bluuebunny/crossref_metadata_2025 huggingface dataset:
Tenascin, laminin, and fibronectin produced by cultured cells
Authors: Erickson Harold P , Briscoe Gina
Published: November 2023

Tissue culture cells synthesize a variety of extracellular matrix (ECM) molecules and incorporate them into a matrix. In most cases, however, the incorporation into the matrix appears to be inefficient, and by far the majority of the ECM proteins are simply secreted into the medium as soluble molecules. One can harvest the ‘conditioned medium’ from confluent cell cultures every 2—4 days and purify the molecules of interest. If a monoclonal antibody is available in large supply, affinity purification on an antibody column is frequently the preferred method. A disadvantage of immunoaffinity purification is that the protein must be exposed to denaturing or chaotropic agents to elute it from the column. Alternative biochemical purification may be essential for proteins that are easily denatured, and is desirable whenever one is testing for unknown and potentially sensitive functions.
Sample preparation
Authors: Primrose William U
Published: November 2023

Over the last few years, there have been rapid and sustained advances in the hardware and software associated with the NMR analysis of biological macromolecules. Larger magnets, faster computers, and new and improved pulse sequences have been combined with isotopic labelling to make NMR the unrivalled method for answering biological questions at the atomic level in the solution state. These dramatic advances have perhaps tended to obscure the importance of correct preparation of the sample, which tends to be a rather poorly documented subject. The purpose of this chapter is to aid you in the correct choice of conditions for preparation of your vital and hard-won sample. This will then allow you to obtain the maximum benefit from the sophisticated spectroscopic methods described in other chapters in this book. The importance of correct handling of the sample cannot be over-emphasized, as no amount of expensive spectrometer and computer equipment can compensate for an intrinsically poor sample. A Formula One racing car will not break any lap records running on two-stroke fuel !
NMR Of Nucleic Acids; From Spectrum To Structure
Authors: Wijmenga Sybren S , Mooren Margret M W , Hilbers Cornelis W
Published: November 2023

Nucleic acids are perhaps the most intensively studied molecules found in nature, which is not too surprising in view of their role as carriers of genetic information. It has been a deep-rooted belief among molecular biologists that structure dictates function and, therefore, these studies have often involved the elucidation of structural aspects of these molecules, be it the linear base sequence or the three-dimensional arrangement of their nucleotide units. In a similar spirit it has been a deep-rooted belief among NMR spectroscopists that ‘ their ’ field of spectroscopy should be able to contribute to the elucidation of the structure and dynamics of molecules, in particular, biomolecules, in a way unparalleled by other forms of spectroscopy. Over the last few years this goal has come within reach thanks to the development of multidimensional NMR techniques which provide the necessary spectral resolution for the successful interpretation of the complicated spectra of biomolecules. Triggered off by the development of two-dimensional NMR, an avalanche of NMR studies on nucleic acids has appeared (1).
Structure Determination from NMR Data I. Analysis Of NMR Data
Authors: Barsukov Igor L , Lian Lu-Yun
Published: November 2023

This chapter deals with the strategies which are used to derive the experimental data required as inputs for calculation of the three-dimensional structure of a protein. The experiments used for spectral assignment described in Chapters 4 and 5 are also used for obtaining the data for structure calculations, although for the latter purpose, a more quantitative analysis is required. The principal structural data derived from NMR are scalar (J) couplings and NOEs, which are translated into restraints on dihedral angles and interproton distances, respectively. It is important to bear in mind that NMR leads to constraints on angles and distances, not to exact values of these parameters. The quality of the solution structure obtained has been shown to be proportional to the number of constraints used in the structure calculations.
Aetiology of type I diabetes: genetic aspects
Authors: Wassmuth Ralf , Kockum Ingrid , Karlsen Allan , Hagopian William , BäRmeier Heike , Dube Syama Lima , Lernmark Åke
Published: November 2023

Ancient literature has indicated that two forms of diabetes could be discerned. One was associated with emaciation, dehydration, polyuria, and lassitude, and the other with stout build, gluttony, obesity, and sleepiness (Bliss 1982). At the beginning of the twentieth century, however, and even after the discovery of insulin in 1921, diabetes was more often than not regarded as a single syndrome. A distinction was later made between juvenile and maturity onset diabetes mellitus but it was not until 1940 that these two forms of diabetes were suggested to be genetically distinct (Rotter et al. 1990). It also became clear that diabetes mellitus is not only insulin dependent or non-insulin dependent. A large number of disorders or conditions are associated with glucose intolerance; their causes range from mono genie disorders to diabetes secondary to a pancreatic affliction or to a peripheral insulin resistance induced by drugs or tumours. A recent survey lists more than 60 distinct genetic disorders associated with glucose intolerance and, sometimes, clinical diabetes (Rotter et al. 1990). Glucose intolerance and diabetes are therefore symptoms rather than defining diagnostic criteria. In order to properly diagnose insulin-dependent diabetes a set of diagnostic criteria are necessary. Such criteria.
Metabolic mapping of local neuronal activity
Authors: Kennedy Charles , Smith, Carolyn Beebe , Sokoloff Louis
Published: November 2023

The radioactive deoxyglucose method was developed to measure local rates of energy metabolism simultaneously in all components of the brain of conscious laboratory animals. It takes advantage of the extraordinary spatial resolution made possible by quantitative autoradiography (1). The dependence on autoradiography prescribed the use of radioactive substrates for energy metabolism, the labelled products of which could be assayed in the tissues by the autoradiographic technique. Although oxygen consumption is the most direct measure of energy metabolism, the volatility of oxygen and its metabolic products and the short physical half-life of its radioactive isotopes precluded measurement of oxidative metabolism by the autoradiographic technique.
Fluorescence—monitoring cell chemistry in vivo
Authors: Duchen Michael R
Published: November 2023

Many molecules, when exposed to light of appropriate energy, absorb photons which excite electrons into a higher energy state. As those electrons relax to their original orbits, photons are re-emitted, a phenomenon known as fluorescence. The detailed spectra of the light that is absorbed and emitted may vary accordinging to other interactions of the molecule-its binding to Ca2+, protons, or other ions, the electrical field it senses, and so on. In recent years, we have seen major developments in fluorescent probes that can transmit information about intracellular events within living cells. The changes in intracellular concentrations of ions and other second messenger systems that play so fundamental a role in cell function have at last become accessible to direct study without having to impale cells with microelectrodes.
Enzyme reactions
Authors: Crabbe M J C
Published: November 2023

This chapter introduces some of the uses for microcomputers in studies of enzyme reactions. In many ways, it follows on from an earlier chapter on the applications of microcomputers in the analysis of enzyme reactions, published in this series in 1984 (1). The intervening years have seen great advances in the facilities offered by microcomputers. Machines with 20 MHz 32-bit CPUs and 4 Mbyte RAM are now commonplace, while in 1984 laboratories were more likely to be using micros with 4 MHz 8-bit CPUs and 32 kbyte of RAM. This increase in speed and storage facilities, not to mention improvements in screen resolution and colour display, have made microcomputers an invaluable tool in any laboratory. Similar advances in networking, laser printing, and X-Y plotting now enable raw data to be manipulated directly into publishable hard copy.
Gas Chromatography
Authors: Baugh P J
Published: November 2023

Gas chromatography (GC) has been one of the most used and successful chromatographic techniques since its inception over 40 years ago and is employed by almost every type of chemical and related industry in research and development, quality control and specifications, environmental monitoring —personnel, and process chemistry, biochemical and biological research establishments, clinical and toxicological departments of hospitals and related institutions, analytical and consultant/public analyst laboratories, water companies and regulatory bodies (National Rivers Authority), and higher education teaching/research institutions at all levels.
Applications of packed and capillary GC
Authors: Baugh Peter J
Published: November 2023

Although it can be said that capillary columns (high performance, HP) have superceded packed columns0 (low performance, LP) in GC applications and industrial uses of GC, the latter for many years has remained the preferred technique for quality control and chemical process monitoring. The reason has not necessarily been a reluctance to change but purely because LP columns could, with high capacity, achieve the performance adequately enough to satisfy the requirements. In such applications it was only necessary to determine the major components and impurities (< 1%) could be ignored.
Synthesis Of Oligo-2’-Deoxyribonucleoside Methylphosphonates
Authors: Miller Paul S , Cushman Cynthia D , Levis Joel T
Published: November 2023

Oligo-2'-deoxyribonucleoside methylphosphonates contain nuclease resistant, non-ionic internucleotide bonds. These oligomers are taken up intact by mammalian cells in culture. In addition to their unique physical properties, these oligonucleotide analogues give sequence-specific inhibitory effects when targeted against specific messenger RNA or precursor messenger RNA in mammalian cells in culture. The use of oligonucleoside methylphosphonates as antisense reagents in cells has been extensively reviewed (1-3). This chapter will describe the automated synthesis, deprotection, and purification of these oligomers, methods used to characterize the oligomers, and a general description of procedures for preparing them for use in cell culture experiments.
Oligonucleotides With Reporter Groups Attached to the 5’-Terminus
Authors: Sinha Nanda D , Striepeke Steve
Published: November 2023

Traditionally, oligonucleotides or DNA have been labelled and detected via use of P-labelled ATP and enzymatic phosphorylation. Although of high sensitivity, this technique has intrinsic hazards, expense, and problems associated with the short half-life of this isotope. Recent advances have shown that high sensitivity detection can be achieved by alternative markers based on fluorescent, chromophoric, or chemiluminescent detection. Incorporation of these labels can be achieved by enzymatic (1-8) or chemical (9-23) methods. One of the simplest and most useful methods involves introduction of primary amino or sulphyrdryl (thiol) groups to the 5’-terminus of the chemically assembled support-bound oligonucleotide (14-23). Subsequently fluorophores, chromophores, biotin, or alkaline phosphatase may be added. Further examples are discussed in Chapter l0.1With this approach the position of the amino or thiol group is defined and unambiguous, and there is negligible interference in subsequent hybridizations. Additionally, the 3’ -terminus of the oligonucleotide chain is free for enzymatic manipulations as required in Sanger’s sequencing and PCR amplification applications (14, 24-26). The attachment of other groups to the 5’-terminus of oligonucleotides is described in Chapter 12.
The European Community at the cross-roads
Authors: Vilar Dr Emilio Rui
Published: November 2023

When I have to address an audience like this one, I always remember the story of a famous speechmaker, who was able to be at the same time brief and accurate. Invited to speak about the great problems of mankind before a huge audience, he said: “The major problems of mankind are ignorance and indifference” and became silent. Among the audience someone asked his neighbour: “What did he say?” “I don’t know and I don’t care” was the reply.
Improvement of malting barley quality
Authors: versen John Ing
Published: November 2023

The current trend in one of the major malting barley growing areas of the world—Central Europe, is that more and more feed barley is grown on expense of especially row spring barley types. To revert this development and to breed even better malting barley for the future a research programme to “Adapt Barley to Industrial Needs”—(ABIN) was initiated in 1988. Within the ABIN project the most modern techniques are employed to solve some of the problems which faces the maltster and brewer, when present day barley is used as raw material in the industry. The lecture will present the background, overview and current status of the ABIN project.
Germination profile—a new term in barley analyses
Authors: Riis Preben , Bang-Olsen Kirsten
Published: November 2023

Production of high quality malt requires that the barley germinates fast, completely and uniformly. Based on an extension of the BRF method to measure Germlnatlve Energy, we propose two supplementary measures of quality: Germination Index, a measure of germination rate, and Germination Homogeneity, a measure of the variance of germination. To evaluate the feasibility of these measures, series of micromaltings were performed using barley with artificially reduced vigour. The results of these micromaltings show that the three measures for germination, for which we use the unifying term Germination Profile, can be a valuable tool in controlling maltings.
Untersuchungen zur Wasseraufnahme und Wasserverteilung der Gerste auf der Grundlage von rasterelektronenmikroskopischen Analysen (REM) der Endospermstruktur
Authors: Rath F , Schildbach R
Published: November 2023

Precise knowledge of the physiology of water uptake and the factors influencing it, is a requirement for the exercise of influence achieved during the malting process and also for the development of new criteria for selection in barley breeding.
Sulphur compounds in brewing
Authors: Clarke B J , Burmeister M S , Krynicki L , Pfisterer E A , Sin Y O , Sime K J , Hawthorne D B
Published: November 2023

Routine monitoring with high sensitivity of the concentration of a range of volatile sulphur compounds in beer has now become feasible. With the development of the chemiluminescent detector the ready quantitation of at least six sulphur compounds in a single analysis is possible, the major components detected are dimethyl sulphide and hydrogen sulphide.
Yeast physiological condition and fermentation performance
Authors: Boulton C A , Jones A R , Hinchliffe E
Published: November 2023

Current regimes of yeast management may produce variations in physiological condition. In particular perturbations in the ratio of glycogen to sterol may result in fermentation inconsistency unless an appropriate pitching rate and wort oxygen concentration is selected. A procedure is described here in which yeast is subjected to vigorous oxygenation, pre-pitching, the object of which is to provide pitching yeast of standardized physiology. Methods which may be used to assess yeast physiological condition are discussed. Evidence is presented that the tests are poor predictors of fermentation performance when applied to conventional pitching yeast but show good correlation using oxygenated yeast.
Lipoxygénase: Origine, é volution et conséquence dans la fllière malterie- brasserie
Authors: Martel C , Kohl S , Boivin P
Published: November 2023

Flavor stability is an important factor in beer quality. It depends on many parameters like insaturated fatty acids and lipoxidase activity. We carried out, micro-scale, pilot and industrial malting and brewing processes, to follow up lipoxidase activity and by-products of polyinsaturated fatty acids, for different barley varieties.
Role of active oxygens on deterioration of beer flavor
Authors: Kaneda Hirotaka , Kano Yukinobu , Osawa Toshihiko , Kawakishi Shunro , Koshino Shouhei
Published: November 2023

The role of active oxygens during flavor staling in packaged beer was studied by chemiluminescence (CL) analysis using 2-methyl-6-phenyl-3,7- dihydroimidazo[1,2-a] pyrazin-3-one (CLA) which can only react with active oxygens such as ‘O2 and and show a marked luminescence. The generation of CLA-dependent luminescence in beer was accelerated and the maximum luminescence intensities were reached sooner with increasing storage temperatures and/or storage times. In several types of lager beers, the generation of CLA-dependent luminescence showed a good relationship with the staling degree of stored beer flavor. It was, therefore, speculated that active oxygens were produced in packaged beer during storage and played an important role in the deterioration of beer flavor, most likely as an initiator in the free radical reactions.
Assay of S-methylmethionine (SMM) in malt using NIR spectroscopy
Authors: Yan S H , Bodart E , Dufour J -P
Published: November 2023

Lager malt with SMM (dimethylsulfide precursor) content of maximum 19 ppm (5 ppm DMS equivalent) is often asked by the breweries. However average concentrations range from 10 ppm SMM in kilned lager malt to more than 50 ppm in green malt. NIRS with its fast response time would be the ideal investigation method to follow SMM during kilning. Assays of SMM in freeze-dried germinating barley samples were carried out using the NIRS calibration (42 samples) and prediction (41 samples) methods with input data obtained from HPLC analysis. Correlation coefficients of 0.97 (S.E. = 1.81 ppm) and 0.84 (S.E. = 3.5 ppm) were obtained for the calibration and the prediction, respectively. The method has been evaluated to follow SMM evolution during kilning.
The importance of polyphenols in cold break formation
Authors: Crompton I E , Hegarty P K
Published: November 2023

The precipitation of protein-polyphenol complexes is of considerable importance in the brewing process, resulting in the formation of both hot and cold break. Excessive cold break can lead to problems such as off-flavours, poor fining and hindrance of accurate control of yeast pitching rate. Here we describe the chemical characterisation of hot and cold break. The factors which influence the quantity of cold break formed are described. Wort polyphenols are a major factor influencing cold break formation; removal of polyphenols from hot wort prior to cooling minimises the amount of cold break formed.
A new patented apparatus for continuous mashing
Authors: Béndek G , Horváth I , Ullmann† P
Published: November 2023

The advantages of the system are: low investment and operating cost, stable product quality. Drawings show the structural details of the vessels. The invention can be utilised in various other industries as well, where constant temperature and variable liquid level is required.
Crossflow flitration of beer
Authors: Kiefer Johannes
Published: November 2023

Due to increasing problems concerning the utilization and waste disposal of kieselguhr, the crossflow-membrane-filtration seems to be a possible alternative to beer filtration. Taking into consideration the technological and quality requirements, the criterions for a technical plant for beer filtration are examined under economic and technical points of view.
Factors affecting trub formation during wort boiling
Authors: Reed R J R , Jordan (GB) G
Published: November 2023

Evaporation during wort boiling is used to ensure satisfactory removal undesirable flavour components and the flocculation of heat coagulable protein into a settleable trub. The latter duty can be effected in a more controlled manner by the use of mechanical agitation. A cylindrical laboratory kettle/trub settlement chamber has been developed to examine the effects of agitation, wort composition, hopping regimes and kettle finings on trub settleability. Application to a pilot scale brewery indicates that all evaporation in excess of 2-3% can be replaced by only 0.03 KW/hl power.
Structural order in starch granules and its loss during gelatinisation
Authors: Gidley M J
Published: November 2023

Mature starch granules exhibit significant molecular and ultrastructural order which serves to maintain the hydrophilic starch polysaccharides in a low moisture state during deposition and storage, and to control many technologically-important properties such as gelatinization. A wide range of experimental probes of starch order are available which provide complementary information on distance scales from nm to µm. Gelatinisation in voIves disruption or melting of ordered structures. Quantitative analysis of crystalline and helical order in granular starches and comparison of gelatinisation enthalpies with melting enthalpies for relevant model crystallites, suggests that the enthalpy of gelatinisation is determined primarily by melting of double helices and not loss of crystalline register within granules. The major unresolved questions in granule architecture are the location of amylose molecules and the mechanism by which they are maintained in single chain states. It is suggested that amylose may be arranged orthogonal to the general radial direction of amylopectin structures, possibly chaperoned or shielded by lipid and/or protein.
Microstructure and viscoelastic behaviour of potato starch pastes
Authors: Svegmark Karin , Hermansson Anne-Marie
Published: November 2023

A combination of viscoelastic measurements and light microscopy has been used to investigate the specific properties of potato starch pastes. The influence of various shear and heat treatments has been evaluated in the concentration range 4% to 10%.The microstructure of potato starch pastes that had been subjected to a minimum amount of shear was completely dominated by swollen granules. Practically all of the water was taken up by the granules during gelatinisation. The diffusion of amylase from the granules seemed to be hindered by the extensive swelling of the granules. Shearing completely changed the viscoelastic behaviour and the structural character. The transition in viscoelastic behaviour on shearing corresponded with the point where fragments of the granules were dispersed in a continuous phase of solubilised starch. Autoclaved starch pastes, in which the granules were dissolved by heating, showed a viscoelastic behaviour similar to that of the sheared pastes. A microscale demixing of amylose and amylopectin occurs within the phase of solubilised starch.
Carrageenan/starch interaction in cream desserts
Authors: Nadison J , Doreau A
Published: November 2023

The purpose of this study is to improve our understanding of factors influencing the manufacture of cream desserts. Desserts containing different starch/carrageenan pairs were prepared, the objective being: To determine the limits of tolerance of the products to effects such as shearing forces and temperature during manufacture. To evaluate the organoleptic and rheological characteristics of each dessert. To examine the importance of cooking of the starch on the behaviour of the final product.
Surfactant-induced surface diffusion of protein is a determinant of disperse phase stability
Authors: Clark David C , Wilde Peter J
Published: November 2023

A fluorescence recovery after photo bleaching technique (FRAP) has been developed which facilitates the measurement of the surface diffusion of molecules adsorbed at interfaces. The appearance of surface diffusion of –lacto globulin induced by the emulsifier, Tween 20 in model foam systems composed of single suspended thin liquid films was shown to correlate with observed destabilisation of the bulk foam. Measurements using the same emulsifier-protein system were made at the oil-water interfaces of an aqueous film between two oil droplets. This system serves as a model of a concentrated or creamed emulsion. The FRAP results showed that the transition in surface diffusion in the protein fraction from immobile to mobile occurred at a different emulsifier to protein ratio than found at the airwater interface. These results point to differences in the behaviour of a given protein at oil-water and air-water interfaces.
Basic techniques for the preparation and analysis of chromosomes from bone marrow and leukaemic blood
Authors: Czepulkowski B H , Bhatt B , Rooney D E
Published: November 2023

This chapter is the first of five covering all aspects of the cytogenetic study of the leukaemias and related disorders. As such, it serves to provide basic and general information on the culture and analysis of the tissues most frequently used for such studies, namely bone marrow and blood. Protocols for the use of other tissues and further techniques specific to the different disease types are provided in the following three chapters.
Assay of gene transcription in vitro
Authors: Mason Philip J , Enver Tariq , wilkinson David , Williams Jeffrey G
Published: November 2023

All methods of analysing gene transcription in vitro rely on the ability of nucleic acid molecules to hybridize, one to another. Nucleic acid hybridization can be used to determine the primary structure and concentration of an individual RNA species by a variety of procedures.
Cloning and functional analysis of heterologous eukaryotic transcription factors in yeast
Authors: Becker Daniel M , Fikes John D
Published: November 2023

The study of transcription in yeast has provided many of our current paradigms for understanding the regulation of gene expression in higher organisms. Two things have made this possible: the ease with which, in yeast, one can combine genetic approaches with molecular genetic tools, and a striking evolutionary conservation in transcriptional mechanisms and in the transcription machinery from yeast to mammals. These same two properties make yeast an attractive surrogate organism in which to clone and characterize transcription factors from heterologous eukaryotic species, the subject of this chapter.
Hydatidosis and cysticercosis—larval cestodes
Authors: Craig Philips. , Rogan Michael T , Allan James C
Published: November 2023

Hydatidosis and cysticercosis are the names given to human or animal infection with the larval cystic stages of taeniid tapeworms belonging to the genera Echinococcus and Taenia. The most important species for public health are, in order, E. granulosus (the cause of cystic hydatid disease), T. solium (the cause of neurocysticercosis), and E. multilocularis (the cause of alveolar hydatid disease). These cestodes are transmitted in life cycles involving two mammalian hosts (Table 1) (1, 2). T. solium (with T. saginata) is unique among parasitic zoonoses in that man comprises the sole definitive host in nature. Apart from these species a few rare larval cestode zoonotic infections havebeen reported and these are thecause of polycystic hydatidosis, coenurosis, and sparganosis (see section 5).
Diagnosis of intestinal helminth infections
Authors: Denham D A , Suswillo R R
Published: November 2023

Intestinal helminths may cause minimal gastro-intestinal symptoms but many (e.g. Ascaris) have a major impact on human health compromising the nutritional status of children in developing countries (see Chapter 1)

Several species of nematodes, trematodes, and cestodes live in the human intestine and produce eggs which are passed in the faeces. Eggs of lung and liver flukes also are passed in the faeces. Parasitological diagnosis of most intestinal helminthic infections depends on finding and identifying these eggs. With the Taenia species, however, it is rare to find eggs in faeces as the proglottids are usually passed whole. Similarly, Enterobius vermicularis deposits its eggs on the perianal skin and eggs are seldom found in the faeces. Strongyloides stercoralis produces first stage larvae (L1) instead of eggs.
Histopathology of parasitic diseases
Authors: Francis N
Published: November 2023

Because of widespread international travel the diagnosis of imported diseases is a problem in hospitals outside their own geographical range. Added to this is the expanding population of HIV infected people in whom the diagnosis of opportunistic infections and unusual tissue responses to them is going to present an increasing problem across the UK and Europe. There are also a variety of parasitic infections indigenous to the UK which are discussed.
Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates
Authors: Geer Peter van der , Luo Kunxin , Sefton Bartholomew M , Hunter Tony
Published: November 2023

Phosphopeptide mapping enables the investigator to study the phosphorylation of a protein of interest in detail. The technique can be used to estimate the number of sites of phosphorylation in a protein, to determine the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for the phosphorylation of specific sites. Additionally, comparative phosphopeptide mapping is an invaluable tool for the determination of the identity, or lack of identity, of phosphoproteins obtainable only in trace amounts.
Studies of protein kinase/phosphatase specificity using synthetic peptides
Authors: Pearson Richard B , Mitchelhill Kenneth I , Kemp Bruce E
Published: November 2023

This chapter is concerned with studies of the specificity of protein kinases and protein phosphatases using synthetic peptides. During the past 15 years synthetic peptides have played an extremely important role in helping to elucidate the substrate specificity properties of protein kinases and, to a lesser extent, protein phosphatases. This knowledge has also played an important part in the development of our understanding of the mechanisms of regulation of protein kinases and protein phosphatases by intrasteric control. A brief summary of phosphorylation site motifs will be given, followed by an outline of the techniques involved in the preparation of synthetic peptide substrates, as well as the study of their phosphorylation and dephosphorylation. Frequent problems encountered in these techniques will be discussed under a trouble-shooting section. The approaches taken to design synthetic peptide substrates will be described in detail. Two emerging approaches, including rational enzyme mutagenesis and protein crystallography, have the potential to add to our understanding of the specificity of protein kinases.
Immunocytochemistry II: post-embedding staining
Authors: Ingham C A
Published: November 2023

Post-embedding immunocytochemistry involves carrying out immuno cytochemical techniques on tissue which has been fixed, dehydrated, and embedded in a material suitable for cutting sections for subsequent light or electron microscopy, (e.g. wax, celloidin, resin). It is beyond the scope of this chapter to discuss all the available techniques and so it will concentrate on post-embedding methods applied to material which has been prepared in the conventional manner for electron microscopy, including post-fixation with osmium tetroxide and embedding in epoxy resin. Many antigens do not survive this treatment; their antigenicity may be affected by osmium tetroxide treatment, by resin infiltration (possibly due to interactions of the antigen with epoxy groups), and by heat (1-3). However, amino acids such as -y aminobutyric acid (GABA), glutamate, aspartate, taurine, glycine, and glutamine have been successfully localized using these methods (4).
In situ hybridization histochemistry with oligonucleotides for localization of messenger RNA in neurones
Authors: Gerfen Charles R , Young W Scott , Emson Piers
Published: November 2023

In situ hybridization histochemical (ISHH) localization of messenger RNA transcripts (mRNA) provides a powerful technique for characterizing the bio chemical phenotype of neurones in the brain (1). With the growing list of characterized mRNAs encoding proteins and neuropeptide precursors the utility of ISHH for the study of neural system organization and function has increased at a phenomenal rate. Included in the list of available probes are those directed against a variety of neurotransmitter and neuropeptide receptors, including G-protein coupled receptors and ion channel receptors, proteins of various signal transduction systems, neurotransmitter synthetic enzymes, and neuropeptides. It is thus possible with ISHH techniques to characterize neurones on the basis of their expression of neurotransmitter receptors, signal transduction systems linked to these receptors, and the neurotransmitter/neuropeptides they may use for synaptic signalling. When ISHH is combined with retrograde axonal tracing techniques the additional information of the neuroanatomical connections of neurones may be added to their neurochemical characteristics. Additionally, levels of mRNA transcripts in neurones are sometimes regulated either during development or in the adult by alterations in the functional state of the neurones. Quantitative ISHH techniques provide the ability to measure changes in levels of mRNA levels in neurones to determine the functional organization of neural systems. In this chapter methods of utilizing oligonucleotides for ISHH localization of mRNA transcripts in neurones will be detailed. Both radiolabelled and non radiolabelled oligonucleotide methods, combinations of these methods to detect two mRNAs in single histologic brain sections, and a method for quantifying relative levels of mRNAs in neurones will be described.
Isolation and characterization of transfected cell lines expressing neurotransmitter receptors using a calcium dye and flow cytometry
Authors: Schieren Ira , MacDermott Amy B
Published: November 2023

An important early application of flow cytometry was to detect subpopulations of cells marked with a fluorescent ligand. This methodology was also used to sort out the targeted cells from the whole population. Traditionally, primary antibodies developed against unique cell markers were coupled to fluorescently tagged secondary antibodies and used to identify subpopulations of cells. In many cases, these markers were cell-type specific for receptors on the plasma membrane. For example, antibodies against immunoglobin receptors specific for subpopulations of lymphocytic T cells were usefully exploited as a way to detect changes in the expression of T cell subsets in different disease states (1). However, since in many cases it has proved difficult to generate antibodies to receptors for hormones, neurotransmitters, and neuromodulators, investigators have been motivated to explore other methods, not dependent on antibodies, with which to identify cells expressing specific receptors on their surface.
Cell lines in developmental neurobiology: assays of adhesion and neurite outgrowth
Authors: Pizzey John A
Published: November 2023

Communication between cells is a fundamental requirement for the diverse interactions implicit in normal development and tissue homeostasis. Such communication is generally mediated by membrane-associated molecules which can be classified (1) into three broad functional classes; cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), and cell junctional molecules (CJMs). However, it should be noted that these are not mutually exclusive classifications; for example, the neural cadherin (N-cadherin) is present both on the surface of neural cells as a CAM (2), but is also found in the adherens junctions on some non-neural tissues (3) and thus could be considered a junctional molecule.
Localization of Proteins and Carbohydrates using Immunogold Labelling in Light and Electron Microscopy
Authors: Vandenbosch Kathryn A
Published: November 2023

Interest in inducible and tissue-specific genes has spawned a need for visualization of gene expression within complex, differentiated tissues. Three available techniques address this need by combining molecular probes with anatomical methods. These include in situ analysis of gene expression using reporter genes, in situ hybridization for identification of messenger RNA species, and immunogold labelling for localization of gene products. Together, the three approaches overlay molecular information on an anatomical outline, and are rapidly increasing the sophistication of our knowledge of tissue composition and organization on a cellular level. The development of post-embedding labelling has enabled immunogold labelling to become routine practice for the demonstration of antigens in tissues (1). In this procedure, tissues are chemically fixed and embedded in plastic (with slight modifications from these protocols used for routine ultrastructural preservation). Tissue sections are then incubated sequentially in a primary antibody, specific for an antigen of interest, followed by a colloidal gold conjugate that binds to the primary antibody. The bound gold particles reveal the location of the antigen within the tissue. The combination of high specificity of immunological detection and good resolution by electron microscopy make immunogold labelling a powerful technique. This technique may also be used in combination with light microscopy where ultrastructural resolution is not required.
Preparation and Characterization of Oligosaccharide Elicitors of Phytoalexin Accumulation
Authors: Hahn Michael G , Darvill Alan , Albersheim Peter , Bergmann Carl , Cheong Jong-Joo , Koller Alan , Lo Veng-Meng
Published: November 2023

Plants utilize a variety of defence responses when confronted with invasive micro-organisms (1, 2). These defence reponses include such diverse mechanisms as the synthesis and accumulation of low molecular weight anti-microbial compounds called phytoalexins (3-6), the production of glycosyl hydrolases capable of attacking microbial surface polymers (7), the synthesis of proteins that inhibit microbial degradative enzymes (8, 9), and the modification of plant cell walls by deposition of callose (10, 11), hydroxyproline-rich glycoproteins (12, 13), and/or lignin (14).Biochemical analysis of the induction of plant defence responses has been facilitated by the recognition that cell-free extracts of microbial and plant origin are capable of inducing defence responses when applied to plant tissues. The active components in the extracts are commonly referred to as ‘elicitors’. The term ‘elicitor’ was originally used to refer to molecules and other stimuli that induce phytoalexin synthesis in plant cells (15), but the term is now used to describe molecules that elicit any of the observed plant defence responses.
Analysis of replication complexes of positive strand RNA plant viruses
Authors: Hayes Robert J , Buck Kenneth W
Published: November 2023

Viruses with genomes of positive strand (messenger-sense) RNA are found in animals, plants, and bacteria. Examples include poliovirus, foot and mouth disease virus, flockhouse virus (FHV), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and the bacteriophages MS2 and 013 (1). However, they are most abundant in plants. About 77% (approximately 500) of known plant viruses have positive strand RNA genomes (2). Enzymes that replicate RNA templates (RNA-dependent RNA polymerase, RdRp) apparently do not have proof-reading activities and have error rates of 10-4 to 10-5 (3). This may well have limited the size of viral RNA genomes, the largest of which among plant viruses are the closteroviruses (about 20 kb) and among animal viruses the coronaviruses (about 32 kb). Because of the high error rate, RNA of a given virus may be a population in dynamic equilibrium, with viable mutants arising at a high rate on the one hand and being selected against on the other.
Baculovirus expression vectors
Authors: Stewart Lorna M D , Possee Robert D
Published: November 2023

Progress in molecular virology has given us sufficient understanding of genetic organization and regulation in certain viruses of eukaryotes to enable us to utilize these systems as expression vectors of foreign genes. Analysis of the baculovirus genome has identified genes which are redundant when the virus is grown in cell culture. Furthermore, the promoters associated with these genes are exceptionally active, leading to the production of considerable quantities of protein within virus-infected cells. Consequently, baculoviruses have been used as a vehicle for the production of a variety of foreign gene products. In most cases the yields are very high and provide material suitable for studies of protein structure-function, therapeutic applications, or as vaccines. Foreign genes have also been inserted into the baculovirus genome to enhance the effectiveness of these agents as virus insecticides.
Expression of growth factors in microbial systems
Authors: Dudgeon Timothy J , Clements John M , Hunter Michael G
Published: November 2023

This chapter describes the use of microbial systems for the expression of growth factors. The important parameters which influence the choice of expression system are discussed. Protocols for the construction and expression of a synthetic gene for PDGF-B are shown. We describe in detail the construction and use of a yeast expression system to secrete platelet-derived growth factor B-chain (PDGF-BB), and draw comparison to the different expression routes used for epidermal growth factor (EGF), and transforming growth factor alpha (TGF-a), highlighting the benefits and disadvantages of the various approaches. Table 1 summarizes some of the routes which have been used to express various growth factors. Many of the standard molecular cloning techniques are now widely practised and are not described here. Protocols for these techniques may be found elsewhere (1, 2).
Long-term mouse bone marrow cultures
Authors: Spooncer E , Eliason J , Dexter T M
Published: November 2023

The first description of mouse long-term bone marrow cultures was published 15 years ago. At that time it was the only in vitro technique which supported the self-renewal of haemopoietic stem cells: a situation which still persists to the present day (1).
Isolation and characterization of glycoproteins
Authors: Carlsson Sven R
Published: November 2023

Today, significant information on the structure of a protein can be obtained without the need of having the protein in a pure form. After the breakthrough of cDNA cloning, very few full-length protein sequences have been determined at the amino acid level. Instead, the primary structure is deduced from the sequence of nucleotides in the DNA encoding the protein. From the derived sequence, certain properties of the protein can be predicted. Such sequence-specific information include ER-translocation signals, transmembrane domains, and potential glycosylation sites for N-linked glycans. Furthermore, with refined algorithms for secondary structure and folding predictions, molecular modelling in three dimensions can be made from only primary sequence information, especially in combination with crystallographic or NMR data from homologous proteins.
Isolation and fractionation of N-linked oligosaccharides from glycoproteins
Authors: Kobata Akira , Endo Tamao
Published: November 2023

For many glycoproteins, the carbohydrate moieties endow their polypeptide portion with important physical properties such as conformational stability, protease resistance, charge, and water-binding capacity. Interest in the biological roles of the carbohydrate chains of glycoproteins has been intensified because of interesting examples of their importance as recognition markers for protein targeting, and cell-cell and cell-matrix interactions (1). Furthermore, the importance of carbohydrate chains has been increasingly recognized year by year, as evident from the recent data of recombinant glycoproteins produced in heterologous cell types (2). In order to elucidate the molecular basis of such physicochemical effects and recognition phenomena of the carbohydrate moiety of glycoproteins, information as to the exact structure of the relevant oligosaccharide moieties are indispensable.
Structural characterization of oligosaccharides from glycoproteins
Authors: Kobata A Kira , Takasaki Seiichi
Published: November 2023

The final goal of structural analysis of sugar chains is to determine sequences, linkages, and anomeric configurations of monosaccharides included in oligosaccharides. Various enzymatic and chemical methods have been developed and applied to the structural analysis. One of the useful approaches is to use exoglycosidases which release exposed monosaccharide units of sugar chains. Usually, each of these enzymes has the strict glycon specificity toward monosaccharide species and its anomeric configuration. For example, β-galactosidases can hydrolyse β-galactosides, but not any other β-glycosides. β-Galactosidases do not hydrolyse a-galactosides. There is no glycosidase acting on both α- and β-glycosides. Thus, an exoglycosidase can release a particular monosaccharide with either α- or β-configuration from the non-reducing termini of sugar chains. A typical exception is β-N- acetylhexosaminidases which cleave both β-N-acetylglucosaminyl linkages and β-N-acetylgalactosaminyl linkages.
Structural characterization of N-glycans obtained from metabolically-radiolabelled glycoproteins
Authors: Cummings Richard D
Published: November 2023

Within the last few years tremendous advances have been made in our understanding of the structure and function of glycoconjugates synthesized by mammalian cells. The oligosaccharide moieties of glycoproteins, glycolipids, and glycosaminoglycans are now known to promote specific recognition events important in cell-cell adhesion, cell-matrix adhesion, binding and uptake of extracellular glycoproteins, and intracellular targeting of macro-molecules to organelles. The excitement in this area of research has created a need to improve the analytical techniques for structural characterization of extremely small amounts of glycoconjugates. One highly sensitive and reliable approach involves the structural characterization of metabolically-radiolabelled oligosaccharides. In this technique living cells are incubated with radioactive sugar precursors such as 2-[3H]mannose, 6-[3H]glucosamine, and 6-[3H]galactose, and glycoconjugates synthesized by the cells are radiolabelled by the respective sugar nucleotide donors. The radiolabelled oligosaccharide moieties of glycoconjugates can be then be released, either enzymatically or chemically, separated, and analysed by a variety of techniques. All the techniques of structural analysis of glycoconjugates, except those involving spectroscopic instrumentation, are useful for characterizing metabolically-radiolabelled N-glycans.
Advances in antibody eng1•neer1• ng
Authors: Verhoeyen Martine E , Windust John H C
Published: November 2023

In 1894, Emil Behring reported the discovery of antibodies, then called antitoxins, and their therapeutic effects (1). About 80 years later, Kohler and Milstein established the techniques to reliably produce monoclonal antibodies (mAbs) by hybridoma cell lines (2). This event, together with the availability of increasingly sophisticated molecular biology and protein engineering techniques, opened up the exciting new science area of antibody engineering, and led to the creation of antibodies and antibody-derived molecules designed to suit possible applications not only in the medical but also industrial world.