bluuebunny/crossref_metadata_2025 huggingface dataset:
Population and ecological genetics
Authors: Brookfield J F Y
Published: November 2023

Drosophila melanogaster is a cosmopolitan species that lives as a human commensal. The family Drosophilidae belongs to the Dipteran Sub-Order Cyclorrhapha and is thought to be primitively saprophytic, living on fermenting substrates. However, immense variety now exists in the food sources exploited by different species. Of 3020 species in the 61 genera of the family, 1677 are in the genus Drosophila, a result that must partially reflect incomplete sampling of the others (1). The taxonomic richness of the group potentially allows the comparison of D. melanogaster genes with those of other species at many different levels of evolutionary divergence. The phylogenetic relationships of species of Drosophila are imperfectly known, but a number of subgenera, species groups, species subgroups, and species complexes are recognized between the generic and specific ranks. The melanogaster species group, including also D. simulans, D. mauritiana, D. sechellia, D. yakuba, D. teissieri, D. erecta, and D. arena has a phylogeny that has been well established through study of the polytene chromosomes and DNA sequence divergence. These species are important in some tests for selection outlined below. The species group originated in Africa, and the first bifurcation, separating the ancestor of D. arena and D. erecta from that of the others, occurred between 15 and 20 million years ago.
Methods to map origins of replication in eukaryotes
Authors: Gerbi Susan A , Bielinsky Anja K , Liang Chun , Lunyak Victoria V , Urnov Fyodor D
Published: November 2023

According to the replicon model of Jacob, Brenner, and Cuzin (1), initiation of DNA replication is achieved via the binding of a trans-acting protein (the initiator) to a cis-acting sequence (the replicator). The initiator recruits the replication machinery to the replicator, and DNA synthesis ensues. Studies in prokaryotes, fungi, and viruses of metazoans have provided ample experimental support for this hypothesis, and in such model systems as E. coli, S. cerevisiae, and SV40, a considerable amount of experimental data exist about the nature of replicators and the initiator protein(s) (see refs 2-4 for recent reviews). A useful and interesting property of replicators in these organisms is that they retain function when placed on an episome. The autonomously replicating sequence (ARS) assay that exploits this property allowed researchers to use the power of ‘reverse genetics’ to identify and study the replicators in these systems (for example, ref. 5). Unfortunately, the ARS assay has not been as useful for studying replicators in metazoa, where the efficiency of episome replication seems more related to the length rather than the sequence of the insert (6-8). There are additional complexities: sequences as much as 50 kb away from the origin can influence its activity (9), and proper chromatin organization of the DNA is important for replication initiation (10).
The use of Xenopus laevis interphase egg extracts to study genomic DNA replication
Authors: Walter Johannes , Newport John
Published: November 2023

Cell-free extracts derived from the eggs of Xenopus laevis are a powerful experimental system to study genomic DNA replication in higher eukaryotes (1-3). DNA templates (either plasmid DNA or Xenopus sperm chromatin) added to these extracts are packaged into nuclei and then replicated under normal cell cycle control. This process occurs rapidly and efficiently and can be extensively manipulated in order to elucidate underlying biochemical mechanisms.
Viral in vitro replication systems
Authors: Bullock Peter A
Published: November 2023

It is possible to study the duplication of DNA using a number of in vitro replication systems, including those employing prokaryotic (1) as well as eukaryotic replication factors (2). In many instances, the eukaryotic replication factors were identified by fractionation of cellular extracts that support the in vitro replication of viral DNA (3-5 for reviews). Prominent among the in vitro viral DNA replication systems are those that support adenovirus, papillomavirus, and papovavirus DNA replication. Additional systems for studying viral DNA replication (e.g. herpesvirus, poxvirus, and parvovirus) have been described (2).
Analysis of DNA replication complexes by DNA probing
Authors: Borowiec James A , Gillette Thomas G , Smelkova Natalia V , Iftode Cristina
Published: November 2023

This chapter describes techniques used to characterize complexes formed in solution between replication proteins and DNA. Although general DNA probing or ‘footprinting’ techniques used to probe standard protein:DNA complexes are provided, special emphasis will be placed on those methods most useful to investigators studying the interaction of DNA replication proteins with DNA replication substrates. Thus, the reader will find a variety of DNA probing techniques that can reveal the presence and location of distorted DNA within complexes. We discuss chemical and enzymatic probes useful for examining the disposition of single-stranded DNA (ssDNA) often found in replication intermediates. We also describe the use of interference assays which allow the determination of DNA contacts important for protein binding, and for the characterization of transient complexes formed between replication proteins and DNA.
Microtubule dynamics, molecular motors, and chromosome behavior
Authors: Vernos Isabelle , Karsenti Eric
Published: November 2023

Perhaps because of its aesthetically pleasing and exquisite geometrical shape, the mitotic spindle has fascinated researchers for more than a hundred years. In 1896, Wilson (see ref. 1) had already described accurately the shape of the spindle and the principal phases of mitosis. He described how two centrosomes moved around the nucleus, how fibers grew between the migrating centrosomes and how chromosomes eventually aligned on the 'equatorial plate' of the mitotic spindle. It was already clear that chromosome segregation took place on the fibers of the spindle during anaphase, providing the two daughter cells with equivalent chromosome numbers.
Monitoring of trichothecene producing Fusarium species in brewing cereals using a group specific Polymerase Chain Reaction
Authors: Niessen M L , Vogel R F
Published: November 2023

A pair of PCR primers (Tox5-1 and Tox5-2) was designed and a PCR set up and optimized. With the primer pair used a 658 bp fragment was amplified from DNA isolated from Fusarium species described as producers of trichothecene mycotoxins. Non-trichothecene producers, cereals, and bacteria gave no signal in the assay. Trichothecene producers were detected in samples of contaminated malts. Results showed that the method developed is group specific for the detection of all Fusarium species capable of producing trichothecenes. The method may be used for quality monitoring in the brewing and malting industry.
Enzymes from barley which solubilize β -glucan
Authors: Bamforth C W , Moore J , McKillop D , Williamson G , Kroon P A
Published: November 2023

ß-Glucan solubilase releases ß-glucan and probably pentosans from cell walls making them more accessible to endo-B-glucanases and pentosanases respectively. An improved assay for measuring solubilase activity has been developed. Solubilase is present in raw barley and is relatively heat stable. Four fractions of solubilase activity have been located. Two of the fractions contain feruloyl esterase. Ester linkages involving ferulic acid, probably with the arabinoxylan component of the cell walls, may be disrupted by solubilase.
Proanthocyanidin-free malting barley - a solution of the beer haze problem
Authors: Jende-Strid B
Published: November 2023

Technical stabilization of beer is not necessary if proanthocyanidin-free malt is used for brewing. At Carlsberg a breeding programme for development of proanthocyanidin-free malting barley varieties has resulted in a commercial malting barley variety “Caminant” with a yield performance and a malting quality which is comparable to other malting barley varieties on the market. Production scale maltings and brewing trials with Caminant show, that it is possible to produce malts and beers which live up to the breweries’ specifications. New improved proanthocyanidin-free malting barley varieties are under way. Breweries can avoid the expenses of technical beer stabilization by use of good proanthocyanidin-free malting barley.
Application industrielle de I’ensemencement de ferment au cours du maltage pour 1’amélioration de la qualité sanitaire et biophysique du malt
Authors: Boivin Patrick , Malanda M’Baka
Published: November 2023

Geotrichum, a yeast naturally present in malting, has been used industrially in malting with the aim of controlling the development of the microflora and improving the biophysical and microbiological quality of malt.
Lactic acid starter cultures for prevention of the formation of Fusarium mycotoxins during malting
Authors: Laitila A , Tapani K -M , Haikara A
Published: November 2023

Contamination of barley by mycotoxigenic and active gushing inducing fusaria are of concern to maltsters and brewers. The ability of lactic acid bacteria to restrict the growth of Fusarium graminearum and F. culmorum species was studied in vitro and in laboratory scale maltings. Five from 15 lactic acid bacteria tested notably retarded the growth of Fusarium fungi. Addition of the most active lactobacilli into the steeping diminished the amount of Fusarium contamination and formation of deoxynivalenol and zearalenone during malting when naturally infested barley was used. Formation of mycotoxins during malting was also studied with artificially contaminated barley.
Caractérisation rapide des différentes étapes du maltage par spectroscopie moyen infrarouge en réflexion totale atténuée
Authors: Allosio Nathalie , Robert Paul , Bertrand Dominique , Boivin Patrick
Published: November 2023

To produce a malt of good quality, it is necessary to have a rapid method to control the malting process. However, very few rapid physical techniques are available to maltsters to characterise their products. The feasibility of attenuated total reflectance, a method allowing rapid analysis of wet products with strong absorption such as green malt, has been tested. The application of principal component analysis to spectra scanned in the mid infrared with this method, shows that it is possible to classify samples according to their malting step.
Influence de la composition en hétérocycles azotés de malts spéciaux sur le profil aromatique de la bière
Authors: Herent Marie-France , Vanthoumhout Cedric , Collin Laurence Gijs & Sonia
Published: November 2023

Les malts caramels sont utilisés dans la production de btères spSciales auxquelles ils confèrent une coloration et un ardme caractéristiques. Ce dernier est essentiellement apporté par des heterocycles azotés tels que lés dérives pyrazines et thiazoles.
A new dimension in malting laboratory automation
Authors: Chan Sherman H , Hollaar Kees
Published: November 2023

The increased quality assurance requirements in the malting industry had led to a need for the laboratory automation.
Possibilities to use Agrobacterium tumefaciens as a gene transfer vector in hop
Authors: Gurriaràn M J , Benftez J L , López M A , Ordàs R J , Tamés R S
Published: November 2023

A. tumefaciens promises to be a suitable vector to obtain transgenic hop plants. Slices from organogenic callus were cocultivated with the bacteria. Growth and vir gene induction by in vitro extracts and exudates were studied. The bacteria grew in in vitro extracts, and vir D expression was not induced by exudates. This agrees with the fact that transient gene expression was only observed in those slices cocultivated in the presence of Nicotiana bentamiana leaf discs. The situation could be overcome by addition of supplement vir gene inducers, opening the way to get hop plants tolerant to herbicides or pests.
A new mashing-in system that reduces oxygen uptake
Authors: Yamaguchi lchiro , Ueda Tsutomu , Ujihara Shigeru , Yamada Minoru , Fukushima Shuji
Published: November 2023

In order to improve beer flavour stability, a new mashing-in system that reduces oxygen uptake was introduced into a brewery. This system entails purging ingredients with carbon dioxide, then feeding them from the bottom of vessels. In addition, deaerated water was also used for brewing liquor. Compared with the conventional system, the new system decreased the trans-2-nonenal content of packaged beer while taste test results also indicated that the new system results in less oxidation. In relative terms, feeding ingredients from the bottom was more effective in reducing oxygen uptake than deaerating the brewing liquor.
Studies on the survival of amylose-lipid complexes under mashing conditions
Authors: Wilhelmson A , Vainikka M , Home S
Published: November 2023

The aim of this study was to clarify whether the amylose-lipid complexes remain unhydrolyzed under mashing conditions. As amylose-lipid complexes are difficult to analyze from a complicated matrix such as spent grains, the hydrolysis of these complexes was studied by incubating pure barley starch in malt enzyme extracts with varying enzyme activities. After the incubation, most of the starch lipids were found in the insoluble precipitate. The hydrolysis of amylose-lipid complexes during incubation was strongly influenced by the amylolytic activity of the malt extract. At lower enzyme activities the complexed amylose was more slowly hydrolysed than the free amylose.
Weiterentwicklung der thermischen Brildenverdichtung
Authors: Fohr Markus , Meyer-Pittroff Roland
Published: November 2023

Die thermische Briidenverdichtung mit einem Dampfstrahlverdichter hat sich als Alternative zur mechanischen Briidenverdichtung bei der Wiirzekochung etabliert. Es besteht jedoch noch Entwicklungsbedarf hinsichtlich des Einsatzes regelbarer Dampfstrahlverdichter und, wie auch bei anderen Wurzekochsystemen,
Einfluß einer Vekuumverdampfung vor der Würzekülung auf Bierqualität und Geschmacksstabilität
Authors: Lustig Stefan , Kunst Thomas , Hill Peter
Published: November 2023

During the hot stand of wort in the whirlpool, various compounds are formed by thermally mediated reaction mechanisms, and are only partially removed in the steam. Since these compounds lead to unwanted aromas in finished beer and are known to impair taste stability, enhanced removal by vacuum evaporation from the whirlpool was investigated. A successful plant-scale trial achieved separation of hot trub at reduced pressure of 0.5 bar. The influence of this procedure on the quality of the wort and beer, as well as the taste stability of beer, was investigated.
Utilisation d’1802 pour évaluer l’impact du phénomène d’oxydation durant le brassage et le stockage de la bière
Authors: Collini S , Noel S , Bonte S , Metais N , Bodart E , Peladan F , Dupire S
Published: November 2023

To understand the oxygen incidence in bottled beer, 1802 has been injected in the headspace just before ageing. Although very potent for sulfite, polyphenol and isohumulone degradations, bottled oxygen has been found not to be incorporated in the flavouring fraction, indicating that cardboard aroma is not issued from lipid oxidation. The impact of oxygen during mashing has been evidenced on the final beer. A comparison of trans-2-nonenal contents of worts produced in presence of nitrogen, 1602 or 1802 clearly indicates that three synthesis pathways and two degradation mechanisms modulate the alcenal concentration in the unboiled wort. The nonenal potential measurement after wort filtration appears to be a good indicator in predicting subsequent beer ageing.
Identification of foam-stabilizing proteins under conditions of normal beer dispense and their biochemical and physico-chemical properties
Authors: Douma A C , Mocking-Bode H C M , Kooijman M , Stolzenbach E , Orsel R , Bekkers A C A P A , Angelino S A G F
Published: November 2023

To investigate which proteins are relevant for foam stabilization, the protein composition of beer and ageing beer foam was compared. During foam ageing, a strong accumulation of a relatively hydrophobic 8-18 kD protein group was observed. Protein Z, the most abundant beer protein, showed favourable physico-chemical characteristics for foam stabilization. The 8-18 kD protein group was able to enhance the beneficial visco-elastic properties of protein films composed of protein Z. Methods to quantify the level and ratio of 8-18 kD and 40 kD protein may enable the determination of foam protein quality in beer as well as during malting and brewing.
DNA wrapping in Escherichia coli RNA polymerase open promoter complexes revealed by scanning force microscopy
Authors: Rivetti Claudio
Published: November 2023

Transcription is the first step in the expression of the genetic information encoded in DNA In Escherichia coli, this process is carried out by RNAP, a single enzyme that can exist in two forms: the core enzyme, with a subunit composition α2 β β Mr 380 000, is responsible for the actual RNA synthesis and the holoenzyme, which possesses an additional er subunit that enables RNAP to recognize promoters and to initiate transcription. In prokaryotes, the majority of the promoters are recognized by the 70 kDa IT o factor. IT70-RNAP binds to the promot’r to form a close promoter complex (CPC) which spontaneously isomeriz’S through various int’rm’diatcs into an open promoter complex (OPC) containing an unwound DNA section (for review see refs. 1 and 2). Upon addition of nucleoside triphosphat’s RNAP b’gins RNA synthesis. u factor is released, and the core enzyme moves in a highly processiv’ manner along the DNA until a t’rminator is reached (3, 4). During transcription, RNAP translocates on the DNA, unwinding the DNA ahead and rewinding it in its wake, thereby maintaining the transcription bubble with no net free energy cost.
Laser UV-laser photoreactivity of nucleoprotein complexes in vitro
Authors: Buckle Malcolm , Place Christophe , Pemberton lain K
Published: November 2023

In order to define a kinetically competent nucleoprotein complex, a complete description of all the contacts and conformational changes characterizing both its formation and subsequent function is required. Chemical, physical and biological probes are all used to generate this information. Such probes must, however, report a specific structure that is both structurally unique and also singular with respect to time. The two overall stringent requirements for such probes are that they are first non-perturbing and second, highly specific (1), In this chapter we describe the use of high-energy photons to excite and study nucleoprotein interactions in vitro. The use of a similar system in vivo is described in Chapter 15.
Analysis of DNA-protein interactions by time-resolved fluorescence spectroscopy
Authors: Thompson E H Z , Millar D P
Published: November 2023

In addition to its uses as a probe of macromolecular structure and dynamics, fluorescence spectroscopy is emerging as a powerful tool for studies of DNA protein interactions. Particular advantages of fluorescence-based measurements include: the ability lo observe interactions under conditions of thermodynamic equilibrium; their sensitivity, allowing investigations at concentrations approaching the Kd values of many DNA-protein interactions; the ability to monitor complexation events in solution, accommodating a widC’ variety of conditions: and, most of all. the short time-scale of the emission process. Since the emission of a photon occurs on the same time-scale as a number of dynamic processes including solvent relaxation, intersystem crossing, charge transfer, and quenching-it is highly sensitive to the characteristics of the local environment.
Site-specific protein-DNA photocrosslinking
Authors: Kim Tae-Kyung , Lagrange Thierry , Reinberg Danny
Published: November 2023

Transcription and replication involve multiprotein-DNA complexes with molecular weight masses in excess of 400 kDa (1-5). Understanding transcription and replication will require understanding the arrangement of proteins relative to DNA in these complexes. High-resolution structures of components of the complexes have been determined (1-5). However, the intact, fully assembled, complexes are too large for high-resolution structure-determination by current methods. Therefore, efforts to define the arrangement of proteins relative to DNA within the intact complxes must rely heavily on biochemical methods.
Transcription-coupled and global genoine repair in yeast and huinans
Authors: Tijsterman Marcel , Verhage Richard A , Brouwer Jaap
Published: November 2023

Nucleotide excision repair (NER) is able to remove a wide variety of DNA lesions from the genome. Incision of the damaged strand on both sides of the lesion is followed by resynthesis of excised DNA using the non-damaged strand as a template. The role of the proteins involved in this cut-and-paste mechanism is studied biochemically, and these studies have recently culminated in the reconstitution of eukaryotic NER in vitro using repair components purified from both yeast and human cells (see Chapter 5 for a detailed discussion on the proteins involved). Whereas these in vitro assays allowed the identification of a minimal set of proteins (here referred to as core-NER proteins) required to perform the reaction on naked DNA, analysis of repair in vivo indicated that additional proteins are necessary for the efficient removal of damage from genomic DNA. These factors are believed to be involved in the first step of the NER pathway: the recognition of different types of DNA damage in a genome that is continuously undergoing various cellular processes (e.g. transcription, replication, recombination) and which, in addition, is folded into chromatin with different levels of complexity.
Interaction of DNA virus proteins with host cytokines
Authors: Lalani Alshad S , Nash Piers , Seet Bruce T , Robichaud Janine , Mcfadden Grant
Published: November 2023

It has become evident that large DNA viruses have adapted a variety of strategies to modulate the host immune response to virus infection. Recently, a variety of DNA virus-encoded gene products have been identified with activities targeted towards modulating the cytokine networks of the inflammatory response of their host. Members of the poxvirus family were the first DNA viruses shown to encode secreted versions of cellular cytokine receptors, and currently a dozen virus proteins that interact with cytokines have been identified in both the poxvirus and herpesvirus families (Table 1) (1, 2). Viral cytokine receptor mimics (also known as ‘viroceptors’) are soluble or membrane-bound proteins that may share significant homology to the ligandbinding domains of cellular cytokine receptors, and function by sequestering cytokine ligands from interacting with their cognate cellular receptors.
Chemotherapy of DNA virus infections
Authors: Cane Patricia A , Pillay Deenan
Published: November 2023

In recent years there have been considerable advances in the development of effective antiviral drugs. Much of this progress has been driven by the need to combat human immunodeficiency virus (HIV) infection, but there are currently nearly thirty antiviral agents in clinical development or licensed for use in humans against DNA viruses. These are listed, together with their principal activities, in Table 1. In theory, any of the stages of the replication cycle of a virus (attachment, penetration, uncoating, replication, maturation, and release) are potential targets for antiviral drugs, but in the case of DNA viruses most of the effective drugs act by inhibiting DNA replication. Such drugs are mainly either nucleoside analogues such as acyclovir, ganciclovir, and lamivudine, or phosphate analogues such as foscarnet.
Background on Caenorhabditis elegans
Authors: Hope Ian A
Published: November 2023

The number of researchers using Caenorhabditis elegans as a model system for biological research has grown steadily over the past three decades (see Figure 1). But interest in this species has suddenly soared in response to the success of the C. elegans genome project (1). The genome project was effectively completed in 1998, making C. elegans the first multicellular organism for which the genome has been sequenced. The genome sequence data have emphasized the considerable conservation of biological mechanisms across the animal kingdom that has become apparent through the molecular genetic characterization of biological processes. This conservation confirms the value of C. elegans as a model system, which has contributed and will continue to contribute significantly to our biological understanding of all animals including humans. C. elegans was deliberately selected as a model system (2), primarily because it possessed particularly favourable characteristics for laboratory study and also because it was assumed that similar mechanisms would direct equivalent biological processes in all animal species. This selection has now been shown to be fully justified.
Resins and anchors for solid phase organic synthesis
Authors: Winter Martin , Warrassꝉ Ralf
Published: November 2023

Isolation of the pure intermediates and products in a multi-step synthetic scheme is often the most time-consuming and uneconomical part in the overall process. This effort increases proportionally with the number of synthetic steps. The extent of this problem is greater when several different compounds are to be made in parallel as is the case in combinatorial chemistry. Differences in the physical and chemical properties of members of a chemical library, such as solubility, pKa values, polarity of the building blocks and intermediate products, aggravate to a great extent the standardization of a procedure or may even make it completely impracticable.
Multi-component reactions (MCRs) of isocyanides and their chemical libraries
Authors: Ugi Ivar , Dömlingꝉ Alexander
Published: November 2023

In principle, all chemical reactions correspond to equilibria between one or two reactants and products. In practice, however, preparative chemical reactions proceed irreversibly and, in the absence of competing reactions, extremely high yields of pure products can be obtained (1-3). If more than two (n > 2) reactants are required for the synthesis of a product, usually several (at least n - 1) preparative steps must be carried out. The more preparative steps there are, the more isolation and characterization work must be achieved, and the lower the yields are after each step of the synthetic effort. In contrast with this approach, many products can also be prepared in a one-step, one-pot multi-component reaction (MCR) of three or more starting materials. MCRs are defined as a collection of subreactions taking place simultaneously and/or sequentially, each involving two reacting molecules.
Experimental techniques in fluorous synthesis: a user’s guide
Authors: Curran Dennis P , Hadidaꝉ Sabine , Studer╪ Armido , He§ Mu , Kim Sun-Young , Luo Zhiyong , Larhed* Mats , Hallberg* Anders , Linclau Bruno
Published: November 2023

In discovery research, the ‘reaction’ stage of a synthetic step and the ‘separation’ stage have traditionally been uncoupled. Reactions are executed and then reaction mixtures are purified by using trial and error (or, perhaps more commonly, experience) to select a suitable purification technique. Experimental techniques for combinatorial chemistry and parallel synthesis need to be simple, fast, and efficient, and these needs have precipitated the development of a number of general new approaches for making small organic molecules (1, 2) that couple reaction and separation at strategy level. The goal of strategy level separation is to plan a reaction that can be purified simply by workup (3, 4). In other words, the desired product of a reaction will partition into (or be a part of) one phase, while all the other products of the reaction will partition into another phase. The commonly used phases for organic separations are the gas phase, the water phase, the organic liquid phase, and the solid phase, and these phases can be separated by evaporation, extraction, filtration (used alone or in combination, as appropriate). A number of imaginative and effective ‘workup level’ separation techniques have recently been introduced that take advantage of these different phases (3).
Practical aspects of the experimental design for differential display of transcripts obtained from complex tissues
Authors: Medhurst Andrew D , Chambers David , Gray Julie , Davis John B , Shale Julian A , Mason Ivor , Jenner Peter , Newton Richard A
Published: November 2023

Differential display is a valuable technique for identifying differentially expressed m.RNAs and for cloning their cDNAs (1). Since its introduction in 1992, several technical improvements have increased the successful application of differential display (2-4). Many types of proteins (e.g. growth factors, chemokines, and proteases) have been identified using this technique, implicating their involvement in processes such as apoptosis, cell differentiation, and senescence (5). In addition, differential display constitutes a valuable tool for scientists interested in the identification of novel genes coding for proteins that may be suitable for pharmacological intervention in diseases such as cerebral ischaemia (6) and cancer (7).
Studying gene expression profiles in specialized brain regions by microSAGE
Authors: Vreugdenhil Erno , Datson Nicole
Published: November 2023

The human genome is thought to contain 100 000 genes of which a subset of approximately 15 000 to 20 000 genes is expressed in an individual cell. The set of genes expressed and the stoichiometry of the resulting messenger RNAs, together called a transcriptome, determine the phenotype of a cell, tissue, and whole organism. It is generally accepted that a transcriptome is largely determined by an interplay of hereditary and environmental factors. For example, in the CNS, a challenge from the environment, e.g. a learning or a traumatic experience may lead to an alteration of the transcriptome of target neurons. Thus, transcriptome analysis and subsequent transcriptome comparisons may reveal novel insights in the molecular mechanisms underlying complex processes such learning and memory formation.
Regulation of phosphoinositide-specific phospholipase C isozymes
Authors: Rhee Sue Goo , Poulin Benoit , Lee Sang Bong , Sekiya Fujio
Published: November 2023

The hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a specific phospholipase C (PLC) is one of the earliest key events in the regulation of various cell functions by a number of extracellular signalling molecules (1). This reaction produces two intracellular messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mediate the activation of protein kinase C (PKC) and intracellular Ca2+ release, respectively. Ins(1,4,5)P3 is further converted by the action of several distinct kinases and phosphatases to various inositol phosphates, some of which have been implicated for distinct cellular functions (see Chapter 10). PtdIns(4,5)P2 is a precursor not only for Ins(1,4,5)P3 and DAG but also for the newly emerging messenger PtdIns(3,4,5)P3 that is produced by phosphoinositide 3-kinase (2; see also Chapter 2). PtdIns(3,4,5)P3 accumulates in cells that have been activated through protein tyrosine kinase (PTK)- and G-protein-linked receptors, and has been known to modulate the activities of cellular effector molecules, such as the small GTP-binding protein Rac, the Ser/Thr protein kinase B (PKB; also known as Akt), phosphoinositide-dependent kinase (PDK; also called PKB kinase), Bruton’s tyrosine kinase (Btk), certain isoforms of PKC, and Grp-1 (a guanine nucleotide exchange factor for the small G proteins ARF) (2). PtdIns(3,4,5)P3 also appears to function as a general component of PLC-γ-dependent calcium signalling pathway (see below). PtdIns(4,5)P2 directly modulates the function of protein kinase C, phospholipase D (PLD), and phospholipase A2 (PLA2) (3–5). PtdIns(4,5)P2 regulates actin polymerization and depolymerization processes by binding to actin regulatory proteins, such as profilin, cofilin, gelsolin, α-actinin, and gCap (6; see also Chapter 5). Finally, PtdIns(4,5)P2 binds to pleckstrin homology (PH) domains and thus serves as a membrane docking site for a host of PH domain-containing proteins (7).
Selection of methods for measuring proliferation
Authors: Wieder Robert
Published: November 2023

In the process of defining the phenotype of a cell, one of the most frequent parameters scientists measure is the rate of proliferation. Often, the first decision that confronts us is that of choosing ‘the right assay’. The selection of an assay is critical to the outcome and depends on a variety of factors. These include the experimental conditions, the cell cycle parameters affected by the variable under investigation, and the source and state of the cells or the tissue to be assayed. To select an appropriate assay, we have to be aware of the events that constitute the cell cycle.
Cell growth and cytotoxicity assays
Authors: Skehan Philip
Published: November 2023

This chapter describes non-radioactive assays commonly used to measure changes in cell growth and cytotoxicity induced in a cell population by treatment with agents such as drugs, hormones, nutrients, and irradiation. The focus is specifically on mammalian cell cultures, but the methods can be used with a wide variety of cells, tissues, and microorganisms. Because of the ease and speed with which large numbers of samples can be processed by optical plate readers, most of the protocols which follow are designed specifically for 96-well plates, but are easily adapted to other culture vessels.
Cell growth and kinetics in multicell spheroids
Authors: Durand Ralph E
Published: November 2023

This chapter examines cell proliferation kinetics and mechanisms of cell growth control in a complex tissue culture system: three-dimensional multicell spheroids (1). While spheroids are about to enter their fourth decade of use as an ‘in vitro tumour model’ (2), they are also very useful as a model for some normal tissues due to the co-existence of heterogeneous microenvironments that collectively influence their growth properties (3). We will, therefore, briefly characterize the system, indicate many of the novel growth perturbations that arise, and then address some of their implications vis-a-vis proliferation and quiescence.
Assessment of the role of growth factors and their receptors in cell proliferation
Authors: Bishayee Subal
Published: November 2023

The growth of cells in culture and, most likely , in the animal, is subject to control by various agents, such as extracellular signalling proteins (growth factors). These growth factors mediate their pleiotropic actions by binding to and activating high affinity cell surface receptors. At least five important classes of polypeptide growth factors have been identified and their interactions with cell surface receptors well characterized. These are: (a) epidermal growth factor (EGF) and EGF-like factors, a family of 6-7 kDa polypeptides that share a common receptor (EGF receptor); (b) plateletderived growth factor (PDGF), a disulfide-bonded dimeric protein (25-30 kDa) composed of two non-identical but highly homologous polypeptide chains (in different combinations); (c) insulin and insulin-like growth factors, a class of structurally related 6-7 kDa polypeptides which interact with multiple receptors; (d) transforming growth factor-13 (TGF-13; also acts as a differentiation factor), a family of 25 kDa proteins that act through multiple receptors; and (e) the interleukins and their respective receptors (reviewed in ref. 1). It is recognized that ectopic synthesis or overproduction of these growth factors can lead to an autocrine stimulation of growth and expression of the transformed phenotype (reviewed in ref. 2). In addition, aberrant expression of a growth factor receptor due to gene deletion or mutation and overexpression of a normal growth factor receptor gene can also produce transformation (3). This chapter will focus only on growth factors whose receptors display intrinsic tyrosine kinase activity.
Analysis of growth-signalling protein kinase cascades
Authors: Alessandrini Alessandro
Published: November 2023

One of the main questions in signal transduction is how do cells take an extracellular signal and convert it into an intracellular one. The first step that enables this process involves receptors that bind to specific extracellular signals. Most receptors have intrinsic tyrosine kinase activity or have tyrosine kinase activity associated with them. This results in a series of phosphorylation events involving endogenous Ser/Thr kinases. The final steps in a cell’s response to an extracellular signal may involve the reorganization of the cytoskeleton which may lead to motogenesis (cellular migrations) and the transcriptional activation of specific genes that may lead to mitogenesis and/or differentiation. This chapter will focus on the downstream components in signal transduction and, in particular, the MEK/ERK pathway.
Cytometric analysis of the pRB pathway
Authors: Juan Gloria , Traganos Frank , Darzynkiewicz Zbigniew
Published: November 2023

The term retinoblastoma protein (pRB) pathway (‘pRB pathway’) has recently been introduced to define the components and function of the cell cycle regulatory machinery directly associated with phosphorylation of the retinoblastoma susceptibility gene product (1-6). In addition to the central element, pRB, the pathway includes the cell cycle-dependent kinases which phosphorylate this protein (Cdks), activators of these kinases (several types of cyclins), their inhibitors from both the Ink and Kip families (Ckis), and the products whose activation is controlled by pRB phosphorylation, the E2F family of transcription factors (7-9). The pRB pathway serves as the master regulatory switch triggering the cell’s entrance to and progression through the S phase. In non-tumour cells, the pRB pathway responds to changes in cell environment, primarily to growth factors, hormones, and cell to cell interactions, through the respective receptors and signal transduction pathways.
Isolation and visualization of the principal compon.ents of nuclear architecture
Authors: Stein Gary S , Montecino Martin , Mcneil Sandra , Pockwinse Shirwin , Van wijnen André J , Stein Janet L , Lian Jane B
Published: November 2023

It is becoming increasingly evident that gene expression in vivo is functionally interrelated with nuclear architecture. The linear ordering of protein coding sequences in genes, as well as the representation of flanking and intragenic regulatory elements, only partially accounts for transcriptional responsiveness to the broad spectrum of cues from physiological signalling pathways that modulate gene expression under diverse biological circumstances. Equally important, cataloguing protein-DNA and protein-protein interactions at gene regulatory sequences is necessary but insufficient to explain the transcriptional mechanisms that are operative in intact cells and tissues. Rather, there is growing awareness that gene expression must be understood from the perspective of contributions by multiple levels of nuclear organization.
Induction of differentiation of human intestinal cells in vitro
Authors: Cross Heide S , Kallay Enikö
Published: November 2023

The intestinal epithelium is, in principle, a valuable tissue for the study of cell differentiation, as it is spatially organized around its proliferative units, the crypt (small intestine) or the basal section of the crypt (large intestine). The progeny of clonal undifferentiated proliferating cells express eventually at least four very different phenotypes (1), e.g. enterocytes, mucin-producing goblet cells, endocrine cells, and Paneth cells. The determinants of this phenotypic commitment are largely unknown. During the orderly upward migration of cells from crypt to villus along the crypt length in the colon, the temporal sequence of differentiative events can be well defined. However, this situation unfortunately cannot be recreated in intestinal culture systems, probably because of the absence of supportive tissues, and the inability to recapitulate this complex dynamic process with continuous cell proliferation and cell loss within a few days. Thus, in vitro studies of differentiation markers in normal intestinal cells are difficult to perform.
Proliferation and differentiation of human prostate cells
Authors: Thalhammer Therese , Cross Heide S
Published: November 2023

More than 200000 new cases of prostate cancer are diagnosed anually in the USA. Thus, in incidence and prevalence, prostate cancer is the most common malignancy in American men, and black men in the USA have the highest incidence of prostate cancer in the world. Some epidemiological observations in various populations suggest a vital role for steroid hormones in prostate carcinogenesis. Although androgens are clearly the most influential in this respect, others such as oestrogens, retinoids, and vitamin D compounds might also be important. With advancing age, while there is a decline in serum testosterone levels, a concomitant increase of benign prostatic hyperplasia is observed. However, the incidence of microscopic, latent prostate cancer also shows a very close correlation with increasing age and thus decreasing testosterone.
Senescence and im.mortalization of human cells
Authors: Hubbard Karen , Ozer Harvey L
Published: November 2023

Normal vertebrate diploid cells in culture have a finite life-span (1). The maximum number of population doublings is dependent on cell type and species. It has also been shown to vary as a function of age of the donor organism, resulting in its utilization as a model for the proliferative aspects of cellular ageing. Although cells from all species eventually cease to proliferate and become ‘senescent’, the capability to achieve infinite or continuous cell growth (i.e. became ‘immortal’) by mechanisms that overcome cellular senescence is strikingly dependent on the animal species. Rodent cells readily become ‘spontaneously’ immortal such that there are multiple established fibroblastoid cell lines; for example, mouse 3T3 cells. In marked contrast, normal chicken and human fibroblasts (HF) rarely, if ever, become immortal. The observation that many human tumours are immortal cell lines when introduced into tissue culture suggests that overcoming senescence is a stage in carcinogenesis. One experimental manilpulation that has been successful in facilitating the immortalization of HF has been through the introduction of genes from DNA tumour viruses into otherwise normal cells.
Methods to study actin assembly and dynamics in yeast
Authors: Karpova Tatiana S , Cooper John A
Published: November 2023

Budding yeast (Saccharomyces cerevisiae) is a good model organism for studies of the actin cytoskeleton (reviewed in rd. 1 ). Actin and many components of the yeast actin cytoskeleton are highly homologous to cytoskeletal proteins of higher eukaryotes. Another advantage of yeast is the relative simplicity of the actin cytoskeleton. For example, actin and most of the actin-binding proteins are represented by a single isoform. The genetics of yeast is well developed. The genome of yeast has been sequenced. which implifies the search for new actin-binding proteins, either by homology searches or by testing the phenotypes of mutants with targeted ORF disruption.
Video microscopy: protocols for examining the actin-based motility of Listeria, Shigella, vaccinia, and lanthanum-induced endosomes
Authors: Zeile W L , Purich D L , Southwick F S
Published: November 2023

Cellular locomotion takes on many forms—from macrophage tracking and capturing a bacterium during infection to platelet spreading during blood clotting to tumour cell crawling during metastasis to cells dividing during mitosis or meiosis, but the common element is exquisitely choreographed directed movement. There are literally hundreds of proteins and enzymes involved in cell motility, and many of these proteins have multiple and often redundant roles in promoting specific types of motility (e.g. lamellipodial versus filipodial extension). The challenge then for cell biologists is to unravel the protein-protein interactions and other regulatory signals that constitute each distinct mechanism of vectorial movement. To meet this challenge requires both intuition and invention in the development and systematic application of new experimental techniques. Although this chapter describes the specifics of applying video microscopy, microinjection, and immunolocalization to investigate actin-based motility of intracellular pathogens, it is also true that the strategies presented here will be of value to those interested in other forms of cell motility and studies in cell biology in general. Significantly, video microscopy, microinjection, and immunofluorescence have been used with great success in the study of signal transduction pathways, specifically the role of monomeric GTPases in cytoskeletal remodelling(1).
Metabolic alterations associated with apoptosis
Authors: Costa-Pereira Ana P , Cotter Thomas G
Published: November 2023

Apoptosis is an active form of cell death, induced both by endogenous and exogenous stimuli, which occurs under physiological conditions (1, 2). Morphologically, it is characterized by cellular features such as membrane blebbing, cell shrinkage, and chromatin condensation (3). Intracellular events include various types of DNA fragmentation (4), cytoskeletal alterations, and protease activation (reviewed in ref. 5).
Hidden Markov models for database similarity searches
Authors: Birney Ewan
Published: November 2023

Despite the huge number of genes in an organism, the protein coding genes are thought to be made from a limited number of basic protein structures. Evolution has reused these protein structures, combining them to form different proteins, and altering them in different genes to achieve different functions. The diversity of species, each with its own copies of genes made from the limited number of building blocks, means that, for a protein of interest, a number of different related proteins may be found. In this chapter, I will discuss one set of techniques which can be used to take advantage of this diversity of protein sequence. These techniques are all related to the use of profiles, which are also discussed in Chapter 5. In this chapter, the emphasis will be on the use of hidden Markov models (HMMs) for profile analysis. Some practitioners consider profiles to be a type of HMM.
Predicting secondary structure from protein sequences
Authors: Heringa Jaap
Published: November 2023

Protein structure is intrinsically hierarchic in its internal organization. The highest level in this hierarchy is constituted by complete proteins or assemblies of such proteins, which become subdivided through domains via super-secondary structure to secondary structure at the lowest hierarchical level.
Methods for discovering conserved patterns in protein sequences and structures
Authors: Jonassen Inge
Published: November 2023

The amount of available biomolecular data is exploding: the number of known protein sequences is increasing rapidly while the number of known structures is also increasing, though not as rapidly. A very useful observation in this situation is that common features among proteins can be used to group them into families, and then study the proteins on a family level. By a family we mean a set of proteins sharing some definite biological properties in terms of common function and/or structure, often implying that the proteins have evolved from a common ancestor, i.e. that they are homologous.
Networking for the biologist
Authors: Harper R A
Published: November 2023

Every research worker would like to have the tools on hand to make his job quicker and more efficient, and with the advent of the World Wide Web many of the tasks associated with molecular biology have become freely available online. In the past when a scientist wanted to know something about a particular subject then the first option was to talk to colleagues in the laboratory and ask for their advice. If that was not sufficient then it was off to the library to scan abstracts or the latest journals for the relevant information.