization became a useful tool for gene mapping. However, the relatively coarse map position generated by this method, coupled with the tedious and difficult nature of the technique, limited its widespread application. It is the advent of fluorescence in situ hybridization (FISH) that has revealed the true power of the technology, allowing fast, precise detection, and localization of single copy targets. FISH has rapidly achieved roles as a research tool, and as a diagnostic tool in the fields of prenatal diagnosis, cancer diagnosis, infectious disease diagnosis, and gene dosimetry.

Nucleic acid probe sequences can be hybridized in situ to their complementary DNA sequences within fixed chromosomes, extended DNA molecules, or cytoplasmic RNA or DNA (mitochondrial, bacterial, or viral). This chapter concentrates on metaphase and interphase cytogenetics, with a discussion of hybridization to extended DNA molecules as it relates to gene ordering.
The action and fermentation of carbohydrates in the human colon
Authors: Edwards C A , Parrett A M
Published: November 2023

Carbohydrates that enter the human colon are subject to fermentation by the bacterial flora. The main products of fermentation are short chain fatty acids (SCFA) and gases. These products have a range of physiological activities including effects on motility, cell turnover, as well as acting as sources of energy for the mucosal cells and the body in general. The extent, rate and site of fermentation will determine the physiological action of the carbohydrate in the colon. Easily fermented carbohydrates may have little effect on stool output but more profound effects on cellular metabolism. Fermentation in more distal parts of the colon may have greater effects on stool water even if most of the carbohydrate is used by the bacteria.
Association and segregation in ternary polymer solutions and gels
Authors: Piculell L , Iliopoulos I , Linse1 P , Nilsson1 S , Turquois T , Viebke, C , Zhang W
Published: November 2023

Some consequences of (i) polymer charges and (ii) polymer stiffness for the segregation or association of unlike polymers in mixed biopolymer gels are discussed, in the light of model calculations and experiments on ternary polymer solutions or gels. For segregating mixtures, the occurrence of charges on one of the polymers leads to an increased miscibility, compared to the neutral system. The miscibility decreases again, however, if charges of the same sign occur on both polymers, or if salt is added. For mixed gels of kappa-carrageenan with galacto-or glucomannans, cesium-133 NMR results indicate an association, rather than a segregation, of the unlike polymers. It is suggested that the mixed aggregates consist of a rod-like core of selfaggregated carrageenan helices with disordered mannan chains adsorbed to the surface. Lattice model calculations on the adsorption of a flexible polymer onto a rodlike cylinder indicate a weak increase of the surface coverage with increasing rod radius.
Synergistic gelation of galactomannans or konjac glucomannan: binding or exclusion?
Authors: Goycoolea F M , Foster T J , Richardson R K , Morris E R , Gidley M J
Published: November 2023

Synergistic gels of xanthan or deacetylated xanthan with locust bean gum (LBG) or konjac mannan (KM) melt and set at ∼600C, with no thermal hysteresis. Gel-like rheology persists to very low concentrations (0.02% w/v). Gelation occurs with the xanthan component in either its ordered or disordered form, and is accompanied, with KM as co-synergist, by large enthalpy changes (Aff) in DSC. Gel modulus (G’) and Affincrease linearly with increasing ratio of co-synergist:xanthan up to ∼1:1, with little further change at higher ratios. The heat changes (per gram of xanthan) depend only on mixing ratio, not on total concentration. We interpret these results as conclusive evidence of stoichiometric binding, and suggest that, as indicated by previous X-ray work, final attachment is to the cellulosic backbone of xanthan, rather than to the surface of the 5-fold helix.
Thixotropic carrageenan gels and dairy desserts
Authors: Parker Alan , Tilly Gerard
Published: November 2023

Rapid thixotropic recovery is an important factor in the production of multi-layer dairy desserts. Therefore the thixotropy of gels and model dairy desserts containing i-carrageenan has been studied. The results of thixotropic recovery in a rheometer after: i) gelation under shear and ii) subsequent cold shearing are compared for aqueous gels, milk gels and model dairy desserts. These measurements are compared with the recovery of samples sheared during processing on a pilot plant. Thixotropic recovery was never complete and appeared to be divided into a fast and a slow component. Both the viscosity during geiation under shear and the kinetics of recovery can be scaled, so that results for all shear rates form a single master-curve. For cold shearing of already formed gels the maturation time before shearing was not a significant factor for aqueous or milk gels, but caused significant differences in the final texture of model dairy desserts treated on a pilot plant.
Die moderne Brauereitechnologie: gelöste und ungelöste Probleme. Ein Überblick
Authors: Narzillß L
Published: November 2023

During the last 25 - 30 years, the technology of brewing underwent a very positive development of all the individual production steps of malting and brewing. Future efforts are influenced by a strong consciousness to environment: the breeding of barleys and hops resistant to diseases, the preservation of the quality of brewing water as well as an environment minded processing. However, it is another task of technology to differentiate the individual products. Even though the properties like foam, chemical-physical stability, flavour stability and filtrability have been searched for, there is still something to contribute to the understanding of these phenomena.
Gene elements controlling hydrolase production in barley
Authors: Leah R , Karnahl K , Rouster J , Skriver K , Mundy J
Published: November 2023

To develop malting barley varieties with improved hydrolase production, it is necessary to characterize gene elements controlling hydrolase expression. We have delineated two such DNA elements, one determining aleurone-specificity, the other responsive to gibberellin. These gene elements can be seen as locks which, when recognized by a specific key, a regulatory protein, allow expression of the gene. We are now using the gibberellin responsive DNA element as a probe to find proteins which recognize it. Such protein factors may control the production of numerous hydrolases. They are, therefore, important targets for study and modification in malting barleys.
S. cerevisiae flocculation: identification of specific cell wall proteins
Authors: Costa Manuel João , Moradas-Ferreira Pedro
Published: November 2023

s. cerevisiae flocculation depends on cell wall protein interactions and this work aimed at identifying putative flocculins. Analysis of both SDS and glucanase extractable cell wall mannoproteins revealed differences which fairly correlate with the flocculent phenotype. Highly flocculent cells accumulated SOS-extractable proteins of 54 and 67 KDa, and glucanase extractable high molecular mass proteins (about 1000 KDa).
Novel method for measurement and control of evaporation in the brewhouse kettle
Authors: Forrest I S , Skrgatic D , Coker I A J , Heap J
Published: November 2023

Automatic in-line measurement of evaporation allows the development of alternate energy control strategies and cleaning regimes without the safety hazard of manual sampling. Sound velocity in boiling wort, measured with a surface mounted ultra-sonic transducer at the inlet to an external calandria, was used as a measure of wort gravity. This allowed calculation of evaporation to 0.1%, and thus close control of evaporation. The condition of the heat exchange surfaces could also be monitored and cleaning cycles initiated only as required. The combined savings in steam and detergent produced a payback in under 4 months.
Some technological developments in a packaging line
Authors: Sawahara Kouji , Ishikawa Kazusi , Endo Norio , Mawatari Masao , Iwata Hiroo
Published: November 2023

We have developed new automatic inspectors and bottle-handling systems so that 24-hour packaging lines utilizing minimal manual labour will be realized. Details of action and practical advantages of the systems are: a) a case-inspector and rejection system for rejection of foreign objects in crates returned from marketplace; b) an “automatic filled-bottle inspector” includes our unique high quality CCD camera system and it has been successfully carried out without using manual inspection;
Technological relationship between the brewing of sake and beer
Authors: Nishiya Takamichi
Published: November 2023

Brewing of the traditional Japanese rice wine known as “sake” originates with the introduction of rice cultivation from the Eurasian continent, and has a 2,000 year history. A sake-like drink is even mentioned in Japanese mythology. In Japan, not only the technologies developed for the fermentation of other alcoholic beverages, but also those required for the fermentation of amino acids, nucleic acids and antibiotics are derived from the traditional techniques used in sake brewing. This lecture is a review of the technological relationship between sake and beer brewing. In addition, recent developments in sake brewing biotechnology will be described, such as the application of genetic transfer techniques to koji-moulds and yeasts.
The impact of oxygen absorbing crown corks on shelflife of bottled beer
Authors: Eils H -G , Teumac F , Zenner B
Published: November 2023

Oxygen has a significant influence on the taste stability of beer. Ingress of oxygen (from air) into bottled beer during orage can be prevented by using oxygen absorbing crown corks (Pure Seal , SmartCapR). This fact is proven by a method allowing the evaluation of oxygen values down to 2 ppb. Changes in the flavour stability of bottled beer stored at temperatures of 20°C and 30°C have to be considered differently.
Computer-planned mashing
Authors: Aarts Robert J , Sjöholm Katharina , Pietilä Silja Home & Kirsti
Published: November 2023

A knowledge-based computer system was used to plan temperature profiles for mashing. The system uses malt analysis results and wort specifications as inputs. Several malts and wort specifications were used for testing. The enzyme activities and the degree of modification of the malts varied, and the worts required different fermentabilities. The system planned temperature programs that were used in laboratory scale mashing. Analysis of the worts showed all were within their respective specifications.
Application of ultrasonic wave fields to brewing separation processes
Authors: Davies Gareth B
Published: November 2023

The use of megahertz frequency ultrasonic stationary waves in a novel brewing separation process has been assessed. On a small scale, this technique is capable of removing greater than 981of beer2solids over 4 µm in diameter at flowrates of approximately 1 l.h per cm of paired trans¬ducer area. Process scale-up towards several hundred hectolitres per hour seems to be practical. Preliminary investigations have also been conducted into accelerated sedimentation of yeast and the enhancement of cross-flow microfiltration membrane fluxes. Ultrasonic stationary wave technology promises to provide an alternative, non-invasive method of solid-liquid separation in the brewing industry.
Humoral immune responses and detection during HIV infection
Authors: Cheingsong-Popov Rachanee , Constantine Niel T , Weber Jonathan
Published: November 2023

In common with many infectious agents, HIV infection elicits persistent humoral and cellular immune responses. Shortly following HIV infection, viral replication occurs, resulting in the elaboration of specific proteins and glycoproteins (antigens) to which the efferent humoral arm of the immune system responds with the production of antibodies. The resultant antibodies represent a complex mixture of immunoglobulins which vary in titre, isotype, and idiotype. The response, for the most part, is consistent and predictable, and is well-defined. However, there have been anecdotal reports where there is a delay in the detection of antibody in the sera of some persons for more than three months following infection (1).
Quantification of CD4-positive T lymphocytes
Authors: Bird A G
Published: November 2023

Introduction

The central lesion in HIV-induced immunodeficiency is the functional and numerical decrease in the CD4-positive population of T lymphocytes. Progressive loss of this key lymphoid population is associated with an increasing range and severity of cellular immunodeficiency and predisposition to intracellular pathogens. The number of CD4-positive lymphocytes provides a relatively reliable staging marker of HIV in adults and low levels identify a group of patients at greatest risk of major opportunistic infections (1, 2). Since late 1992, Centres for Disease Control, Atlanta, USA, have used the association of HIV infection and a CD4 count below 200 cells/mm3 as an amended case definition for AIDS. Rises in CD4 cell counts have also been utilized as a surrogate marker of early efficacy of anti-retroviral agents but the results of recent trials have cast some doubt about the validity of this approach in reflecting extended patient survival (3).
Lymphokine responses to HIV infection
Authors: Matear P M , Naraghi A , Deakin S , Meager A , Vyakarnam A , Vyakarnam A
Published: November 2023

Studies of cytokine production in HIV infections are important because cytokines have been implicated in viral pathogenesis leading to AIDS. One of the characteristic features of HIV infection, as in many chronic disease states, is the dysregulated production of several cytokines (see refs 1-5). These cytokines include TNFot, TNFfJ, IFN gamma, IL-6, IL-1, and IL-4. In addition to their effects on HIV replication, TNFot and IFN gamma have been shown to induce HIV replication in macrophages and CD4-positive T cells (1-4, 6, 7). The pattern of cytokines produced by HIV-positive individuals and AIDS patients could also be important in regulating their immune response to the virus, e.g. by selectively activating Thl or Th2 cells (8-10). Additionally, the pattern of cytokines may serve as a disease marker, distinguishing infected individuals who are healthy from those with disease. Recent studies have shown, for example, that asymptomatic HIV-positive individuals produced a predominantly Thl cytokine pattern (high IL-2, high IFN gamma, low IL-4 production) whereas those with disease symptoms produce a predominantly Th2 cytokine pattern (high IL-4, low IFN gamma, and low IL-2) (11).
The use of flow cytometry to study platelet activation
Authors: Michelson Alan D , Shattil Sanford J
Published: November 2023

Many common clinical conditions have been reported to be associated with platelet hyperreactivity and/or circulating activated platelets: coronary artery disease (angina pectoris, acute myocardial infarction) diabetes mellitus stroke exercise emotional stress cigarette smoking pregnancy and pre-eclampsia blood bank storage of platelets for transfusion cardiopulmonary bypass angioplasty deep vein thrombosis hyperlipoproteinaemia essential thrombocythemia adult respiratory distress syndrome However, the role of platelet activation in clinical settings is controversial, in part because the methods used to detect platelet activation (e.g. platelet aggregation and radioimmunoassays of plasma P-thromboglobulin (pTG) and/or platelet factor 4 (PF4) have major methodologic problems (1-6)).
Measurement of protein phosphorylation, kinase activity, and G protein function in intact platelets and membrane preparations
Authors: Wadman Isobel A , Virdee Kanwar , Fernandez Denise S , Wasunna Christine L , Farndale Richard W
Published: November 2023

We have collated in this chapter methods for locating or determining the activity of platelet signalling proteins. SDS-PAGE provides a means of separating the proteins, their labelling is achieved either by covalent or affinity methods, and they are usually detected on film, either by autoradiography or Western blotting. The preparation of platelets and platelet membranes from Blood Transfusion Service concentrates, together with basic electrophoretic methods, are described in Section 1. Methods for protein phosphorylation and the determination of kinase activity are described in Section 2, and for GTP binding proteins in Section 3. We expect that the reader will modify the precise conditions of the methods to suit their particular need; the protocols below will provide a starting point from which these and other assays may be developed.
MHC Volume 1
Authors: Butcher G
Published: November 2023

Immunochemistry is of immense importance to virtually all areas of modern biology and medicine, and the space afforded by two volumes has allowed coverage of a wide range of topics. The familiar and clear Practical Approach format will make a large body of valuable information easily accessible to a diverse readership.
The use of pulsed-field gel electrophoresis to map the human MHC
Authors: Sanséau Philippe , Trowsdale John
Published: November 2023

The major histocompatibility complex (MHC) is located on the short arm of chromosome 6 in the distal portion of the 6p21.3 band. It consists of three linked gene clusters covering 4 Mb of DNA. The class I and class II regions encode highly polymorphic families of cell surface glycoproteins involved in immune regulation. The class I region which covers -1500 kb, contains at least 40 genes including the classical transplantation antigens (HLA-A, -B, and -C). This region is located at the telomeric side of the MHC. The class II region encodes the classical antigens (HLA-DP, -DQ, -DR) but also novel class II genes like HLA-DM, and the TAPs and LMPs involved in antigen processing. The class I and class II regions are separated by the class III region which spans - 1100 kb. This part of the MHC contains a large number of genes unrelated to the classical transplantation antigens, although some are involved in the immune system, such as the complement genes C2, C4, and Bf, the tumour necrosis factor (TNF) A and B genes, and the heat shock protein HSP70 genes.
Sequence analysis of major histocompatibility molecules
Authors: Lawlor Da Yid A , Laud Purnima
Published: November 2023

The GenBank database for nucleotide sequences contains over 1500 000 entries and 1.5% are derived from loci of the major histocompatibility complex (MHC). Parham noted that in 1986 approximately 12000 amino acid residues had been determined for human class I molecules by protein and DNA sequencing methods. He attributed the exponential growth of sequence information (fivefold increase in database size in less than three years) to the replacement of protein sequencing with the more efficient DNA sequencing procedures (1). At today’s rate, the derived amino acid information for all MHC molecules increases by that amount every two weeks. Table I lists the MHC sequence entries in GenBank for the various classes and species.
Peroxides and other products
Authors: Brown R K , Kelly F J
Published: November 2023

The only definitive way to demonstrate excessive free radical activity in vivo is by electron spin resonance as already discussed in Chapter 2, but clearly this is currently inapplicable in clinical practice due to the nature of the spin traps that must be used. Instead investigators must rely upon the measurement of established markers of free radical activity in biological fluids and tissues. Traditionally, most markers of oxidative injury utilized reflect free radical attack on polyunsaturated fatty acids, with the classical route of attack involving lipid peroxidation, generating hydroperoxides, endoperoxides, long-lived aldehydes and the end-products malondialdehyde, ethane, and pentane.
Mechanisms of oncogene perturbation
Authors: Imreh Stephan , Szekely Laszlo , Wiman Klas G , Klein George
Published: November 2023

Viruses have been implicated for a long time as possible aetiologic agents in the genesis of human and animal tumours. Isolation of various DNA and RNA viruses that induced tumours in experimental animals led to the belief that all of them carry genetic elements, oncogenes, that are responsible for tumour induction. However, it became apparent that the oncogenes of the RNA tumour viruses have been acquired from the host cell genome, were not necessary for viral replication, and were maintained in the virus stocks under sustained selection for tumorigenicity. In contrast the oncogenic DNA viruses were found to encode proteins that are essential for viral replication and are also responsible for tumour induction. Viruses of different types are believed to contribute to the aetiology of approximately 15% of all human tumours (1).
Oncogenic cytoplasmic protein tyrosine kinases
Authors: Roche Serge , Courtneidge Sara A
Published: November 2023

Phosphorylation on tyrosine residues accounts for only 0.05% of total protein phosphorylation in mammalian cells, yet protein tyrosine kinases play important roles in the intracellular transduction of extracellular signals. They are involved in various biological responses including cell growth and differentiation, cell motility, and cell secretion. Protein tyrosine kinases can be divided into two groups: receptor and non-receptor tyrosine kinases. The receptor class comprises a family of cell surface proteins, which act as receptors for a variety of ligands. They are composed of an extracellular domain which contains the ligand binding site, a transmembrane domain, and a cytoplasmic domain that contains catalytic activity. This class of kinase is detailed in Chapter 3. Members of the second class of tyrosine kinases do not have extracellular sequences and do not span the plasma membrane. They can be located in the cytoplasm, on the inner part of the plasma membrane, or in the nucleus. The cytoplasmic tyrosine kinases have been sub classified into a number of families based upon their structural homology.