bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic nucleotides: measurement and function
Authors: Butt Elke , Walter Ulrich
Published: November 2023

The activation of human platelets is inhibited by a variety of agents which exert their effects through distinct mechanisms. Examples include inhibitors of thromboxane A2 generation (e.g. aspirin), inhibitors of thrombin (e.g. hirudin), scavengers of ADP (e.g. apyrase), and physiological and pharmacological cyclic nucleotide-elevating agents (1,2). Vascular endothelial cells, under basal conditions and in response to numerous vasoactive agents, synthesize and release prostacyclin (PGI2) and endothelium-derived relaxing factor, nitric oxide (EDRF; or NO), two of the most important physiological platelet inhibitors. PGI2 and EDRF/NO increase the intracellular messenger molecules cAMP and cGMP, respectively, in human platelets and other target cells. The inhibition of platelet activation caused by PGI2 and EDRF/NO is mediated by cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK), respectively. As shown in Table 1, human platelets contain par ticularly high concentrations of cAMP-PK and cGMP-PK (3). The type III cGMP-inhibited phosphodiesterase (cGI-PDE) is an additional important target for cGMP. Elevation of cAMP mediated by cGMP inhibition of the cGI-PDE contributes to the well known synergism between cGMP- and cAMP-elevating platelet inhibitors (4). In human platelets, established cAMP-PK substrates include the small molecular weight G protein rap lB, the subunit of glycoprotein lb (GPib ), the focal adhesion protein VASP, caldesmon, myosin light chain kinase (MLCK), cGI-PDE, and actin-binding protein (ABP) (2). Only the focal adhesion protein VASP is an established substrate for cGMP-PK in platelets although other substrates, perhaps also rap lB and GPib , may exist. The physiological effects of cyclic nucleotide-elevating platelet inhibitors are terminated by cyclic nucleotide-degrading phosphodiesterases (PDEs) and by protein phosphatases which dephosphorylate the cAMP-PK and cGMP-PK substrates. In human platelets, the major PDEs responsible for cAMP or cGMP hydrolysis are type III cGI-PDE and type V cGMP binding, cGMP-specific PDE (cGB-PDE), respectively (4,5).
Isolation and functional properties of antigen presenting cells
Authors: Stockinger Brigitta
Published: November 2023

T cell recognition of antigen depends crucially on degradation of protein and binding of protein fragments to MHC molecules which ‘present’ these fragments to T cells. The term ‘antigen presentation’ is used to summarize the complex events of antigen internalization, processing of antigen into peptide fragments, peptide transport, and MHC/peptide interaction until the final appearance of peptide/MHC complexes on the surface of antigen presenting cells. Two separate pathways exist using largely non-overlapping sources of antigen and distinct intracellular compartments for binding to different MHC molecules. Most biosynthesized, intracellular antigens are degraded in the cytosol and peptides are translocated into the endoplasmic reticulum (ER), where they bind to MHC class I molecules and after transport to the cell surface are recognized by class I restricted cytotoxic T cells. The MHC class II presentation pathway does not make use of peptides in the ER since the peptide binding site of MHC class II is blocked by the invariant chain. Instead MHC class II molecules are re-routed to endosomal compartments where the invariant chain is degraded, so that peptides from exogenous proteins that have been internalized by antigen presenting cells (APCs) or from endogenous sources of protein that can enter these compartments bind to invariant chain-free MHC class II for recognition by class II restricted T cells.
The 20S proteasome and antigen presentation
Authors: Seelig Angela , M.-Kloetzel Peter
Published: November 2023

The 20S protesome is the major cytosolic proteinase complex of eukaryotic cells. This 700 kDa multisubunit enzyme complex is involved in ubiquitin independent proteolysis and is also central to the ATP/ubiquitin-dependent intracellular protein degradation pathway where it represents the proteolytic core of the ‘26S’ proteinase complex (1, 2). Thus, proteasomes are involved in the turnover or activation of transcription factors (3, 4), in cell cycle control (5), and the generation of antigenic peptides which are presented at the cell surface by MHC class I molecules (for review see refs 6 and 7).
Methods for studying the role of the major histocompatibility complex in NK cell cytotoxicity
Authors: Pena J , Solana R , Borrego, F , Rolstad B
Published: November 2023

Our knowledge about how T cells recognize foreign cells has been increased through studies of the interactions between the T cell receptors (TCR) and antigenic peptides presented by class I or class II molecules. The role of the MHC is now equally challenging in NK cell research, as it has been shown that NK cells can recognize certain MHC class I specificities. The receptors that NK cells utilize to recognize class I antigens have recently been identified, and named KiR (killer inhibitory receptors). An attractive idea supported by experimental data is the ‘missing self’ hypothesis (1, 2) which envisages NK cells as a surveillance mechanism against cells showing reduced or aberrant expression of certain MHC class I molecules regarded as ‘self’ by the immune system (for reviews see refs. 3 and 4).
Superoxide dismutase
Authors: Goldstein Sara , Czapski Gidon
Published: November 2023

The superoxide anion radical (02) is considered to be a highly toxic entity in many biological systems (1–4). It is formed in normal metabolism as well as through the action of many drugs, poisons, and radiation (1–4). It is also involved in radiation damage, DNA damage, phagocytosis, ageing, cancer, etc. Superoxide dismutases (SODs) are metalloenzymes that catalyse very efficiently the dismutation of superoxide ions into oxygen and hydrogen peroxide. It has been widely recognized that these enzymes, which are present in almost all living organisms, provide a defence system that is essential for their survival under aerobic conditions. The deleterious role of superoxide during ischaemia (5), which occurs in organ transplantation and many surgical interventions and during heart and brain events, suggests that SOD may have a potential clinical use. This led many investigators to search for SOD mimics, a search which arose because human SODs have short metabolic half-lifes (<10 min), and do not penetrate into the cells.
Protein translocation into the endoplasmic reticulum
Authors: Romisch Karin , Corsi Ann
Published: November 2023

In eukaryotic cells all non-organellar protein synthesis starts on free ribosomes in the cytoplasm (1). Nascent chains destined for the secretory pathway are marked as such by an N-terminal signal peptide (2, 3). The signal peptide is recognized and bound by a ribonudeoprotein complex, the signal recognition particle (SRP), as soon as it emerges from the ribosome (4). Binding of SRP to the signal peptide retards or stops translation (5) and allows for targeting of the SRP-nascent chain-ribosome complex to the endoplasmic reticulum {ER) membrane (6). Interaction of SRP with its receptor in the ER membrane subsequently leads to its dissociation from the nascent chain (5, 7). Translation resumes and the secretory protein is translocated into the ER where its signal peptide is cleaved, and glycosylation, folding, and oligomerization take place (8-11). In the past two decades a large body of work has focused on the elucidation of the first step of the secretory pathway, translocation into the ER. The results allow us to draw a detailed picture of the components involved and their respective functions.
Biogenesis of constitutive secretory vesicles, secretory granules, and synaptic vesicles-£acts and concepts
Authors: ohashi Masato , bauerfeind, rudolf , Huttner wieland b.
Published: November 2023

Vesicle-mediated secretion from animal cells utilizes three types of secretory vesicles (1, 2) (Fig. 1). The first type is the constitutive secretory vesicle (CSV), which is thought to be common to all eukaryotic cells and which mediates the continuous secretion of newly synthesized proteins. The second type is the secretory granule, in neurones also referred to as the large dense core vesicle, which occurs only in cells capable of regulated protein secretion and which mediates the stimulus-dependent release of stored proteins and biogenic amines. The third type is the synaptic vesicle of neurones, which mediates the release of neurotransmitters but lacks secretory proteins, and its counterpart in certain endocrine cells, the synaptic-like microvesicle (SLMV). In this chapter, we will summarize current knowledge, and describe concepts concerning the molecular machinery involved in the biogenesis of these vesicles, its regulation, and the protein sorting processes that occur in the course of vesicle formation.
Time-resolved fluorescence immunoassay
Authors: Barnard Geoff
Published: November 2023

Today, immunoassay methodology is applied in many branches and disciplines of scientific investigation. The procedures are essentially of two types, competitive and non-competitive, and historically these have been typified by radioimmunoassay (RIA) and immunoradiometric assay (IRMA), respectively (see Chapter 1). In recent years, however, there has been the widespread adoption of procedures using alternative detection systems that avoid the use of radioisotopes (e.g. enzymes, luminescent compounds etc.). In particular, over the last 10 years, it has become well established that time resolved fluorescence immunoassay (FIA) can offer unique advantages over most other non-radioisotopic procedures (1).
The RAS/RAF/ERK signal transduction pathway
Authors: Macdonald Susan G , McCormick Frank
Published: November 2023

The importance of RAS proteins in human cancer was established in 1982, when the first human oncogene was recognized to be a mutant version of a normal H-RAS allele (1). These early observations prompted exhaustive efforts to identify effectors of RAS proteins, and so to understand their precise molecular function. These efforts have led to the conclusion that a major effector pathway for RAS proteins is the MAP kinase cascade (2--4; and Fig. 1). Recently, the existence of other effector pathways has been inferred, and will be discussed below. Here we summarize biochemical and biological properties of the mammalian RAS--RAF-MEK-MAP kinase signal transduction pathway and discuss questions about the pathway that remain to be answered.
Toxoplasma as a model genetic system
Authors: Sibley L David , Howe Dank , Wan kiew-lian , Khan shahid , Aslett martin a. , Ajioka James W
Published: November 2023

Several important biological attributes make Toxoplasma well suited for genetic studies. First, the parasite is easily maintained in the laboratory, having the ability to grow productively in virtually any vertebrate cell line. Second, it is possible to produce mutants and to propagate clones indefinitely. Phenotypic analysis of mutants is aided by the fact that replicative stages are haploid. Third, it is possible to conduct classical genetic studies and rudimentary genetic and physical linkage maps are available. Finally, molecular genetic studies are supported by DNA transformation using a variety of selectable markers. These properties make Toxoplasma unique among the world of parasites where biological and technological limitations have generally limited application of genetics.
Biogenesis of specialized organelles: glycosotnes and hydrogenosotnes
Authors: Sommer Jurg M , Bradley Peter J , Wang c. C , Johnson Patricia J
Published: November 2023

Compartmentalization of cellular functions into organelles is a characteristic of all eukaryotic cells. The development of highly specialized intracellular organelles in response to unusual host environments is most strikingly demonstrated by two structures found in certain parasitic protozoa: the glycosomes of trypanosomatids and the hydrogenosomes of trichomonads. These unique organelles represent a major cellular difference between host and parasite and have thus been studied extensively as possible targets for chemotherapeutic agents. Both of these organelles participate in the production of ATP, although in very different ways. The glycosome increases the rate of glycolysis by at least an order of magnitude, while the hydrogenosome participates in substrate-level phosphorylation, converting pyruvate to acetate, CO2, and molecular hydrogen in the process. The evolutionary origin of these organelles has been a matter of much speculation in the past, but recent molecular studies of their biogenesis have confirmed that the glycosomes are derived from an ancestral microbody related to the peroxisome, while the hydrogenosomes of trichmonads may share a common progenitor organelle with mitochondria.
Promoters, sigma factors, and variant RNA polymerases
Authors: Helmann John D
Published: November 2023

Transcription, the copying of genetic information from its DNA repository into functional RNA molecules, is catalyzed by RNA polymerase (RNAP). In this chapter we review the structure and function of bacterial RNAP, discuss the DNA determinants of promoter strength, and consider several examples of alterations to RNAP which regulate gene expression.
Switch systems
Authors: Saunders J R
Published: November 2023

Bacteria of many species are frequently observed to exhibit profound phenotypic changes, typically affecting colonial appearance or behavior. Most often, such changes are evident in pathogenic bacteria, where genetic switches modulate expression of a variety of virulence determinants (1). Such variation is the result of a diverse range of genetic mechanisms that are manifested in what are somewhat imprecisely grouped together as either phase variation or antigenic variation. Phase variation results from quantitative changes in gene expression that may range from what are, in essence, simple on-off genetic switches, through to modulation of the volume of expression of functional gene product at either the transcriptional or translational level. In contrast, antigenic variation is evident as qualitative changes in the nature of one or more surface components. In the main, such variations are detected by sequence changes involving more complicated switches that lead to the expression of an antigenically altered protein. Antigenically variable components have been studied largely because of their importance in bacterial virulence. However, equivalent changes in surface-exposed components of non-pathogenic bacteria almost certainly occur and may have particular selective advantages in their particular environments. Moreover, qualitative changes in a variable gene product could take place without necessarily having effects on the immunological properties of the variable component concerned.
Evolution of prokaryotic regulatory systems
Authors: Baumberg Simon
Published: November 2023

The preceding chapters provide a wealth of detail about the often complex and intricate mechanisms found to control gene expression in present-day prokaryotes. This brief concluding chapter, as a contrasting afterword, consists principally of unanswerable questions, of the type: ‘How did things get the way they are?’.
Enzyme-linked immunoassays
Authors: Kemeny D M
Published: November 2023

Enzyme-linked immunoassays are in widespread use in all areas of biological science today. Their popularity stems from their ease of use and safety. In this chapter I will review the current status of ELISA including assay format, reagent choice, assay optimization, and quantification. Other variations on the method such as the ELISA plaque assay, signal amplification systems, and affinity measurement will be addressed by other authors in these volumes. My comments will be restricted to assays set-up and developed in the user’s own laboratory and to plastic microtitre plate-based assays (which apply equally to plastic tubes or pins) as these are the most widely used. However readers should be aware of their limitations and alternatives which have been reviewed extensively elsewhere (1,2).
Photoluminescence immunoassays
Authors: Hemmilä I A
Published: November 2023

In biospecific assay technologies the detection of a label is most often based on measurement of emitted light. The emission can originate, for example, from radiation (radioimmunoassays, radioluminescence, RIA, IRMA), from a fluorescent compound (fluoroimmunoassays, FIA, IFMA), or the fluorescent end-product of an enzymatic reaction (fluorometric EIAs, ELFIA), from a luminogenic label (luminoimmunoassay, LIA), or luminogenic substrate after enzymatic triggering (enzyme-linked chemiluminescence immunoassay, ECLIA), from a phosphorescent compound (phosphoro-immunoassay), or from photoluminescent lanthanide chelate (time-resolved fluoroimmuno-assay). In the case of lanthanides the emission emanates directly from the long life-time excited ionic state of the metal and is today classified with the generic term luminescence (the term fluorescence being defined as emission emanating from excited singlet level of TI-electrons and hence is restricted to aromatic hydrocarbons). In immunoassays the lanthanide emission is measured with a fluorometer using time resolution. These assays are generally named fluoroimmunoassays, or more specifically time-resolved fluoroimmunoassays, and to avoid confusion, never luminoimmunoassays.
Ligand blotting
Authors: Soutar Anne K , Wade David P
Published: November 2023

Ligand blotting is a method of detecting or identifying proteins on the basis of their specific biological function. It is similar to immunoblotting (Western blotting; see Chapter 1) in that cell proteins are solubilized, fractionated by polyacrylamide gel electrophoresis (PAGE), and transferred electrophoretically (blotted) to a nitrocellulose or similar membrane. However, specific proteins can then be identified on the membrane by their ability to bind a particular ligand, rather than with a specific antibody as in immunoblotting. The ligand can be another protein or peptide, a nucleic acid or, indeed, any molecule that binds with high affinity and for which a detection system is available. So far, the method has been particularly useful for studies on membrane proteins, such as cell surface receptors, which can be difficult to assay in cell extracts.
Prokaryotic in vivo expression systems
Authors: Appelbaum Edward R , Shatzman Allan R
Published: November 2023

Bacterial expression systems are commonly used for production of heterologous gene products of both eukaryotic and prokaryotic origin. This chapter will discuss strategies and provide protocols for the expression of heterologous proteins in Escherichia coli, which is the bacterial system that is most widely and routinely used for this purpose. While many specialized vectors are available for gene expression in other Gram-negative and Gram-positive bacteria, such systems are usually employed for the genetic analysis and manipulation of specific organisms, rather than for high level expression of proteins to be purified and used for research. The emphasis of this chapter is on protocols that are suitable for producing proteins in the laboratory for research purposes. There are additional considerations in the selection of systems for large scale production (i.e. hundreds of litres or more of culture) and for production of proteins for clinical investigation or commercial use. In this chapter we briefly review some of the major factors to consider in selecting an E. coli expression strategy and provide protocols that have been successful in our laboratories in a variety of expression applications. Additional details and extensive literature citations are available in several recent reviews (1-6).
Genotnic imprinting in the mouse: possible final analysis
Authors: Cattanach B M , Beechey C V
Published: November 2023

The concept of a marking or imprinting of genes/ chromosomes in the germline such that maternal and paternal copies behave differently in the zygote has its routes in the Sciara and coccid insect work of Crouse (1), and Brown and Nelson-Rees (2), and has since been adopted to cover a number of seemingly related but diverse phenomena in various plant and animal species (3, 4).
Linkage mapping of plant and animal genomes
Authors: Miller Ross
Published: November 2023

The human genome project has provided increasingly detailed physical (1-3) and genetic maps (4). A large number of other genomes are also being mapped: animal genomes under study include those of mouse (5), rat (6), sheep (7), pig (8-10), cow (11,12), dog (13), chicken (14), the pufferfish Fugu (15), zebrafish (16), honey bee (17), Drosophila (18), and the nematode C. elegans (19), whilst plant genomes being studied include rice (20), wheat (21), maize (22), Arabidopsis (23), spruce (24), tomato (25) and potato (25). These lists are intended merely to illustrate the breadth of the field rather than to be exhaustive.
Genomic libraries and the development of species-specific probes
Authors: Warner J P
Published: November 2023

The development of standard methods for reliable cloning and propagation of DNA fragments of various sizes has transformed our understanding of gene structure and regulation. The ability to generate representative libraries of sequences comprehensively covering the genome regardless of size, complex ity, or type of organism has enabled population geneticists to undertake fine resolution analysis of sequence variation. The purpose of this chapter is to describe general protocols for the construction and screening of genomic libraries and for the development of species-specific probes. Although the construction of libraries was traditionally regarded as technically demanding, modern high-quality enzymes, commercially prepared packaging extracts, competent cells, and complete cloning kits have made genomic cloning universally accessible. A high quality representative library of clones avail able for rapid re-screening whenever new sequences suitable for population analysis are described is ample reward for the time and effort invested in the initial library construction.
PCR protocols and population analysis by direct DNA sequencing and PCR-based DNA fingerprinting
Authors: Hoelzel A R , Green A
Published: November 2023

The polymerase chain reaction (PCR) has become a mainstay of molecular ecology and population genetic research in the brief time since its invention (1). By this technique, defined segments of DNA can be amplified to micro gram quantities from as little as a single template molecule. Although the procedure is in some ways deceptively simple, and the reaction can entail complex biochemical interactions, it is in most applications a fast, relatively inexpensive and easy way to generate ample material for further analyses. For this reason its application is now widespread and facilitating an ever greater diversity of research. From early applications to previously intractable or difficult procedures, such as the pre-natal diagnosis of sickle-cell anaemia, PCR has now been applied to a great variety of problems in molecular biology, including cDNA cloning (2) and mutagenesis (3). It is a natural for population genetic studies where large numbers of individuals need be screened at as fine a resolution as is practical and feasible. The combination of PCR amplifica tion and DNA sequencing has been especially powerful for these studies.
Automated DNA detection with fluorescence-based technologies
Authors: yid Victor A Da , menotti-raymond Marilyn
Published: November 2023

The use of fluorescence-based technologies in DNA detection systems has truly come of age. Automated DNA sequencing instruments, which were once relegated to core sequencing facilities at a few large institutions, are now found at thousands of laboratories throughout the world. The capabilities of these instruments have been expanded to encompass new technical develop ments such as microsatellites (1,2), random amplified polymorphic DNA (RAPD) markers (3,4), variable number tandem repeats (VNTRs; 5) and single-strand conformational polymorphism (SSCP; 6). The amenability of this technology to automation has made it especially useful in large-scale sequencing and genotyping projects; however, high sample throughput can be accomplished without expensive robotic systems. DNA storage, polymerase chain reaction (PCR) amplification, DNA sequence reactions, and sample loading can all be accomplished in a format compatible with a standard 96-well plate. This allows for the use of multi-channel pipettes to facilitate sample processing and multi-channel syringes for sample loading. This chapter will assess current applications of fluorescence-based DNA detection systems with emphasis on the two most widely used applications, DNA sequence analysis and microsatellite genotyping.
Radiolabelled Liposomes for Imaging and Biodistribution Studies
Authors: Goins Beth A , Phillips William T
Published: November 2023

The development of liposome-based agents for diagnostic and therapeutic applications requires an understanding of the blood clearance kinetics and tissue bio-distribution of the agents after administration into the body. Non-invasive biodistribution studies of liposomes labelled with gamma (photon)-emitting radionuclides performed with scintigraphic imaging is a powerful approach for studying liposome-based agents in vivo. This methodology permits determination of the distribution of liposomes at multiple time points in an individual animal or patient (1, 2). While invasive studies of liposomes labelled with beta-emitting radionuclides such as carbon-14 may be suitable for animal studies, human studies with non-invasive photon imaging is a significantly more efficient approach for liposome-based drug development.
Comprehensive mutation detection with denaturing gradient gel electrophoresis
Authors: Lerman L S , Beldjord Cherif
Published: November 2023

Since the basic laboratory procedures for denaturing gradient electrophoresis and the introduction of G,C clamps have not changed significantly from previous descriptions, and they depart only slightly from conventional gel electrophoresis, we provide guidance and references to published complete instructions. However, the computational preparations for mutation searching are critical to efficient application of the method. We provide a detailed introduction to the computational tools, their use in planning a search, and their role in interpreting results.
Mutation detection using T4 endonuclease VII
Authors: Youil Rima
Published: November 2023

The ideal method for analysis of genes implicated in human disease would be a simple, easy to perform procedure that utilizes standard technology available in most laboratories. It should offer high throughput capacity even when performed manually. The procedure should also lend itself to automation to allow for increased throughput, particularly for laboratories that routinely handle large numbers of samples. The ability of the scanning technique to localize the point of mutation would also be highly advantageous.
DNA detection and sequence distinction through oligonucleotide ligation
Authors: Landegren Ulf , Samiotaki Martina , Kwiatkowski Marek , Parik Juri , Nilsson Mats , Hagberg, Anette , Barbany Gisela
Published: November 2023

Synthetic oligonucleotides and DNA ligases are combined in a number of methods for gene analysis. Generally, two oligonucleotide probes are designed to hybridize immediately next to each other on a target DNA or RNA strand. Ends of the oligonucleotide probes, brought close by hybridization, can be covalently joined by a DNA ligase, provided that the probes are properly base paired to the target sequence at the junction. This joining act thus refiects the presence of the target sequence. Below, a method will be presented to use oligonucleotide ligation-assisted analysis to distinguish known variants of amplified DNA sequences.
Calcium and pH imaging in living cells
Authors: Parton Richard M , Read Nick D
Published: November 2023

Calcium and pH imaging, involving the use of ion-sensitive fluorescent dyes, have had a profound impact on our understanding of signal transduction in animal, plant, and fungal cells. This chapter provides an introduction to the techniques involved in using the light microscope to image and measure intracellular free calcium and pH within living cells and tissues. ‘Second messengers’ are intracellular mediators which provide important links in signal-response coupling within cells and act in both signal propagation and amplification (1). The most common and ubiquitous second messenger within eukaryotic cells appears to be Ca2+ (2-4). The role of pH has received far less attention as a second messenger. Nevertheless, a variety of cellular responses to stimuli involve changes in cytoplasmic pH (5-8). In addition, pH homeostasis is important in regulating numerous other aspects of cell metabolism (9-11).
Analysis of macrophage lytic functions
Authors: Varesio Luigi , Bosco Maria Carla , Carta Luca , Musso Tiziana
Published: November 2023

Mononuclear phagocytes, comprising circulating monocytes and tissue macrophages, are phylogenetically ancient cells deriving from the primordial amoeba that was enclosed into a complex pluricellular organism. During the course of evolution, macrophages retained some of the basic features of the amoeba, such as mobility and phagocytosis, and acquired new immunoregulatory properties as a result of the interaction with theemerging immune system. Engulfing bacteria and fighting with competitors are among the primordial functional activities of macrophages that pre-existed the development of the immune system and that are triggered by classical environmental stimuli such as bacteria, toxins, organic molecules, pH, osmotic changes, etc. The need to communicate with the immune system and with the neighbouring cells caused profound modifications in the number and nature of the signals recognized by macrophages, that learned to recognize distress signals derived from lymphocytes, such as lymphokines and antibodies, or from tissues, such as hormones, adhesion molecules, and matrix proteins.
Crystallization of membrane proteins
Authors: Howard Tina D , Mcauley-Hecht Katherine E , Cogdell Richard J
Published: November 2023

As yet, unfortunately, there are no high-resolution structures for any membrane transporters. This probably represents the major barrier to our understanding the precise molecular details of how this important class of membrane proteins actually work. An excellent example of the urgent need for structural information comes from the extensive studies on the lac-permease from Escherichia coli (1). Almost every amino acid in this transporter has been changed by site directed mutagenesis (H. R. Kaback, personal communication) and yet the exact details of how it works are still a mystery. It has been argued that the crystal structure of a transporter may not be such a ‘holy grail’, since it will only give a static picture and transport is, by definition, a dynamic process. However, the recent success in understanding how ATP is made by examining the structure of the mitochondrial F1-ATP synthetase (2, 3) illustrates how, when a structure can be ‘caught’ in several functional states, the dynamics of the reaction can be inferred. Since for many transporters there are specific inhibitors which can ‘lock’ the protein in different functional conformations, there is an excellent chance that when the structure of a transporter is determined it will be possible to deduce the mechanism.
Cryopreservation and rederivation of embryos and gametes
Authors: Glenister Peter H , Rall william F
Published: November 2023

This chapter describes optimized cryopreservation protocols for the long-term storage and maintenance of pre-implantation embryos and gametes from mouse models of human genetic disease, and of normal biological processes. It outlines the theory and practical application of cryopreservation procedures and discusses the quality control requirements to ensure the availability of mouse models for future study. Comprehensive reviews of the history, theory and application of germplasm cryopreservation are available elsewhere (1,2).
Raising antibodies
Authors: Kane* M M , Bankst J N
Published: November 2023

Antibodies are essential components of immunoassays, and the versatility of immunoassays results from the unique features of these proteins. Antibodies can recognize and bind to their antigen with high affinity and specificity, even when they are part of a complex mixture, enabling sensitive measurements of antigens or antibodies in a variety of matrices.
Phosphorylation. site analysis by mass spectrometry
Authors: Mitchelhill Ken I , Kemp Bruce E
Published: November 2023

The determination of phosphorylation sites on proteins is a vital part of their characterization, and an important step in understanding the structural basis of how phosphorylation modulates their functions. With the recent explosive growth in the use of mass spectrometry in the structural analysis of proteins and peptides, this approach has also become the method of choice for the elucidation of post-translational modifications, including phosphorylation. Many developments have taken place since early studies using fast-atom bombardment mass spectrometry (FABMS) in 1985 (1). Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF), electrospray ionization (ESI) triple quadrupole mass spectrometry and its low flow rate version, nano-ESI mass spectrometry, are currently the most widely used forms of instrumentation in these studies. They are both sensitive and highly precise, and this chapter will be confined to these instrumentation types, with which we have practical experience. Hybrid instruments e.g. Q-TOF (quadrupole time-of-flight) are now available, or in advanced stages of development, from several manufacturers, as well as both electrospray and MALDITOF instruments with Fourier transform detection at the frontiers of precision. Other mass spectrometric analytical methodologies, including ion-trap methods, have been applied to phosphorylation site analysis. It is now essential for all laboratories involved in protein phosphorylation studies to have access to mass spectrometry if they are to contribute to understanding this pre-eminent regulatory mechanism.
Cloning and expression of cDNAs encoding protein kinase subunits
Authors: Carling David
Published: November 2023

Protein kinases form a large family of enzymes that play a key role in the control of a diverse variety of cellular processes, including the regulation of metabolic pathways, cell differentiation, and cell proliferation. Changes in the normal activity of protein kinases can lead to the development of a large number of different disease states. A critical step in our understanding of the precise regulatory role of protein phosphorylation within the cell, and the lesions that occur within this regulation, is the identification and characterization of all the protein kinases present in the organism. Whilst this may seem an ambitious goal at present, the rapid progress being made in several key areas of biology suggests that it will be achievable in the not too distant future. The completion of the Saccharomyces cerevisiae genome sequencing project has revealed that budding yeast contain 113 protein kinase genes, over half of which have known or predicted functions (1). Sequencing of the human genome is well underway, and it has been estimated that in humans there are at least 1000 genes encoding protein kinases (2), although in this case the percentage for which there is a known function is much smaller. Advances in gene cloning and manipulation have made analysis of mammalian genes, both in vitro and in vivo, a routine procedure. In order to elucidate the exact function of a specific protein kinase, isolation of the cDNAs encoding the protein kinase subunit(s) is an important and fundamental goal for the researcher. This chapter discusses the methods available for isolation of cDNAs encoding protein kinase subunits, including the new approach of ‘in silica’ cloning which has been made possible by the large-scale DNA sequencing projects currently being carried out around the world. Having isolated the cDNA, methods for expression of the protein kinase in heterologous systems are also described.
Crystallization of protein kinases and phosphatases
Authors: Kobe Bostjan , Gleichmann Thomas , Teh Trazel , Heierhorst Jörg , Kemp Bruce E
Published: November 2023

X-ray crystallography is the most powerful method for determination of macromolecular three-dimensional structures, and it has made enduring contributions to the understanding of the function of protein kinases and phosphatases. Determination of protein crystal structures requires single crystals that are suitable for high resolution X-ray diffraction analysis (Figure 1).Crystallization will only occur within a limited range of conditions that need to be empirically determined. Despite numerous successful crystallization experiments, our understanding of protein cry st allogenesis and macromolecular crystal growth processes remains limited. Crystallization of proteins can be broken down into two aspects: screening (based mainly on trial and error),where one attempts to obtain crystals; and optimization, where one strives to improve the diffraction quality of the crystals.
Kinetic analysis of nuclear receptor interactions
Authors: Cheskis B , Freedman L P
Published: November 2023

Steroid/nuclear receptors modulate transcription by virtue of their binding as part of a multiprotein complex to specific DNA sequences in promoter regions of target genes. This process directs a cascade of protein-protein and protein-DNA interactions affecting gene expression. Careful biochemical analysis or protein-protein and protein-DNA interactions is critical to the understanding of how genes are regulated by this family of ligand-modulated transcription factors. It is also a key to comprehending how small molecules. the cognate ligands for these receptors, affect receptor activity by directing the mode and amplitude of gene transcription.
Hormone-resistance syndromes
Authors: Gurnell M , Chatterjee V K K
Published: November 2023

The nuclear receptors comprise a superfamily of ligand-inducible transcription factor which exhibit a conserved structural organization with distinct functional domains. Members of this family include the glucocorticoid (GR), androgen (AR), oestrogen (ER). mineralocorticoid (MR), and progesterone (PR) receptors, together with those for thyroid hormone (TR), vitamin D (VDR). retinoids (RAK RXR). eicosanoids (PPAR), and an expanding number of ‘orphan· receptors whose ligand and function remain to be elucidated.
Bacteriology of the genital tract
Authors: Ison Catherine A , Davies Alun J , Hawkey Peter M
Published: November 2023

Whilst the diagnosis of genital infections by means of bacteriological examination is straightforward in those infections where an organism has a dear pathogenic role, such as is the case in gonorrhoea and syphilis, other infections are encountered where the contribution of diagnostic bacteriology is less dear. Culture of specimens from patients with genital infections may yield organisms such as Gardnerella spp., Listeria spp., and anaerobic bacteria, which, although capable of causing infections under suitable conditions, do not do so invariably. Unless an organism is generally recognized as a pathogen, its presence in large numbers does not provide evidence of pathogenicity.
Antimicrobial susceptibility testing
Authors: Brown Derek F J , Andrews Jenny , Winstanley Trevor , Alasdair P. MacGowan
Published: November 2023

In vitro antimicrobial susceptibility testing is undertaken in clinical laboratories to predict the likely in vivo response of the infecting organism to a selected range of antimicrobial agents. Susceptibility tests are designed to give a result interpreted as susceptible (sensitive. S), intermediate (moderately susceptible. moderately resistant, I), or resistant (R). A patient infected with a susceptible organism should respond to treatment with the manufacturer’s recommended dosage regimen. whereas one infected with a resistant organism is unlikely to respond. For an organism categorized as intermediate there is uncertainty whether or not the patient will respond to standard doses, but they will be more likely to respond to higher doses or if the agent is concentrated at the site of the infection, such as in the urinary tract. However, intermediate is a term which clinicians find unhelpful. and in practice the intermediate and resistant categories are combined and reported as resistant.
Quality control and quality assurance
Authors: Hyde W A , Curley P K
Published: November 2023

The late European Committee for Clinical Laboratory Standards produced a document on quality control for microbiology, which stated (1): “Internal quality control is the set of procedures undertaken by the staff of a laboratory for the continual evaluation of laboratory work and the emergent results, in order to decide whether they are reliable enough to be released to clinicians or epidemiologists.
Transition states in protein folding
Authors: Daggett Valerie , Fersht Alan R
Published: November 2023

Until the end of the 1980s the approach to protein folding concentrated on the gross characterization of folding intermediates and the refolding of well-known proteins such as bovine pancreatic trypsin inhibitor, ribonuclease A, and lysozyme. These studies utilized primarily circular dichroism and fluorescence, and chemical trap ping methods exploiting disulphide bridges. One of the major developments in the 1990s has been to focus on entire folding pathways at the level of individual residues and atoms. This became possible because of developments in three areas.
Protein folding coupled to disulphide-bond formation
Authors: Creighton Thomas E
Published: November 2023

Understanding in detail the mechanism of protein folding in vitro requires all the conformational states encountered during folding to be known, including the rate limiting transition state. On initiating refolding, different proteins adopt a wide variety of different conformations very rapidly, before folding to the native state. Some protein:-, remain unfolded, others adopt partly folded conformations, while many adopt a compact but disordered state that has come to be known as the ‘molten globule’ (MG) (1). These relatively stable partly folded and MG conformations are frequently assumed to be crucial for directing the folding process, and much effort has gone into characterizing them (Chapter 6). This is usually difficult to achieve, since they are present only transiently. Determining the kinetic roles of such intermediate species, as to whether they are on-pathway intermediates, kinetically trapped, or simply the initially preferred conformational state of the unfolded protein under refolding conditions, is especially difficult with a complex process like protein folding. There is little direct evidence about the kinetic roles of most folding intermediates that have been detected (2, 3).
Case study 1: The folding process of apomyoglobin
Authors: Wright Peter E , Baldwin Robert L
Published: November 2023

The folding of the globular protein apomyoglobin is characterized by well-populated intermediates which occur both transiently, as kinetic intermediates in folding, and at equilibrium near pH 4. If the structure of these intermediates could be determined, and if their relation to each other as part of a kinetic scheme could be rigorously established, then a significant contribution would have been made to understanding the folding process of this model protein. Further, characterization of these inter mediates as persistent conformational species on a folding pathway would constitute an important clement to bring to the ‘classical’ versus ‘new view” debate.
Case study 2: Folding of the collagen triple-helix and its naturally occurring mutants
Authors: Baum Jean , Brodsky Barbara
Published: November 2023

Collagen comprises a family of extracellular matrix molecules responsible for the integrity and mechanical properties of connective tissue, including bone, tendon, skin, cartilage, and cornea (1-3). At least 19 distinct human collagens are known, the most abundant being found in fibrils with an axial 67 nm periodicity. In addition, collagen like domains are crucial to ligand binding and self-association in host-defence proteins such as Clq, mannose-binding protein, and the macrophage scavenger receptor (4, 5). Mutations in collagen are the cause of various connective tissue diseases, including osteogenesis imperfecta and hereditary aortic aneurysm (6-8). Altered folding of the collagen molecule and of fibril assembly have been implicated in the aetiology of these diseases, putting collagen in the context of protein folding and aggregation diseases (9-11).
Protein degradation and proteinases in yeast
Authors: Hilt Wolfgang , Wolf Dieter H
Published: November 2023

Degradation of intracellular proteins has been proven to be essential for cellular life. Adjustment of protein levels by degradation initiated by extracellular or intracellular signals is a central mechanism to allow cells to adapt to changing environmental conditions or intracellular programmes. In eukaryotic cells two major proteolytic systems exist to exert these functions: the proteasome and the lysosome. Proteasomes are multisubunit protein complexes localized in the cytoplasm and nucleus of all eukaryotic cells (1-4). The 26S proteasome is a complex structure consisting of two 19S regulatory complexes and the 20S proteasome (Figure 1). The proteolytic activity of the 20S proteasome depends on an amino terminal threonine residue located in the inner space of the cylinder in three of the seven β-subunits (5).
Carbohydrateis in medicine
Authors: Mcauliffe Joseph C , Hindsgaul Ole
Published: November 2023

Renewed interest in the biology and chemistry of carbohydrates over the past two decades has dramatically accelerated the development of carbohydrate-based drugs (1--4). Specifically, the characterization and cloning of carbohydrate-processing enzymes, improved synthetic techniques and the ability to analyze cell-surface glycans have revealed the significant medical potential of this class of biomolecules (5-7). Currently, carbohydrate therapeutics are used to treat diabetes, epilepsy, thrombosis and arthritis, with further developments promising therapies for reperfusion injury, influenza and cancer. Carbohydrates are also being used to modify the pharmacological profile of other drugs, including recombinant proteins. Such hybrid molecules often display superior efficacy and reduced side-effects when compared with the parent drug. A variation on this theme involves the use of carbohydrates to specifically target drugs to certain cell types. The use of such carbohydrate-based agents for drug and gene delivery is being actively pursued, with several candidates already in clinical trials. This chapter will focus primarily on drug candidates that either are composed mainly of carbohydrates or are carbohydrate derived; however, the use of carbohydrates to modify and deliver other classes of drug will also be discussed.