bluuebunny/crossref_metadata_2025 huggingface dataset:
Polypeptide Neurotoxins as Probes for Certain Voltage-Dependent K+ Channels
Authors: Dolly J Oliver
Published: November 2023

A paucity of specific probes for membrane-bound, neuronal proteins has restricted their molecular characterization, particularly where functional assays applicable to broken cell preparations are not available. Thus, the discovery of toxins acting electively on nicotinic acetylcholine receptor or Na+ channel proteins, for example, has resulted in the elucidation of their detailed structural and functional properties.
Gel-Filtration Assays for Solubilized Receptors
Authors: Hulme E C
Published: November 2023

There are two basic strategies which can be applied to the assay of soluble receptor-ligand complexes. The first is to render the complex insoluble, and then to apply the filtration or centrifugation techniques which are described elsewhere in this book to separate bound from free ligand (Chapters 6, 7); a variant on this approach is to absorb the free ligand, and then to assay the residual binding (Chapter 8). The second is to use solution technology, namely equilibrium dialysis, or gel-filtration, to assay the levels of bound and free ligand.
Receptor-Binding Kinetics
Authors: Prinz Heino
Published: November 2023

The first steps involved in receptor-ligand interactions are always second order binding reactions. They may, or may not, involve conformational changes of the receptor protein. The velocity of binding may be extremely rapid for neuroreceptors but can be much slower for hormone receptors. This diversity leads to a variety of experimental methods employing mostly fluorescent or radioactive ligands. Likewise, the analysis of kinetic experi ments is performed on very different levels lepending on the type and quality of the data. Usually, and as a first app oximation, simple second order association reactions are assumed, which are sufficient for the analysis of most receptor-binding kinetics (1). Possible deviations from these schemes may lead to new insights into the binding mechanism and are, therefore, the focus of this chapter. It should be remembered that disproving a given mechanism is much simpler than proving another one.
Opioid Receptors
Authors: Demoliou-mason Catherine D , Barnard Eric A
Published: November 2023

Evidence for the heterogeneity of opioid receptor sites in the CNS has been provided by a variety of studies. At least three classes of binding sites have been identified and designated as μ (morphine), δ (enkephalin) and x (dynorphin). The assignment of these sites as distinct receptor subtypes has been supported by a variety of studies (reviewed in reference 1): specific ligand competition, selective protection from irreversible inactivation, differential anatomical distributions, the differing pharmacological profiles of various opiate and opioid peptide ligands, and differential affinity cross-linking studies (2).
α2-Adrenergic Receptor Purification
Authors: Regan John W , Matsui Hiroaki
Published: November 2023

Despite recent progress in the purification and cloning of α2 -radrenergic receptors (1,2), the purification of sufficient quantities of receptor for structural characterization remains a formidable challenge. This is due to the low natural abundance of α2 -adrenergic receptors which necessitates a substantial purification (100 000-fold) to obtain a pure preparation of the receptor. With the advent of the molecular cloning of the gene for the human platelet α2 -adrenergic receptor and the possibility of designing high-yield expression systems, this purification problem will be reduced but probably not eliminated. A useful and presently essential tool for the purification of α2 -adrenergic receptors is affinity chromatography.
Structural deductions from receptor sequences
Authors: Green N M
Published: November 2023

Receptors are a class of proteins, expressed mainly on the extracellular surface of the plasma membrane, which transmit effects of chemical stimuli to the cytoplasm. This may involve gating of ions by the receptor, modulation of systems coupled to GTP-binding proteins, direct activation of a protein kinase domain of the receptor, or internalization of the stimulating ligand (virus receptors). A few receptors for lipid-soluble molecules (steroids, retinoic acid) exist as soluble cytoplasmic proteins, but neither these nor the virus receptors will receive explicit consideration here. The methods discussed are nevertheless generally applicable to all types of protein.
Preparation of G-proteins and their subunits
Authors: Sternweis Paul C , Pang Iok-Hou
Published: November 2023

The actions of many hormones that interact with receptors on the cell surface are mediated by a family of GTP-dependent regulatory proteins (G-proteins). The G-proteins are stimulated by a host of receptors and regulate a variety of intracellular activities (adenylyl cyclase, phospholipases, ion channels, and a cGMP-dependent phosphodiesterase). Membership in this family is still growing through the use of molecular genetics and more sophisticated techniques for detection and purification of the proteins from diverse sources. The details of the structural complexity and mechanisms of action of the identified G-proteins have been reviewed (1, 2). A brief discussion follows.
Reconstitution of hormone-sensitive adenylate cyclase and tyrosine kinases
Authors: Cerione Richard A
Published: November 2023

A prerequisite for structure-function studies of purified cell surface receptor proteins is the development of methods for functionally inserting these proteins into well-defined phospholipid vesicle systems. A great deal of progress has been made over the past several years in achieving the reconstitution of the components of the P-adrenergic receptor-coupled adenylate cyclase system (1-4). The development of these reconstituted systems has served two important purposes:

(a) it has enabled a direct assessment of whether the different macromolecular components (i.e. the P-adrenergic receptor, the guanine nucleotide regula tory protein, and adenylate cyclase itself) were purified in a completely functional form; and

(b) it has opened the way to mechanistic studies aimed at probing the detailed pathways responsible for both the stimulatory and inhibitory regulation of this signal-transduction system.
Polyphosphoinositide turnover
Authors: Oldham Kenneth G
Published: November 2023

Receptor-stimulated phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate (Ptdlns 4,5-P2) results in the formation of two second messengers: diacylglycerol, which exerts its effect by activating protein kinase C; and myo-inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which acts by mobilizing calcium from cellular stores. In the original simple picture of calcium mobilization by Ins 1,4,5-P3, Ins 1,4,5-P3 is sequentially degraded, by a series of phosphatases, to inositol which is then reincorporated into phosphatidylinositol (Ptdlns) by the enzyme Ptdlns synthase-the so-called inositol phosphate cycle.
Molecular pharmacology of ion channels using the patch clamp
Authors: Gurney Alison M
Published: November 2023

Many receptors are coupled to membrane ionic channels. Indeed, direct activation of channels by neurotransmitters is the most rapid kind of receptor mediated response known. In other instances, ion-channel activity is modulated by intracellular messengers, liberated when transmitters, hormones, or drugs bind to receptors on the extracellular surface of the plasma membrane.

Much of our understanding of how ion channels function in cell membranes, and how they are influenced by receptor activation, has been provided by voltage clamp experiments. These simply involve setting the voltage across the membrane at a known level while measuring ionic flux as an electric current. Introduction of the patch clamp has refined the technique to the extent that the current flowing through an individual channel in a small patch of cell membrane can be resolved.
Interleukin-2 and the interleukin-2 receptor complex
Authors: Diamantstein T
Published: November 2023

Interleukin 2 (IL-2), originally designated as a T cell growth factor, is a glycoprotein with a molecular weight of 15.4 kD. The genes encoding the human, murine, and bovine IL-2 were cloned, their nucleotide sequences determined, and corresponding amino acid sequences deduced.-3 Human IL-2 consists of 133 amino acids and possesses an intramolecular disulfide bridge (between position 58 and 105) essential for the biological activity of the molecule. The position of the cysteine residues, 58 and 105, are also preserved in other species. These results demonstrate the importance of the folding of the molecule for its biological activity. IL-2 is an α-helical protein consisting of antiparallel smaller helical segments (designated A, B, C, D, E, and F), with helix B being interrupted by proline and divided into two sections, B and B′. The specific mutagenesis of human IL-2 genes and the use of antibodies directed against specific sites of IL-2 make it possible to define the sections important for IL-2 receptor binding. The regions responsible for binding IL-2 to the α-chain of the IL-2 receptor (IL-2R) are situated in specific areas of the helices B, B′, E, and F. Specific sections of helices A and F were defined as the areas responsible for IL-2 binding to the β chain of the IL-2R. The 0- and N-bound sugars give IL-2 microheterogenicity with regard to size and loading, but do not influence its biological activity.
The development of regimens for combination immunotherapy
Authors: Creekmore S P , Longo, D L , Urba W J
Published: November 2023

Immunotherapy regimens based on interleukin-2 (IL-2) are a central feature of many current investigations of biological therapy for cancer. Originally described as a factor necessary to support T cell proliferation,1 IL-2 is now known to potentiate several cell types that may have antitumour activity: large granular lymphocytes (LGLs) such as natural killer (NK) and lymphokine-activated killer (LAK) cells, cytotoxic T cells,5 and monocyte/ macrophages. In addition, antitumour activity may result from secondary effects through cytokine regulatory interactions, either directly from the administered T cell, or indirectly through cytokine production by IL-2 stimulated LGLs or other IL-2-responsive cells. Clinical trials of IL-2 in combination with adoptively transferred LAK cells have shown antitumour responses in 20-40 per cent of patients with melanoma and renal cell cancer. As a single agent, without the adoptive transfer of exogenously generated cells, IL-2 has also been shown to induce responses in such patients. However, antitumour responses are usually partial, and require levels of IL-2 dose intensity that are accompanied by substantial toxicity and expense, although improvements in these areas have been achieved with greater experience. Attention is being addressed, therefore, to making immunotherapy more effective and less toxic.
VII collagen, entactin, and laminin) found in basement membranes
Authors: Hudson Billy G , Gunwar Sripad , Chung Albert E , Burgeson Droberte
Published: November 2023

Basement membranes are specialized extracellular matrices that play key roles in diverse biological processes. The processes include: orchestration of embryonic development, maintenance of tissue architecture during remodelling and development, and ultrafiltration of blood. They appear as an amorphous sheet-like structure that is positioned between a cell layer and a thick collagenous stroma, as exemplified by the dermal—epidermal junction of human skin (Figure IA) or between two layers of cells, as exemplified by the renal glomerular basement membrane (Figure I B). In general, basement membranes are composed of several macromolecular constituents, including type IV collagen, laminin, entactin, and heparan sulfate proteoglycan (perlecan) which interact to form a supramolecular structure ( Figure IC). In the case of skin, this suprastructure is connected to the underlying stroma by anchoring fibrils that are composed of type VII collagen.
Structure and function of the steroid receptor zinc finger region
Authors: Freedman Leonard P
Published: November 2023

Steroid hormone receptors are ligand-inducible transcriptional regulatory proteins. Like many eukaryotic transcription factors, nuclear hormone receptors appear to be organized into relatively discrete functional domains (1), and it is this common domain organization that groups these receptors into a superfamily of functionally and most likely structurally related proteins (reviewed in Chapter 1). Because of this modularity, domains for ligand binding (located in the carboxy-terminal region), DNA binding (located toward the central or amino terminus), nuclear localization (within both the DNA and ligand-binding domains), and transcriptional modulation (localized to more variable regions of the receptors, including the N-terminus) (Fig. 1), can all confer their specific functions when linked to unrelated, nonreceptor proteins (2-7). This has led to the efficacy of examining in detail the properties of a particular receptor functional domain, such as the DNA-binding domain, and of avoiding, at least initially, the potential physical/chemical complexities of studying a full-length native receptor.
The Use Of Cell-Free Extracts Of Xenopus Eggs For Studying Dna Replication In Vitro
Authors: Hutchison C J
Published: November 2023

Cell-free extracts of Xenopus eggs provide the only available systems that support the initiation, elongation, and completion of eukaryolic chromosomal replication in vitro (1, 2). These extracts have been used extensively to study the control of entry into S phase (3, 4), the dependency upon completion of S phase as a requirement for entry into mitosis (5, 6), and the role or the nuclear envelope in the initiation and cell-cycle control of S phase (7-10). Although the exploitation of these extracts reflects the current interest in cell cycle control mechanisms, it also reflects their limitations. The extracts lend themselves to analysis of periodic enzyme activities and to immunochemical dissection. However, they are difficult to fractionate. This has restricted those of us who are interested in the enzymology of DNA synthesis to testing for the involvemenl of previously identified proteins at replication forks (11, 12). Nevertheless, these extracts still represent powerful experimental tools, whose preparation and manipulation will be reviewed.
Xenopus Egg Extracts As A System For Studying Mitosis
Authors: Felix Marie-Anne , Clarke Paul R , Coleman Julia , Verde, Fulvia , Karsenti Eric
Published: November 2023

The ultimate goal (or dream) for a biochemist is to isolate the individual components of a complex system, for example all the enzymes involved in cell-cycle control, to mix them back together, and finally show that the system functions again as it did in the context of the whole cell. This is a very difficult task, however, and no highly complex process has been brought to this point yet. A simpler way to analyse complex systems is to use functional complementation assays. In organisms like Drosophila or yeast, this is feasible using a genetic approach. A single function is eliminated by mutation of a gene and then restored by complementation with the corresponding, fully functional gene. This approach has limitations however, as it is not always possible to locate exactly where and when, in a cascade of events, a gene product function is required. Complex in vitro systems that closely mimic the in vivo situation are required to fill in the gaps between genetic approaches and the reconstitution of functional processes from purified components in vitro. Frog egg extracts belong to this category of systems.
Maturation-Promoting Factor And Cyclin-Dependent Protein Kinases
Authors: Doree Marcel , Lorca. Thierry , Picard Andre
Published: November 2023

The cell cycle has two main phases: interphase and mitosis. In interphasc the cell nucleus remains intact and chromosomes are replicated. In mitosis the cytoplasmic network of microtubules breaks down and is reassembled into the mitotic spindle, whilst the chromosomes condense and attach to it (metaphase). Then the spindle segregates the chromosomes (anaphase), ensuring that each daughter nucleus will have one copy of every chromosome. At the end of mitosis the cell divides to form two complete daughter cells that are genetically identical to the parcnl.
Telomerase
Authors: Kipling David
Published: November 2023

In three ciliates and two mammals it has been shown that terminal repeat sequences are synthesized by a specialized DNA polymerase called telomerase (reviewed by Greider 1990; Blackburn 1992). This chapter investigates to what extent the known properties of telomerase are consistent with various features of the terminal repeat sequences of many species, including the majority for which telomerase has yet to be biochemically identified. These structural features have been described in detail in Chapter 3.
Genome rearrangements and telomeres
Authors: Kipling David
Published: November 2023

What is the substrate for telomere addition in vivo? Much of what we know of this comes from analysis of the process of programmed genome fragmentation and de novo telomere addition in ciliates such as Oxytricha and Tetrahymena and the nematode Ascaris. The unusual life cycle of these organisms will be reviewed. The analysis of de nova telomere addition suggests that telomere sequence addition occurs onto A+ T-rich sequences which would not be predicted to be telomerase substrates in vitro.
Human telomere loss, ageing, and cancer
Authors: Kipling David
Published: November 2023

The telomere is a specialized structure which causes the natural ends of a linear chromosome to behave differently from simple double-strand breaks. This concept was first elaborated by Muller (Chapter 2) on the basis of the inability to recover terminal deletions in Drosophila. In what way does such a double-strand break behave differently from a telomere? Such breaks in maize, resulting from the action of dicentric chromosomes, cause repeated rounds of breakage-fusion-bridge cycles (McClintock 1941, 1942). Thus the idea developed that without a telomere a chromosome end was prone to fusion and recombination. Another consequence is that a break will activate a cell cycle checkpoint and arrest cell division. In yeast the action of components of the RAD9 pathway cause cell cycle arrest in response to DNA damage (Hartwell and Weinert 1989), so that a cell with a damaged genome does not exacerbate the problem by going through division and instead is allowed time to repair the break. A single break in the yeast genome, for example caused by the HO endonuclease, is sufficient to cause cell cycle arrest (Brown et al. 1991; Benneti et al. 1993). If a cell with linear chromosomes is to continue division then telomeres must somehow not activate such checkpoints.
Structure and maintenance of Drosophila telomeres
Authors: Kipling David
Published: November 2023

Drosophila melanogaster has a telomeric sequence organization and mechanism of sequence addition dramatically different from other species which have been analysed. From what has been reviewed up to this point one might think that all eukaryotic chromosomal telomeres consist of a tandem repeat array based on a short sequence, probably synthesized by telomerase. Candida albicans is unusual, as its terminal repeats are 23 bp in length, but it is still possible that they are synthesized by telomerase. However, Drosophila provides the most clear exception to this type of telomere structure and synthesis (reviewed by Biessmann and Mason 1992; Mason and Biessmann 1993).
Telomeres and mammalian genome analysis
Authors: Kipling David
Published: November 2023

This chapter reviews the cloning and sequence organization of human telomeres, their integration with the genetic map, and how their structure compares with those in the mouse. Evidence for true terminal deletions, clustering of VNTR (variable number of tandem repeats) loci in proterminal regions, terminal map expansions, and telomere–telomere exchanges in humans will be reviewed, as will the ability to manipulate chromosome structure experimentally by telomere-associated chromosome fragmentation. Some internal (TTAGGG)n arrays may be the remnants of ancestral telomere fusion events, which will be illustrated by the evolution of human chromosome 2. Telomeric sequences can be used to investigate a number of aspects of chromosome structure and behaviour, such as fragile sites, and in particular can reveal cryptic translocations in patients with apparently normal karyotypes. Finally, a potential link between the unusual genetic behaviour of (TTAGGG)n repeat arrays, as exemplified by those of the mouse, and the instability of short repeated sequences which underlie some human genetic diseases will be discussed.
Atninoacyl-tRNA synthetase-tRNA recognition
Authors: Arnez John G , Moras Dino
Published: November 2023

Aminoacylation is the first and key step in the translation of genetic messages into proteins. The reaction brings together amino acids and their cognate tRNAs and is catalysed by aminoacyl-tRNA synthetases (aaRSs). In the reaction the amino acid is esterified to one of the hydroxyl groups of the 3'-terminal adenosine; the energy in the overall reaction is supplied by the hydrolysis of ATP.
Interaction of 5S RNA -with TFIIIA
Authors: Pieler Tomas
Published: November 2023

Among the structural motifs which identify DNA- and RNA-binding proteins in eukaryotes, zinc finger modules are unique in defining the nucleic acid-binding domain in proteins with a demonstrated ability to form sequence-specific com plexes with either RNA or DNA or even with both classes of nucleic acids (1). Transcription factor IIIA (TFIIIA) is the founding member of the zinc finger protein superfamily, which is comprised of several hundred members in vertebrates (2, 3). TFIIIA is one of the most abundant proteins in immature Xenopus laevis oocytes, where it is found primarily in a complex with 5S ribosomal RNA (4). The key structural feature in TFIIIA comes from nine copies of a sequence motif that has been termed zinc finger, since two pairs of invariant cysteines and histidines mediate the three-dimensional (3-D) folding of two antiparallel !3-sheets and one a helical element, mainly by coordination of zinc (Figure 1). The zinc finger duster in TFIIIA is not only responsible for the specific recognition of 5S RNA, but also promotes sequence-specific binding to the internal control region of the 5S RNA gene, thereby defining the first step in the formation of an active transcription complex (5, 6).
Assaying inositol phospholipid turnover in plant cells
Authors: Brearley Charles A , Hanke David E
Published: November 2023

The inexorable growth in the complexity of inositol phospholipid and inositol phosphate science, principally in the animal field but also in yeast, has served to emphasize our lack of understanding of even the fundamentals in plant cells. The fragmented nature of plant inositol phosphate research is a primary problem (1). The lack of the plant equivalent of the avian or human erythrocyte, which features throughout this chapter as a model system in which virtually all the methods described here were developed or tested, holds back the efforts of many plant scientists. Thus, there is much to be gained from seeking a simple and, most importantly, rigorous description of the identities of the numerous inositol phosphates and inositol phospholipids present in plant tissues before proceeding to the rather more complicated task of investigating their function.
The purification and assay of inositide binding proteins
Authors: Theibert Anne B , Prestwich Glenn D , Jackson Trevor R , Hammonds-Odie Latanya P
Published: November 2023

A valuable strategy towards understanding the physiological roles of inositol polyphosphates (InsPns) and inositol lipids (PtdlnsPns) will be the identification, characterization, isolation, and molecular biological and genetic manipulation of their intracellular receptors. Several experimental approaches have been used to isolate InsPn and PtdlnsPn binding proteins, including conventional chromatography and reversible ligand binding, InsPn affinity column chromatography, and covalent modification of binding proteins with photoactivatable ligands. These approaches have led to the identification of an unexpected variety of InsPn and PtdlnsPn binding proteins, whose functions are only now being uncovered.
Mass assay of inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate
Authors: Challiss R A John
Published: November 2023

Elucidation of the phosphoinositide cycle (by which PtdIns(4,5) P  2 is hydrolysed, and Ins(l,4,5) P  3 and DAG are metabolized for re-incorporation into cellular the phosphoinositide pool), and studies which have highlighted the emerging roles of PtdIns(3,4,5) P  3 and higher inositol polyphosphates in cellular regulation, have been achieved largely by employing radioisotopic labelling strategies. Although radiolabelling methods continue to be invaluable for addressing many of the unresolved questions concerning phosphoinositides and cellular regulation, methods for the mass determination of key cycle intermediates can offer a number of clear advantages in terms of speed/throughput, reproducibility and cost. In this chapter methods for the mass assay of two critical phosphoinositide cycle intermediates are described.
The Ins(1,4,5)P
3 receptor
Authors: Mikoshiba Katsuhiko
Published: November 2023

Inositol 1,4,5-trisphosphate (Ins(l,4,5)P  3) is the second messenger derived from the hydrolysis of phosphatidylinositol bisphosphate via activation of phospholipase C. The Ins(l,4,5)P  3 binds to its specific receptor, which is an Ins(l,4,5)P  3-induced Ca2+-releasing channel located on the endoplasmic reticulum. In this way the Ins(l,4,5)P  3 signal is converted to a Ca2+ signal.
Affinity methods for isolating RNA binding proteins
Authors: Jackson Richard J
Published: November 2023

The need or wish to purify a particular RNA binding protein usually originates from the following type of scenario: At this stage it becomes important to purify and characterize the particular RNA binding protein(s). However, it is worth appreciating that purification is usually not an end in itself. Most commonly the aim is limited to obtaining sufficient protein in sufficient purity for amino acid sequencing, which then allows either the protein to be directly identified if it is already in the databases, or the isolation of cDNA clones leading ultimately to expression as recombinant protein. Thereafter all further work, including the raising of antibodies, exploits the recombinant protein if at all possible. The only reason, though it is an important one, why purified protein from the original source would still be required is to verify that the recombinant protein exhibits the same properties and binds to the RNA element with the same specificity and affinity.
A three-hybrid system to detect and analyse RNA:protein interactions in vivo
Authors: Zhang Bejlin , Kraemer Brian , Sengupta Dhruba , Fields Stanley , Wickens Marvin
Published: November 2023

RNA:protcin interactions arc critical in diverse cellular processes, such as splicing. translation, early development, and infection by RNA viruses. For example, the replication cycle of human immunodeficiency virus (HIV-1) relics on the interactions between reverse transcriptasc and the RNA genome to generate viral DNA, between Tat and TAR to produce mRNA, between Rev and RRE to export mRNA, and between viral proteins and the RNA genome to assemble viral particles. Few general methods are available to detect and analyse RNA:protein interactions in vivo. In vitro.
Prevention Of Unwanted Proteolysis
Authors: North Michael J , Beynon Robert J
Published: November 2023

To many biochemists and cell biologists, proteinases are not so much interesting enzymes or useful tools but persistent nuisances. while many proteins seem to be immune to the ‘ proteinase problem’ , the frequency with which artefacts arise indicates that a cautious approach is justified. Moreover, the availability of accurate mass from new mass spectrometric methods may well bring to light more examples of unwanted proteolytic degradation, especially terminal trimming that might have previously remained undetected through the low resolution of SDS-PAGE.
Neuronal promoter analysis by in vitro transcription using nuclear extracts from rat brain
Authors: Schwartz M L , Schlaepfer W W
Published: November 2023

We present a reliable method for making nuclear extracts from rat brain and for using these extracts in an in vitro transcription assay. Our nuclear extract technique allows for the isolation of nuclear proteins from post-mitotic neurones that presumably contain all of the factors required for the transcriptional regulation of neurone-specific genes. The use of brain as a source of nuclear extract is in contrast to the more commonly used cultured cell lines where expression of neurone-specific genes may be poor or lacking in tissue specificity. In vitro transcription using nuclear extracts from expressing (brain) and non-expressing (liver) tissues provide a direct assessment of promoter strength and level of tissue-specificity apart from the effects of higher order DNA structure (i.e. chromatin). In vitro transcription is also amenable to DNA competitor studies and to the addition of purified or partially purified protein factors and antibodies. In this way, true neurone-specific transcriptional activator or repressor sequences or factors can be characterized. The approach is rapid, reliable, and easily quantifiable. Finally, advancements in the field of nucleosome reconstitution have allowed the coupling of in vitro transcription to nucleosome assembly systems and provide a novel and promising approach to the understanding of neuronal-specific gene expression.
Cell culture
Authors: Blake K , Stacey A
Published: November 2023

The ability to culture cells in vitro has enabled enormous advances to be made in biology, particularly in virology. Many cell types can be routinely pro liferated and cryopreserved, allowing consistent, characterized, contaminant free cells to be used both as a research and diagnostic tool. However, despite the utility of cell culture, researchers must recognize that cells grown in vitro may not behave as cells in the intact organism. For example, the proliferation of cells in vitro is not necessarily conducive to full differentiation, where cell growth is normally absent. The potential loss of differentiated characteristics will inevitably affect properties and therefore the utility of cells. Cell behaviour will also be affected by the loss of cell-cell, and cell-matrix interactions, and loss of tissue architecture. The goal of attaining a greater range of in vivo characteristics is becoming more achievable with the increased availability of growth and differentiation factors, and the development of three-dimensional culture systems which allow cells to assume their correct polarities, shapes and interrelationships. The future perspective looks promising, and cell culture systems may be becoming available which enable in vitro growth and study of currently non-cultivable viruses (Chapter 2).
Antiviral drug testing
Authors: Oxford J S , Kelly L S , Davies S , Lambkin R
Published: November 2023

The clinical effectiveness of acyclovir, and more recently penciclovir, against herpes simplex type I and II infections (1) has led both to great expectations of further discoveries of antiviral compounds and to a change in the scientific direction of clinical diagnostic laboratories. Most such laboratories are at present used primarily to diagnose viral infections, either by culture or serology, or, more often, by detection of viruses using rapid molecular techniques such as PCR. The advent of the new generation antivirals such as protease inhibitors of HIV-1 (2), and neuraminidase inhibitors of influenza (3-5), together with new molecular methodologies, now requires clinical laboratories to quantify virus, usually by PCR or by molecular probing. Also, the therapeutic benefit of antiviral therapy has been enhanced by the use of PCR to analyse viral genes and search for drug-resistant mutations (6). Nowhere is this more important than with HIV, where, on the one hand, a choice of antivirals exists, and on the other hand the introduction of combination chemotherapy has led to a substantial increase in demand for scientific information on which to base clinical decisions.
Mutagenesis of RNA virus genomes
Authors: Barclay Wendy S , Almond Jeffrey W
Published: November 2023

Since the pioneering experiments of Burnett (1) and Cooper (2) in the 1950s and 1960s, the isolation of RNA virus mutants has been a cornerstone of studies aimed at determining the function of virus genomes and their encoded proteins. Not only have the phenotypes of individual mutants been informative about the replication and biology of the wild-type virus, but, collectively, the exploitation of mutants in complementation and recombination assays has provided useful information on the number of genes and the sizes of genomes. Nowadays the generation of mutants is a routine procedure exploited widely in structurefunction studies and for the development of designer viruses as vaccines, vectors, and reporter systems for studies on gene expression and genome replication.
Detection and analysis of host gene targets for oncogenic retroviruses
Authors: Neil J C , Terry A
Published: November 2023

Unlike the DNA viruses, where oncogenic representatives are found in disparate families, only two RNA virus families, the Retroviridae and the Flavoviridae, have so far been implicated in neoplasia. The mechanisms by which the latter operate are largely unknown and the prototypic example, hepatitis C virus, appears to be an indirect cause of cancer, for which the roles of specific gene products are as yet undetermined. In contrast, the Retroviridae have received much attention as causes of cancer. Animal retroviruses have been studied for many years, mainly as model carcinogens; their chief contribution has been through the identification of oncogenes that play a role in cancers of diverse aetiology. This field of work is the main focus of this chapter. However, it should be noted that these agents can be important causes of disease in their natural host species, and that since the discovery of human retroviruses there is much wider interest in retrovirus pathogenesis and methods of their control.
Direct assessment of conformational changes in G protein-coupled receptors, using fluorescence spectroscopy
Authors: Gether Ulrik , Kobilka Brian
Published: November 2023

G protein-coupled receptors (GPCRs) represent the largest class of transmembrane signalling molecules. The mechanism by which hormones and neurotransmitters activate intracellular signalling cascades through these homologous proteins is of great interest. While GPCRs all share a common seven-helix transmembrane structure, domains involved in ligand binding are nearly as diverse as the chemical structures of the known agonists. Small molecular weight ligands bind to sites within the hydrophobic core formed by the transmembrane (TM) a-helices, while binding sites for peptides and protein agonists also include the amino terminus, and extracellular hydrophilic loops joining the transmembrane domains (1). This diversity in ligand binding suggests that different classes of receptors may have unique mechanisms of activation. However, when one considers that all these receptors function by activating a highly homologous family of G proteins, it is reasonable to propose that agonist binding is linked to G protein activation through changes in the conformation of the transmembrane domains, and that what we learn about receptor activation from one class of receptors will apply to most, if not all, members of this large receptor family. It is generally assumed that binding of the agonist to the receptor induces a set of finely orchestrated changes in the tertiary structure of the receptor, that are recognized by the associated G protein a-subunit.
Probing the structure of receptor-binding sites by the substituted-cysteine accessibility method
Authors: Simpson Merrill M , Liapakis, George , Javitch Jonathan A
Published: November 2023

The substituted-cysteine accessibility method (SCAM) provides an approach to map systematically the residues on the water-accessible surface of a binding site, such as the binding-site crevice of a G protein-coupled receptor (GPCR) which binds ligand within the transmembrane portion of the receptor. Consecutive residues in putative membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. The water-accessible residues are identified by assessing the reaction of charged, hydrophilic, sulfhydryl reagents with the engineered cysteines.
Post-translational acylation of signal transducing polypeptides: palmitoylation of G protein-coupled receptors
Authors: Bouvier Michel , Petäjä-Repo Ulla , Loisel, Thomas , Bélanger Charlène
Published: November 2023

In recent years, a number of proteins involved in signal transduction have been found to be acylated by fatty acids or prenyl derivatives. Three major classes of fatty acylation have been characterized. These are: (a) the Nterminal myristoylation of glycine residues through amide linkage; (b) the prenylation of cysteine residues by the formation of thioether links to farnesyl or geranylgeranyl moieties; and (c) the palmitoylation which occurs through the thioesterification of cysteine residues. In contrast to myristoylation and prenylation, which typically occur co-translationally, palmitoylation is a genuine post-translational modification (1). The chemical reactivity of sulfhydryls and thioester linkages have led several investigators to propose that protein palmitoylation may represent a reversible modification which could undergo dynamic regulation. In fact, rapid turnover of the covalently attached palmitate has been reported for several proteins, and this turnover is modulated by external stimuli (2-4).
G protein-coupled receptor phosphorylation and desensitization
Authors: Tobin Andrew B , Rae, Angela , Budd David C
Published: November 2023

Phosphorylation of G protein-coupled receptors (GPCRs) has been linked with receptor desensitization, primarily through work on the 13-adrenergic receptor(l-3). Extensive studies have demonstrated that agonist stimulation of the !3-adrenergic receptor results in phosphorylation of the receptor by two kinases; protein kinase A (PKA), and the receptor-specific kinase !3-adrenergic receptor kinase (GRK-2) (1-3). Each of these kinases phosphorylates distinct sites on the receptor. PKA sites are at RRSS motifs in the third intracellular loop and C-terminal tail (4), and GRK-2 sites are in an acid serine/threoninerich region of the C-terminal tail (5). Phosphorylation at both PKA and GRK-2 sites results in receptor desensitization (1). In the case of GRK-2- mediated desensitization the mechanism has been demonstrated to involve the intermediary protein !3-arrestin, which binds to the phosphorylated receptor and in doing so is able to ‘displace ‘ the Gas-protein, thereby uncoupling the receptor from adenylyl cyclase (6). Cloning studies have revealed that GRK-2 is a member of a protein kinase family called the G protein-coupled receptor kinase (GRK) family (7). There are at present six members of this family (GRK-1 to GRK-6); each is thought to be able to mediate agonist-stimulated phosphorylation of certain GPCRs (7).
Construction and analysis of receptor-G protein fusion proteins
Authors: Wise Alan
Published: November 2023

It is now well established that a multitude of diverse extracellular signals such as neurotransmitters, hormones, odorants, and light elicit intracellular responses via vectorial signal transduction mechanisms across the lipid bilayer, following binding to specific cell surface receptors possessing seven transmembrane-spanning domains. Agonist-occupation of these receptors leads to activation of effector proteins, and the concomitant mobilization or production of second messengers that initiate the desired physiological response within the cell. In all eukaryotic organisms the family of heterotrimeric G proteins plays a pivotal role, acting as intermediates between such cell surface receptors and cytoplasmic or membrane-bound effector molecules, leading to the regulation of humoral, neural, metabolic, and developmental functions. Examples of intracellular effectors which are regulated by G protein activation include adenylate cyclase, phosphoinositidase C, cyclic GMP phosphodiesterases, phospholipase A2, and a number of ion channels (1, 2).
Analysis of the polyphosphorylated inositol lipids of Saccharomyces cerevisiae
Authors: Dove Stephen K , Michell Robert H
Published: November 2023

The yeast Saccharomyces cerevisiae offers several advantages to the researcher interested in determining the functions of phosphoinositides in eukaryotes. The ease with which this yeast can be genetically manipulated, coupled with knowledge of the entire nucleotide sequence of its genome, means that certain approaches can be used which cannot yet be applied to other organisms. For instance, genes that encode proteins involved in the synthesis, degradation, or effector functions of phosphoinositides can be deleted or rendered partially defective, and the phenotypes of the mutants determined. In this way, the contribution of an enzyme or lipid to the operation of a signalling system can rapidly be gauged. By screening for suppression of the phenotypes of such mutants, genes whose actions lie upstream or downstream of a mutated gene can be identified and characterized. In addition, genetic complementation studies are used to test whether the function(s) of an inactivated yeast gene can be replaced by expression of an apparently orthologous gene from another organism (such as a mammal).
Detection, isolation, and quantitative assay of mitogenactivated protein kinases in intact cells and tissues
Authors: Anderson Neil G
Published: November 2023

The landmark discovery of the insulin-stimulated protein kinase, originally termed microtubule-associated protein 2 (MAP-2) kinase, by Sturgill and colleagues in 1987 (1), was of fundamental importance in initiating the huge advances since made in our understanding of how messages received by receptors on the cell surface are transduced to the cell interior to produce specific responses. Although originally isolated from insulin-stimulated 3T3- Ll adipocytes, MAP-2 kinase was subsequently shown to be present in all cells, to be activated in response to an enormous variety of extracellular stimuli, and to have been evolutionarily conserved from yeast to man (see ref. 2 for a review). Appreciation of the ubiquitous nature of MAP-2 kinase led to it being renamed mitogen-activated protein (MAP) kinase. Since then, a number of related protein kinases have been isolated. These all have similar structures and mechanisms of activation, but co-exist in cells as members of parallel signalling pathways stimulated by distinct types of extracellular stimulus. Three major classes of MAP kinase have, so far, been characterized in detail.
Equilibrium dialysis and rate dialysis
Authors: Honoré Bent
Published: November 2023

Studies on the interaction of low molecular mass molecules, i.e. ligands, with high molecular mass protein molecules play fundamental roles in biomedical research. Among important examples to be mentioned are drug binding to plasma proteins, especially albumin (1), vitamins to their transport proteins, as well as binding of various substances, e.g. hormones and transmitter substances to receptor proteins in their target cells. Ideally the questions to be answered from such studies are how many ligand molecules, N, may bind to a given macro molecule and with what affinities, K1, K2, .. , KN under the given set of conditions specified by, e.g. pH, temperature, ionic strength, and buffer composition.
Protein-ligand interactions studied by FTIR spectroscopy: methodological aspects
Authors: Jackson Michael , Mantsch Henry H
Published: November 2023

Many techniques exist for the study of protein-ligand interactions, with each technique providing unique and/or complementary information. For example, calorimetric studies provide information relating to the energetics of protein-ligand interaction (Volume 1, Chapters 10 and 11), while X-ray diffraction techniques provide information on the molecular cordinates of atoms in the protein-ligand complex (see Chapter 1). Between these extremes lies a bewildering array of methods varying in technical sophistication, cost, and information content.
Electrospray ionization mass spectrometry
Authors: Hill T J , Derrick P J
Published: November 2023

Characterization of protein-ligand interactions involves the determination of stoichiometry, affinity, and conformational changes associated with such interactions. Mass spectrometry is rapidly emerging as an important and powerful new tool in this area to complement the wealth of other techniques described in these volumes. The development of ionization methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), applicable to biomolecules in solution or as solids has allowed the routine analysis, with picomole (10™12 M) to attomole (10™18 M) sensitivity, of proteins, peptides, oligonucleotides, carbohydrates, and other classes of biomolecule. Extending beyond simple molecular mass determinations, mass spectrometry is now readily applied to the sequencing of peptides, proteins, and DNA as well as for the study of protein aggregation, folding, and non-covalent interactions in general.
NMR studies of protein-ligand interactions
Authors: Lian Lu-Yun
Published: November 2023

The interactions between pairs of molecules are crucial in many molecular recognition processes in biology, the main examples being found in complexes of proteins with other proteins (e.g. in signalling processes), with small molecules (e.g. enzyme-substrate or protein-drug interactions), and with nucleic acids (e.g. protein-RNA complexes, transcription factors with DNA). Like many other bio-physical techniques, nuclear magnetic resonance (NMR) provides information about protein-ligand interactions. However, unlike many of the other techniques such a fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance, NMR provides information on many different aspects of these interactions, ranging from structures to dynamics, kinetics and thermodynamics. Recent developments in NMR technology (1, 2), together with the routine use of stable isotopes (13C,15N,2H), have enabled the use of a much greater variety of NMR-based techniques for studying protein-ligand interactions, and involving much larger protein systems; these techniques have been used in a many different basic and applied research environments (3, 4). Examples of the latter are evident, for example, in the use of NMR as a method for high-throughput screening in drug development programmes in the pharmaceutical industry (5-7).
Quantification and mapping of protein-ligand interactions at the single molecule level by atomic force microscopy
Authors: Roberts Clive J , Allen Stephanie , Davies Martyn C , Tendler Saul J B , Williams Philip M
Published: November 2023

This chapter illustrates the recent application of atomic force microscopy (AFM) to the measurement of the forces that bind protein-ligand complexes. The experiments highlighted will demonstrate the potential of AFM to quantify the adhesion force between individual molecular pairs and how this can lead to a deeper understanding of protein structure and function. The possibility of spatial mapping active surface bound proteins though their ability to bind ligands will also be illustrated.

An understanding of the recognition mechanisms is not only of importance in understanding protein structure, folding, and function but has practical implications for the development of novel devices in areas such as sensors, screening, and drug discovery.