bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of cell polarity
Authors: Hughson Elaine J , Hirt Robert P
Published: November 2023

The epithelial sheets of metazoa, such as those lining the cavities of the lung, the kidney, or the digestive tract, can be compared to the plasma membrane of unicellular organisms (1). They are the site of complex exchanges between the outside world and the internal milieu which are essential to maintaining a constant body fluid composition. Epithelial cells, therefore, in addition to forming a simple barrier, are the site of numerous vectorial transport processes which are the bases of functions such as water and ion homeostasis, metabolite uptake and secretion (2, 3), and ultimately, protection against pathogens (4).
Transcription on chromatin templates
Authors: Luse Donal , Felsenfeld Gary
Published: November 2023

As it has become clear that the coding regions of transcriptionally active chromatin are packaged in nucleosomes (1, 2, 3), it has also become important to determine how such a histone-covered template can accommodate to the passage of RNA polymerase. For this purpose, it has proved useful to employ ‘model’ systems, in which templates reconstituted from specific DNA sequences and histone octamers are transcribed by either prokaryotic or eukaryotic polymerases. Experiments with prokaryotic enzymes such as the T7 or SP6 polymerases give us information about the intrinsic behaviour of histone octamers during transcription; they obviously cannot reveal possible contributions to the reaction that might be made by a eukaryotic polymerase, or by specific factors capable of modifying nucleosome stability that might be present in eukaryotic cells. Experiments with eukaryotic polymerases have the limitation that in using purified enzymes these specific factors may have been excluded. Despite these limitations, much valuable information about the properties of chromatin templates has been obtained with both kinds of polymerases.
Epigenetic regulation in mammals
Authors: Tilghman Shirley M , Willard Huntington F
Published: November 2023

The term ‘epigenetic’ when applied to mammalian genes refers to different stable states of phenotypic expression usually thought to be due to differential effects of chromosome or chromatin packaging, and determined or influenced by control mechanisms which can be manifested over exceptionally long distances. Both the recognition of epigenetic states and the design of tractable experimental approaches to analyse them require an appreciation for mammalian gene expression in its dynamic setting. One must consider those aspects of chromatin assembly, structure, and replication (see Chapters 1-3) that determine whether, when, and at what level genes are expressed, the series of dynamic events that occur at each cell division, when and under what circumstances epigenetic states are stably transmitted through meiosis or mitosis, and when and under what circumstances epigenetic states are reversed in the course of normal development or as a result of mutation or chromosome rearrangement.
Isolation and growth of hyperthermophiles
Authors: Sharp Rirhard J , Raven Neild. H
Published: November 2023

This chapter discusses the problems encountered in the isolation and cultivation of hyperthermophilic microorganisms and presents procedures and guidance for working with the more common hyperthermophilic. We define hyperthermophilcs as organisms with growth optima of> 75°C. This excludes thermophilic Bacillus and Thcrmus species which have recently been discussed in other reviews (1, 2). The chapter will describe procedures which have been applied to the growth of both hypcrthcrmophilic Archaea and Bacteria.
Fermenter Vent-Gas Analysis
Authors: Salmon Peter M , Buckland Barry C
Published: November 2023

Almost any microbial process involves the consumption or evolution of gases, and vent-gas analysis therefore provides a convenient means to monitor microbial activity. In contrast to other monitoring techniques for which the culture medium must be placed in direct contact with a sampling device or probe, vent-gas analysis poses no risk to sterile operation and has consequently become indispensable in both research and manufacturing (1).
Preparation of single cell suspensions
Authors: Cheetham K M , Shukla N , Fuller B J
Published: November 2023

The cell, as the basic functional unit within a tissue or organism, holds the potential for allowing us to probe the numerous interrelated processes which comprise overall function, or to manipulate specific pathways for scienlif1c or commercial gain. As such, the rapid expansion over the last two decades in cell applications in fields such as biochemistry, molecular biology, and biotechnol ogy has led to an equivalent effort in devising methods for cell isolation and handling. Although many important studies have been performed on continu ously growing cell lines (which may often he commercially available), many other investigations can only be performed on cells from selected intact tis sues, expressing particular differentiated functions. Thus the ability to liberate high yields of viable cells from the structural constraints of their native surroundings is often the first hurdle to overcome in experimental biology. Section 2 describes these methods of dispersal. These are then complemented by the need to ensure that the process has yielded individual cells which have retained metabolic and functional capabilities. Section 3 describes methods of viability assessment. Section 4 provides methods for separating viable and non-viable cells from the isolate, which invariably is a mixture of populations containing the desired cell type. Methods of cell characterization arc described in Section 5. In the current chapter, we have addressed these particular prob lems, giving a variety of examples from currently used techniques.
Applications of Antisense Technology in Plants
Authors: Kuipers Anja G J , Jacobsen, Evert , Visser Richard G F
Published: November 2023

In 1984, Izant and Weintraub (1) published the first experiments on the antisense effect of gene constructs in eukaryotic cells. Since then, the inhibition of gene expression via antisense RNA has developed into a technique that is widely applied in molecular biology. In the field of plant biotechnology and plant molecular biology Ecker and Davis (2) were the first to describe a transient assay system in which carrot protoplasts were simultaneously electro-porated with sense and antisense CAT genes. This resulted in the inhibition of CAT activity of up to 95% as compared to the control which only contained a sense CAT gene. These initial studies were followed by experiments with various plant species, in which the expression of a wide variety of genes has been effectively inhibited, either transiently with in vitro transcribed antisense RNAs, or by the introduction of antisense genes via transformation (reviewed in refs 3-5).
Antisense Molecules and Ribozymes: Medical Applications
Authors: Strauss Michael
Published: November 2023

The application of antisense oligodeoxynucleotides as tools for specific inhibition of gene expression was an extremely important step in the development of molecular medicine (reviewed in ref. 1, see also Chapters 3 and 11). From the first moment of experimental investigation of the power of these molecules it was obvious that, at some stage, this class of molecules would find its way to clinical application as therapeutic drugs (2). After several years of successful use in cell culture experiments, antisense molecules including synthetic oligos and expressed RNA are increasingly applied in animal experiments. Most of the studies carried out in cdl culture and animals so far have been aimed at the investigation of the consequences of the loss of a particular function. The antisense strategy has the advantage over the powerful method of gene knock-outs of allowing transient suppression of gene expression. Thus. it allows one to inactivate, for a short period of time, those functions which would be lethal if inactivated at the gene level (see also Chapter 12 for another approach to the problem of lethality). Since the power of the antisense approach first became obvious, studies focusing on the treatment of diseases have been initiated over the last few years. It seems only a matter of time before this strategy can be applied clinically to the treatment of particular diseases, e.g. infectious diseases like hepatitis B and C, or HIV infection, and even some forms of cancer. This chapter will give a brief overview of the different fields of medical application of antisense molecules, and will provide the reader with protocols for the experimental use of these molecules in cell culture and animal studies.
Measuring antibody affinity in solution
Authors: vadi-ohaniance Lisa dja , Goldberg michele. , friguet bertrand
Published: November 2023

Determination of the affinity of a monoclonal antibody (mAb) for its antigen (Ag) is of considerable importance. It gives a quantitative indication of how strong the interaction is between the Ab and the Ag. It is the basic experi mental parameter in a variety of studies, such as the analysis of mAb/Ag binding mechanism (1, 2) or the use of mAbs as conformational probes (3, 4). Hence the need for convenient and rigorous methods to determine the affin where Kd is the equilibrium dissociation constant, [complex] is the concentra tion of saturated antigen or mAb sites, [Ag] is the concentration of free anti genic sites on the antigen, and [Ab] is the concentration of free binding sites on the antibody. These concentrations are binding site concentrations in the solution at equilibrium. This has three important consequences, as described in Sections 1.1-1.3.
Isolation and fractionation of chromatin and linker histones
Authors: Thomas Jean O
Published: November 2023

Nuclei from different sources usually have a common pattern of core histones (components of the histone octamer) as revealed by SDS/polyacrylamide gel electrophoresis, but in some cases they have distinctive linker histone variants that may correlate with particular states of cell differentiation and transcriptional activity (more subtle Hl subtypes present in many cell types are not resolved in SDS gels). Nuclei from different sources may also differ in nucleosome repeat length. Procedures are described for the isolation of nuclei from rat liver, and from chicken erythrocytes and sea-urchin sperm which contain extreme HI variants and have repeat lengths longer than the canonical 200 base pairs of rat liver and many other tissues. A method is also described for the isolation of nuclear populations from various mammalian brain cell types; the distinctive feature here is that neurones from the cerebral cortex have a shorter repeat length than the glial cells from the same tissue or cerebellar neurones, and only half the Hl content.
Electron microscopy of chromatin
Authors: Thoma Fritz
Published: November 2023

Electron microscopy (EM) has been used to investigate how DNA is folded in nucleosomes, chromatin fibres. and higher-order structures. In contrast to biochemical and biophysical techniques, which produce averaged information of a whole population of structures, EM allows one to visualize and analyse individual molecules or chromatin complexes; averaged information must then come from the analysis of a significant number of complexes.
Cytokinesis
Authors: Goldberg Michael L , Gunsalus Kristin C , Karess Roger E , Chang Fred
Published: November 2023

Cytokinesis is a multi-step process that proceeds linearly with time according to the following general framework. It begins with a signal from the mitotic apparatus that must be transmitted to the cellular cortex at the cleavage site. Receipt of the signal at the cortex sets off a complex set of reactions that transduce the signal into changes in the actin cytoskeleton and lead to formation of an actomyosin-based contractile ring. Mechanical processes in the ring and its interaction with the cortex result in cytokinesis. The distinctions between these steps are probably largely artificial. It is not clear, for example, whether some of the molecules we describe as transducing a cytokinetic signal are in fact part of the signal itself. Even this seemingly innocuous (though admittedly simplified) view of cytokinesis is not free of controversy, and some kinds of cytokinesis almost certainly do not conform to this pattern. Many questions remain unanswered and are addressed in the following sections.
Prediction of barley which causes premature yeast flocculation
Authors: Nakamura Takashi , Chiba Kazuhiro , Asahara Yumiko , Tada1 Setsuzo
Published: November 2023

During fermentation of wort produced from certain malts, premature yeast flocculation occurs leaving fermentable sugars in solution. Premature yeast flocculation results in a low attenuation. We have developed a novel fermentation method to predict barley which causes premature yeast flocculation. A barley extract obtained by hot-water extraction using an enzyme mixture of BÂ¬ glucanase, protease, a-amylase and B-amylase was added to a synthetic medium. The premature yeast flocculation activity of this extract was evaluated by a fermentation test. Analysis could be carried out within four days and was applicable to either dormant or water sensitive barley.
Production of aromatic hop varieties starting from ‘‘in vitro”
Authors: Vanhame! S , Putman R , Timmermans P , Everaert E , Cooman L De , Beeumen J Van , Oord R Van Den , Debergh P
Published: November 2023

The objective was to increase the return of the aroma hop Saaz starting from in vitro cultures. Saaz hops were cultivated on a hop garden in our brewery, in cold greenhouses, hothouses and plastic tunnels. Fingerprinting of various crops showed that they are identical with the original variety; chemical and sensorial analysis of pilot brews showed less differences. The influence of nutritive ingredients, length of the day and temperature on flower growth were evaluated. The formation of bitter acids and essential oils were studied in function of the developmental stage of the hop cones and cultivation method.
Enzymes from the biosynthesis of hop α- and β-acids
Authors: Fung S Y , Zuurbier K W M , Paniego N B , Scheffer J J C , Verpoorte R
Published: November 2023

The hop enzyme catalysing the first step has been purified; it is a new enzyme closely related to the enzyme chaicone synthase from plant flavonoid biosynthesis. Work on hop enzymes of the later stages is still in progress. Knowledge from the study of hop acid biosynthesis could lead to interesting applications in hop breeding.
Differential behaviour of cis- and trans-iso-α-acids
Authors: Hughes P S , Menneer I D , Walters M T , Marinova G
Published: November 2023

Recent analytical developments have allowed the routine separation of iso-a-acids. By applying such methodology, it has become apparent that there are differences in the behaviour of c/s- and trans-iso-a-acids. Thus, trans-iso-a-acids are lost to a greater extent during beer production and on subsequent storage, and are also partitioned into beer foam to a greater extent than their cis- counterparts. Whilst it is difficult to explain these differences on hydrophobicity grounds, it is possible that there is a thermodynamic rationale. These differences in behaviour are of practical significance, particularly in terms of optimizing hop acid utilization, bitterness delivery and foam quality.
The effect of different cereal coloured malts on reducing power during the brewing process
Authors: Griffiths D J , Maule A P
Published: November 2023

This project was designed to investigate the relative reducing power of barley, wheat, rye and oat crystal malts, the relationship with degree of colour formation and the reducing power levels during the brewing process when these malts were used. A specially designed 3 kg capacity mini-roaster was used to produce the crystal malts. Reducing power was measured using the Chapon rapid determination method (iron II dipyridyl complex) on a Tannometer. Indications are that measurement of reducing power during the brewing process will provide useful data towards maximising beer stability by selection of raw materials.
Modernization, rationalization and automation of the brewhouse operation at the Carlsberg Breweries in Copenhagen
Authors: Munksgaard Jannik
Published: November 2023

The aims of the modernization-project were several. It was intended to improve wort quality, improve the brewhouse yield, improve the efficiency and reduce the consumption of utilities. The project was undertaken under timerestrain, as full production had to be maintained throughout the rebuilding phase, which should be finalized before taking over production in connection with the general rationalization of Carlsberg’s Danish operation. The main items of the project were the installation of new mash-filters and a control system covering the whole brewhouse operation. However, the brewhouse was almost entirely renewed. The project succeeded on all expectations, though not without problems during the implementation.
Microbiological contaminations in immobilized yeast bioreactors for primary fermentation
Authors: Haikara A , Virkajarvi I , Kronlof J , Pajunen E
Published: November 2023

Microbiological purity in beer fermentation is a prerequisite, particularly in continuous fermentation. In contrast to secondary fermentation a wide range of micro-organisms was able to attach and grow in immobilized yeast bioreactors for primary fermentation. Wort bacteria with high specific growth rates were the most hazardous ones, producing off-flavours such as dimethyl sulphide. A Saccharomyces wild yeast caused both superattenuation and formation of phenolic off-flavours. Lactic acid bacteria were neither able to multiply nor to produce off-flavours. Aseptic treatment of wort and yeast, efficient monitoring also by means of chemical analysis and appropriate cleaning procedures are suggested for hindering microbiological contaminations.
Purification and characterization of yeast 3-methyl butanal reductases involved in the removal of wort carbonyls during fermentation
Authors: Ncdcrvelde L Van , Verlinden V , Philipp D , Debourg A
Published: November 2023

Aldehydes are known to impact negatively on beer flavour and flavour stability and yeast reductase activity is essential in the removal of the worty off-flavours during fermentation. In order to characterize the 3-methyl butanal reductases recently identified in yeast, a 3 steps purification procedure has been developed and the pure enzyme is identified by a specific detection after electrophoresis on acrylamide gel. The obtention of this purified enzyme has allowed a detailed study of the enzyme affinity and the determination of the N-terminal amino acid sequence is underway and will lead to the cloning of the gene encoding this enzyme.
The effect of the divalent ions magnesium and calcium on yeast fermentation performance in conventional (12°P) and high (20°P) gravity worts in both static and shaking fermentations
Authors: Rees Elizabeth M R , Stewart Graham G
Published: November 2023

Investigations into alleviating the drawbacks of high gravity brewing have shown that the addition of certain compounds, including metal ions, can optimize yeast performance under the conditions exerted by high gravity worts. In the present study it was observed that the addition of calcium or magnesium resulted in changes in the fermentation parameters in the ale and lager strains employed. These results emphasise the need to monitor the calcium to magnesium ratio in brewer’s wort, and for an awareness of the inter-actions that occur between these ions during fermentation.
Beer maturation in a continuously operating bioreactor using a flocculating brewer’s yeast strain
Authors: Mafra Isabel , Teixeira J M Machado Cruz& J A
Published: November 2023

The use of a flocculating yeast for beer maturation - one of the main areas of concern in today’s brewing industry - is assayed. Experiments made in a 6 litre continuous internal loop gas-lift bioreactor with a sedimentation zone using a brewer’s yeast flocculating strain show that this system can be applied for beer maturation. For a residence time of 32 hours, the quality of the final beer is not affected and the desired reduction of VDKs to levels below the threshold value is obtained. The operational stability of this procedure was confirmed by one month of continuous operation.
New techniques for the evaluation of interactions in beer foams
Authors: Hughes P S , Wilde P J
Published: November 2023

The heterogeneous and dynamic nature of beer foam presents a significant scientific challenge to scientists. Applying techniques such as microconductivity and dilational rheology, the behaviour of individual iso-a-acids and beer protein fraction has been studied. From these model studies it was apparent that the amphipathicity of hop-derived bitter acids is key to their foam¬ stabilizing potency. From brewing trials, it is apparent that foam assessment methods were sensitive hop bitter acids in various ways. Thus, Ross & Clark and microRudin values are adversely affected by high proportions of trans-iso-a-acids. This study indicates the importance of hop acid distribution for optimal foam activity.
Practice on the improvement of beer flavour stability by new technology and quality management
Authors: Takemura Osamu , Oka Kaneo , Uchicta Masaaki , Kawasaki Yasutsugu , Kakimi Yoshihiko
Published: November 2023

We have practiced a new methodology in the quality management for the flavour stability of beers produced in over 25-year-old breweries using new technology. The flavour stability has been improved almost two times better than before by adopting the following three technologies:

process changes for the reduction of active oxygen generating activity of beers, the enhancement of active oxygen quenching activity of beers and the minimization of oxygen in beers; 2) introduction of rapid prediction methods for daily analysis of flavour stability using electron spin resonance spectroscopy; 3) quick action in process control in response to the results of its daily analysis.
Untersuchungen zum Bieraroma und der Geschmacksstabilität mit verschiedenen Malzschilttungen hergestellter dunkler Biere
Authors: Forster C , NarziB L , Back W
Published: November 2023

Dark beers were brewed with varying ratios of different dark malts, black malt as well as coloured beer. In dark beers brewed with specially kilned dark malts according to a specific withering scheme high concentrations of flavour compounds were found which show a strong increase during the staling of pale lager beer. In dark beers, however, these substances are in high concentrations responsible for an agreeable sweet malty flavour. The time after kilning at which dark malt is used for brewing has a great influence on malt flavour as well as on staling of dark beer. Black malt has a totally different flavour composition and results in dark beers of little malt flavour and low flavour stability.
A modern approach to the sensory evaluation of beers
Authors: Hegarty Paul K
Published: November 2023

Flavour assessment is sometimes seen to be time consuming and unreliable and there is a need for robust methods. Products should be optimised using external consumer research and subsequent internal assessment used to ensure that the target is met. The rigid demands of sensory analysis must be balanced with considerations of cost and sample number to be assessed. Methodology reported reduces the need for duplicate sample assessment and dependence on complex statistics. An intercollaborative study showed that flavour results are not currently comparable between breweries. Development of international flavour methods is possible but benefits must be balanced against the cost of their development and maintenance
The relationship between sensory data and the composition of beer
Authors: Hughes P S , Menneer I D
Published: November 2023

Sensory evaluation tends to be an expensive procedure and prone to high noise levels. Thus, it is desirable to be able to model sensory parameters from analytical data alone, and understand the key variables which need to be controlled. Studies on the effects of gas composition on beer flavour revealed that high levels of nitrogen gas in beer significantly affect perceived hoppy, bitter and astringent attributes, whilst the composition of the base beer is unaltered. Time-intensity (Tl) assessment of beers brewed with different hop products revealed that bitterness Tl curves are dependent on the hop acid profile.
Optical systems for the in-line process control of consumer sensitive beer parameters
Authors: Sharpe F Richard , Engineer Sam J , Ormrod Ian H L
Published: November 2023

Optical in-line process control systems for assessing consumer-sensitive beer parameters are reviewed. Existing in-line optical techniques available for the measurement of colour and haze are discussed. Eighteen months experience of an in-line near infra-red analysis of alcohol in a 2.5m hi plant where multiple beer qualities are brewed diluted and packaged is reviewed highlighting advantages and issues. The development and commercial experience with a new in-line bitterness measurement are described together with a simple laboratory instrumental technique for bitterness analysis. It will be demonstrated how both these latter techniques can measure colour simultaneously.
An investigation into styrene in beer
Authors: Daly Brian , Collins Eddie , Madigan David , Coakley Dan Donnelly Mairead , Ross Paul
Published: November 2023

Certain yeast strains possess a decarboxylase activity which converts ferulic acid to 4-vinyl guaiacol (4VG), a marker compound for phenolic off-flavour (pof). A similar mechanism may be responsible for the conversion of cinnamic acid, a constituent of wort, to styrene. To explore this two analytical techniques were developed for the analysis of styrene. Fermentations using Por strains were shown to form 4VG and styrene. Formation of 4VG was mirrored by the intensity of styrene formation. With Pof strains neither 4VG nor styrene was produced. Polymerase chain reaction analysis showed that the pof gene was conserved in terms of size in all strains, and was surprisingly present in Pof strains.
Der Einflu8 der Kurzzeiterhitzung auf die Bierqualität
Authors: Wackerbauer Karl , Zufall Carsten
Published: November 2023

The impact of flash pasteurization on beer quality using different temperature-time combinations has been examined with trials of 15, 80 and 500 pasteurization units at temperatures of 60°, 72° and 84°C each. In contrast to the common opinion that high flash pasteurization temperatures are equivalent to a stronger damage to the product, a flash pasteurization made at 60°C led to a more pronounced stale flavour and higher aldehyde content and a lower flavour stability compared to higher temperatures. The time during which beer is kept at pasteurization temperature seems to play a decisive role in this context. Whilst 72°C turned out to be the most favourable temperature for 15 PU, beer quality preservation during flash pasteurizations of 80 and 500 PU was best at a temperature of 84°C. It could thus be demonstrated, that the pasteurization unit is not suitable as a measure for product damage through thermal treatment.
Monitoring the reliability of sensory profiling data by comparing flavour profiling results with results from an ‘overall difference test’
Authors: Daems V , Delvaux F
Published: November 2023

To evaluate the effect of specific production parameters on the sensory properties of beer, 15 test beers were tasted by a sensory profiling panel, and by a so-called “difference-panel”, scoring the overall difference between pairs of beers, without identifying specific attributes. The comparison of Biplots and MDS-plots showed that beers were grouped the same way, based on their overall flavour impression (difference testing) or based on specific flavour characteristics (profiling). This can be seen as an indication of the reliability of the profiling data and the relevance of the descriptive terminology.
The use of hydraulic mechanisms for the development of high flux crossflow microfiltration systems
Authors: O’Shaughnessy C L , Durosinmi-Etti 0.
Published: November 2023

The main reasons that crossflow microfiltra!ion has not been widely accepted in brewing is due to low filtrate productivity and potential loss of quality components. Studies aimed at understanding the fouling mechanisms involved have shown that filtration performance is influenced by raw materials, distribution of particulates within the beer and the filter membrane material. Three novel techniques for enhancing filtrate productivity without loss of beer components have been demonstrated, warmer filtration, pressure ramping (gradually increasing the pressure during a filter run) and backpulsing (the momentary reversal of filtrate flow to dislodge the fouling layer). The latter is the most effective.
Cold stabilised beer - an economic reality
Authors: Walker Grahame , Gaub Reiner
Published: November 2023

Membrane filtration has been available to brewers for some years as an alternative to pasteurisation. This technique is attractive as it provides good security and the natural fresh flavour of the beer is retained. Early systems were manually operated and required careful management to be economic. The CFS (Cluster Filter System) is a fully automated membrane filter for microbiological stabilisation of beer. The unique patented (EP87116109.7 US5417101) cluster arrangement allows groups of cartridges to be integrity tested and cleaned independently. This ensures that cleaning is optimised and if necessaIy the unit can be operated with one or more clusters isolated. This poster will review the design and operation of the CFS including independent studies of microbiological and economic performance.
Recovery of CO2 with relatively high air content
Authors: Swinkes Wim , Haffmans Bert , Crommentuyn Leon
Published: November 2023

With respect to the recovery of CO2, the following requirements apply, which to date, have not been met: increase of the recovery quota, simplification of complicated changeover of the fermentation tank, higher volumetric efficiency of the CO2 recovery plant, lower concentration of 02 and by-products of fermentation in the recovered CO2• A new CO2 recovery plant at Bavaria Brewery, Lieshout (capacity 3,000 kg CO/h} meets these requirements. By means of careful design, the concentration of CO2 in the vent gasses is reduced to 65 vol.-%. This results in a total efficiency of 86 % or a CO2- related efficiency of 90 %, and consequently an extremely.efficient recovery.
Microcalorimetry of protein-DNA interactions
Authors: Cooper Alan
Published: November 2023

This chapter describes the application of current DSC and ITC calorimetry methods to the study of protein-DNA complexes and related protein-nucleic acid interactions, together with the minimal thermodynamic background necessary to interpret the experimental data. Microcalorimetry is a potentially non-invasive technique for studying biomolecular stability and interaction in solution. Since almost all chemical or physical changes involve uptake or release of heat energy, calorimetric measurement of this heat effect can be used as a general. relatively non-specific analytical probe of the extent or magnitude of the pa11icular process under investigation. In this respect, calorimetric methods are just like other analytical methods (fluorescence, UV/vis, etc.) that rely on intrinsic properties of the molecules concerned, and it is not necessary to understand the fundamental thermodynamics lying behind the heat effects. However, for those that need it, calorimetry also provides direct measurements of real thermodynamic quantities that relate to the fundamental forces at work in the process and which can give additional analytical information.
Solid-phase DNAse I footprinting
Authors: Sandaltzopoulos Raphael
Published: November 2023

Classical DNase I footprinting is used to map the DNA targets of sequence-specific binding proteins (1). An end-labelled DNA fragment encompassing a specific target sequence is incubated with the DNA-binding protein to form a protein-DNA complex. Mild digestion of the fragment with DNA as I gives rise to a distinct fragmentation pattern which c;in be visualized owing to the end-labelling. Tightly bound protein will protect its target sequences from DNase I cleavage. The direct comparison of the cleavage pattern derived from the DNA/protein complex with that derived from free DNA reveals the sequences that are protected by the hound protein. Ordinary footprinting protocols are laborious, with most of the time needed being spent on the preparation of the labelled template and purification of DNA fragments prior to gel electrophoresis.
Digitization and quantitative analysis of footprinting gels
Authors: Smith Judith
Published: November 2023

Quantitative footprinting is used in a variety of experimental procedures, including general nuclease protection, band shift analysis. drug-DNA binding, ribosome cleavage and protein component analysis. The 3-step process of quantitative gel analysis is achieved by gel electrophoresis follow’d by digitization of the gel, and subsequent computer analysis. Electrophoresis produces a pattern of more or less well-separated bands which correspond to the individual components of the sample. Digitization of the gel enables the pattern to be subjected to quantitative analysis, and hence the determination of the contribution from each individual band. This fundamental technique is used by marry experimentalists for many kinds of quantitative gel analysis, but where approximate area calculations may be sufficient for some anc1lyses, footprinting experiments need to be able to detect minute differences between band intensities, for which a high resolution analysis is required.
Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP)
Authors: Ekwall Karl , Partridge Janet F
Published: November 2023

The fission yeast (Schizosaccharomyces pombe/S. pombe) is a unicellular ascomycete, that is a well-studied model organism for cell biology and molecular genetics.
Isolation of vertebrate metaphase chromosomes and their analysis by FISH
Authors: Craig Jeff
Published: November 2023

This chapter concentrates on the in situ hybridization of DNA probes to metaphase chromosomes isolated from vertebrate cells. Fluorescence in situ hybridization (FISH) has a number of applications within the field of chromosome structure, ranging from the simple mapping of specific genes or markers (1-3), mapping transgene integration sites (4-7), through to analysis of the distribution of particular classes of DNA sequence across the genome, e.g. repeat sequence families (6--10), CpG islands (11), fractions of defined base composition (12), and scaffold or matrix-associated sites (13). Whole chromosomes can also be highlighted by chromosome painting probes (14, 15, and Chapter 6), and interspecies hybridization can highlight regions of conserved synteny (16, 17). There are also other novel applications of FISH in studies of chromosome structure, these either analyse karyotype changes or explore the higher order organization of chromosomes (18-24).
Analysis of mammalian interphase chromosomes by FISH and immunofluorescence
Authors: Bridger Joanna M , Lichter Peter
Published: November 2023

The analysis of chromosome structure and organization in mammals has traditionally been performed on metaphase chromosomes. This is especially true for the localization of specific DNA sequences and chromosomal-associated proteins longitudinally along the metaphase chromosomes (1). It is not yet clear to what extent the structure and organization of metaphase chromosomes is different from, or related to, the structure and organization of the same chromosomes in interphase nuclei. Furthermore, different proteins may contribute to the maintenance of chromosome structure and organization during mitosis and interphase.
Fluorescence in situ hybridization in whole-mount tissues
Authors: Dernburg Abby F
Published: November 2023

This chapter outlines techniques for the investigation of chromosome organization using fluorescence in-situ hybridization (FISH) to DNA in whole-mount tissues. Emphasis is placed on procedures that preserve global tissue morphology. Nuclear architecture is not static, but may vary with time, cell type, and stage of development or differentiation. Therefore, many questions demand the examination of chromosome organization in a relevant developmental, spatial, and temporal context. These techniques were designed to permit the investigation of a variety of such questions, including chromosome pairing (1, 2), interaction of chromosomal DNA with proteins or other nuclear structures (3), and the global arrangement of chromosomes within the nucleus (4). Whole-mount FISH also allows direct karyotyping of gametes or embryos ( (5), Figure 1).
Control of transcription by adenovirus-E1A proteins
Authors: Zantema Alt , Van Der Eb Alex J
Published: November 2023

Human adenoviruses induce productive infection in their natural host, but in rodent cells they usually cause only abortive infection. Some abortively infected rodent cells will undergo transformation, in which case adenovirus (Ad) DNA is integrated into the host genome and at least some of the Ad genes are expressed. Early region 1 (E1) of Ad DNA plays an essential role, both in lytic infection and transformation. In either case, it affects the cell by altering cellular gene expression and, in the case of lytic infection, it also stimulates transcription of other Ad genes. The E1 region consists of two transcriptional units, E1A and E1B, which respectively encode two related proteins of about 45 kDa and two unrelated proteins of 19 kDa and 55 kDa. E1A is the gene that induces most of the alterations of transcription and is able to immortalize cells (1). When E1B is also present, a distinct morphological transformation takes place and transformation occurs more efficiently due to the fact that the E1B region protects the cells from apoptosis, one of the (side)-effects of E1A (2): the E1B-55 kDa protein inactivates p53 and thereby blocks the p53-dependent apoptosis (for a review on the role of p53 see ref. 3), while the ElB-19 kDa protein binds to and inactivates the proapoptotic Bax, Bak and Bhk/Bik proteins (4 – 8).
Mutagenesis of DNA virus genomes
Authors: Leppard Keith n.
Published: November 2023

DNA viruses encompass a wide range of virus types which can infect bacteria, plants, insects, and birds, as well as mammals including man. Mutant strains of these viruses have been essential to our understanding of how individual viral genes contribute to the process of infection but, for a variety of reasons, far more mutant strains have been isolated of some viruses than of others. DNA viruses which have been subject to extensive genetic characterization include various bacteriophages, and mammalian viruses such as simian virus 40, adenovirus types 2 and 5, herpes simplex virus type 1, and vaccinia virus. Although there are considerable similarities between the techniques used to isolate mutant viruses from different host systems, this chapter deals exclusively with the study of mammalian viruses.
Transformation
Authors: Jin Yishi
Published: November 2023

The technique of transformation was introduced to C. elegans in the early 1980s, when tRNAs isolated from sup 7(st5) amber suppressor mutants were injected into the gonads of tra 3 animals to suppress the sex transformation phenotype of an amber suppressible mutation, tra 3(e1107) (1). Soon after, DNA mediated germline transformation was evolved both in integrative and non integrative forms (2, 3).
Gene expression patterns
Authors: Mounsey Andrew , Molin Laurent , Hope Ian A
Published: November 2023

The first step after identifying a C. elegans gene of interest, either through a molecular approach or through sequence homology, is often to determine the gene ‘s expression pattern. Expression pattern information is relatively easy to obtain and in combination with the sequence data can provide a valuable guide to subsequent strategies of investigation. For example, when and where a gene is expressed may direct the search for a mutant phenotype for that gene. Perhaps the most frequently used technique for the study of gene expression in C. elegans involves reporter genes. Plasmid expression vectors have been constructed containing reporter genes specifically designed for examining gene expression in C. elegans. A gene fusion is created between the C. elegans gene of interest and the reporter gene, by standard recombinant DNA techniques. The resulting recombinant plasmid is microinjected into C. elegans to generate stable transgenic lines (see Chapter 5). Expression of the reporter gene in the transformants is driven by regulatory elements of the C. elegans gene, and the distribution of the protein product of the reporter gene is discerned in the intact animal.
Combinatorial Chemistry
Authors: Fenniri Hicham
Published: November 2023

Combinatorial Chemistry is a genuine practical guide covering all the major areas of combinatorial chemistry from an experimental and conceptual point of view. Being one of the most powerful of modern technologies, combinatorial chemistry has had implications to many areas of chemistry and biology and the current approaches to drug, catalyst, receptor, and materials development and discovery are all included in this volume. It also contains protocols on solid, liquid, and solution phase synthesis and expedient methods of library screening and evaluation. The use of automation and robotics is also explained. It is written at a level easily accessible to novices and will enable readers to use combinatorial techniques to the best advantage.
Organic reactions on solid support — an overview
Authors: Bennett William D
Published: November 2023

These tables document the rapidly expanding number of traditional solution phase reactions that have been successfully adapted to solid phase synthesis over the past two years. Details about the resins and solvents used and the yields obtained are also included. Peptide and oligonucleotide synthesis reactions are well documented and are therefore not included in these tables unless they illustrate new resins, reagents, or synthetic strategies. Reflecting the growing interest in using polymer-supported reagents for solution phase synthesis and work-up, tables of scavengers, catalysts, and reagents on insoluble supports are also featured.
Multi-step solution phase combinatorial synthesis
Authors: Boger Dale L , Goldberg Joel
Published: November 2023

The use of combinatorial chemistry for the discovery or optimization of novel chemical leads has emerged as a powerful method for the acceleration of the drug discovery process (1-4). Initially explored with peptide or oligonucleotide libraries and related structures, subsequent efforts have been directed at exploiting the diversity and range of useful properties embodied in small molecule libraries (1, 5).
Automated solution phase synthesis and its application in combinatorial chemistry
Authors: Sugawara Tohru , Cork David G
Published: November 2023

The early human diet was characteristically extremely varied, and a wide range of plant species and plant organs were consumed. Foods of animal origin included those taken opportunistically, such as invertebrates, amphibians, reptiles, small mammals, birds and their eggs, and the scavenging and hunting of larger mammals. Each of these types of food have characteristic nutritional compositions. Comparison of these compositional features shows that an adequate diet could be obtained in many different ways. The selection of food providing fat had substantial advantages in reducing the amount of plant foods to be gathered, in the satiety provided and in supplying essential micronutrients. Obtaining adequate water and energy would probably be the main physiological drives. Many plant foods contain natural toxicants, and would only have been suitable as major items in the diet once cooking had been developed, and the preference for sweet tastes would have protected humans from eating bitter, toxic plants
Combinatorial discovery and optimization of electrocatalysts
Authors: Reddington Erik , Yu Jong-Sung , Chan Benny C , Sapienza Anthony , Chen Guoying , Mallouk Thomas E , Gurauꝉ Bogdan , Viswanathanꝉ Rameshkrishnan , Liu╪ Renxuan , Smotkinꝉ Eugene S , Sarangapani S
Published: November 2023

Combinatorial methods have been used for over a decade to discover interesting new organic and biological molecules (1). Although no products discovered by a combinatorial technique have reached the market place, some are now in clinical trials (2). While it may seem that combinatorial chemistry is relatively new, its origins are in fact nearly three decades old. Hanak used a combinatorial method, which he called ‘the multiple-sample approach’, to discover new superconducting materials (3). The ‘high Tc’ superconducting alloys he discovered had a critical temperature of less than 10 K, and were quickly forgotten as they found no practical use. Also forgotten was Hanak’s novel, but efficient approach to materials discovery. He identified these materials in a period of five months without the benefit of desktop computers or elaborate array fabrication devices. Partially inspired by organic and biochemists, many inorganic materials chemists have implemented .combinatorial schemes of their own within the past five years. During this time, several
Phosphatidylinositol 3-kinase and membrane trafficking
Authors: Stenmark Harald
Published: November 2023

The eukaryotic cell is divided into a number of structurally and functionally distinct membrane-bounded compartments. Each compartment contains its distinct set of molecules, and an elaborate molecular machinery ensures that proteins, lipids, and sugars can be transported between compartments while maintaining organelle identity. Figure 1 shows schematically the most important stations of the intracellular membrane trafficking pathways in mammalian cells. The first station of the exocytic pathway is the endoplasmic reticulum (ER), the membrane of which is continuous with the nuclear envelope. Membrane traffic from the ER to the Golgi apparatus occurs via a vesiculotubular intermediate compartment (IC) (1). From the Golgi apparatus, molecules can be further transported to the trans-Golgi network (TGN). Here, cargo molecules are sorted to distinct domains of the plasma membrane as well as to the endocytic pathway (2). The endocytic pathway begins with the formation of vesicles from the plasma membrane. The endocytic vesicles fuse with early endosomes (EE). Like the TGN, the EE is an important sorting station, which shunts molecules to alternative destinations, including the plasma membrane and later endocytic compartments (3). Late endosomes (LEs) frequently contain internal vesicles and are sometimes referred to as multivesicular bodies (MVBs) (4, 5). These organelles appear to have a two-way communication with the TGN: they receive lysosomal enzymes, complexed to mannose 6-phosphate receptors, via TGN vesicles, and they recycle ligand-free mannose 6-phosphate receptors back to the TGN (6). LEs may fuse with primary (or dense-core) lysosomes, to form secondary lysosomes. Here, the degradation of luminal material occurs very efficiently. There is evidence that primary lysosomes may be reformed from secondary lysosomes through a selective membrane recovery (7).