bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis and Interpretation of Binding at Equilibrium
Authors: Wells James W 
Published: November 2023

Abstract
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 experiments 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.
Lymphokine-activated killer cells: regulation of activity
Authors: Ortaldo J R 
Published: November 2023

Abstract
Interleukin-2 (IL-2) has been reported to play a major role in many types of immune responses. This lymphokine induces proliferation of T lymphocytes and cells mediating NK activity, as well as production of lFN-γ; it also results in the induction oflymphokine-activated killer (LAK) cells with cytotoxic activity against fresh tumour cell preparations and NK-resistant cell lines. Cells mediating LAK activity induced by IL-2 are potent cytotoxic cells that may have an important role in the immunotherapy of solid tumours.
Tumour escape mechanisms
Authors: Guillou P J 
Published: November 2023

Abstract
The introduction of interleukin-2 administration, with or without adoptive cellular immunotherapy of one form or another, represents a novel approach to the immunotherapy of several different tumour types. It is disappointing, therefore, that the almost universal success of this treatment in experimental animal tumour systems2 has not been parallelled by the clinical experience of this approach. Indeed, even with those tumour types such as malignant melanoma and hypernephroma which do appear to respond to IL-2 therapy, response rates of 20-30 per cent would seem to be the best that can be expected, others such as colorectal cancer seeming to fare less well (response rates of 17 per cent or thereabouts), and yet others not responding at all. There has been no recorded correlation between the degree of lymphocytotixicity generated in response to IL-2 and clinical response. This and other data suggest that the therapeutic effects of IL-2 may be indirect. While there is doubt about such fundamental issues as the mode of action oflL-2 and the necessity or otherwise of adoptive reinfusion of in vitro activated LAK, TIL or A-LAK cells, the design of further modifications of current therapeutic protocols involving IL-2 can only be empirical. This review aims to outline some of the mechanisms which tumours may exploit to subvert the possible immunological mechanisms of tumour cell lysis induced by IL-2. The assumption is made, perhaps unjustifiably, that IL-2 activated lymphocytotoxicity plays at least some part in the therapeutic effect, or is an epiphenomenon of the final common pathway whereby the effects are brought about.
Open paper and poster sessions
Authors: Gallagher G , Guillou P 
Published: November 2023

Abstract
After interleukin- I, interleukin-2 (IL-2) is arguably the most important immunological mediator, given its ability to support the growth and differentiation of many cell types, in addition to its activity as a T cell growth factor. While the bias of the short paper and poster sessions was towards the role of IL-2 in cancer treatment, other important aspects of this molecule’s function were described.
Isolation and characterization of ribonucleoprotein complexes
Authors: Lamond Angus I , Sproat Brian S 
Published: November 2023

Abstract
Detailed analyses of a wide variety of RNA processing reactions in vitro have shown that the processing machinery usually either comprises ribonucleoprotein (RNP) complexes or is dependent upon the formation of RNP complexes during the reaction mechanism. Major experimental goals for the analysis of RNA processing mechanisms therefore include.
Analysis of pre-mRNA splicing in yeast
Authors: Newman Andrew 
Published: November 2023

Abstract
Many simple but powerful techniques have been developed to investigate gene expression in Saccharomyces cerevisiae, and a number of these have been employed in the analysis of RNA splicing. Test substrates can be transcribed from expression cassettes introduced into yeast by transformation or transplacement, and splicing can be monitored by RNA analysis or assay of a suitable reporter gene product. Gene disruption and inducible expression systems have been invaluable for investigating the roles of components of the splicing machinery in yeast. Many of the genes for yeast splicing factors have recently been isolated by molecular cloning, which is facilitated by the compact nature of the Saccharomyces genome and the fact that these genes are present as single copies. Splicing of yeast mRNA precursors can also be studied in vitro, since for Saccharomyces a simple method has been developed for making cell-free extracts capable of splicing synthetic pre-mRNA substrates. Such studies have shown that the splicing pathway in yeast is very similar to that of higher eukaryotes and that there is considerable conservation of structure and function between splicing factors in yeast and mammalian cells. However, yeast is an extremely valuable system for the detailed analysis of splicing since it allows some approaches which are not possible with mammalian cells, particularly the genetic selection and isolation of splicing mutants.
Role of heat-shock proteins in steroid receptor function
Authors: Pratt William B 
Published: November 2023

Abstract
Because of their intrinsic interest as hormone-regulated transcriptional enhancers, the steroid receptors have attracted the curiosity of people with diverse backgrounds in biology. In recent years, the tools of modern molecular biology and protein chemistry have been used to define both steroid receptor primary structure and supramolecular structure. The demonstration that steroid receptors form stable interactions with heat-shock proteins (hsp) has contributed significantly to the understanding of steroid hormone action, and the study of receptor-associated proteins has evolved as a subfield of nuclear receptor research. It should not escape the notice of the general scientific community that this structural knowledge has made the receptor proteins themselves into powerful tools for probing the mechanisms of basic biological processes, such as protein folding, protein transport, and regulation of protein function. This chapter addresses the way in which the study of hsp-mediated receptor folding is leading to the development of some concepts that may have wide application in other biological systems.
The Telomere
Authors: Kipling David 
Published: November 2023

Abstract
Telomeres are specialised structures at ends of linear chromosomes. They are involved in a wide range of fascinating genetic phenomena ranging from novel biochemistry to a potential role in human ageing and cancer. This is the first book of its kind to cover this exciting and rapidly expanding field of molecular biology and genetics. Its aim is to introduce the concepts and central questions involved in the study of telomeres, along with the body of experimental data.
Telomere proteins
Authors: Kipling David 
Published: November 2023

Abstract
Telomere function requires not only telomeric DNA sequences but also various interacting proteins such as telomerase (reviewed by Zakian 1989; Blackburn 1991; Henderson and Larson 1991). Protecting a telomere from fusion and recombination may not be a direct result of the DNA sequence but rather a result of the action of telomere binding proteins. In S. cerevisiae the RAP1 protein, in concert with a range of other factors, is speculated to form a heterochromatin-like domain at the telomere, making the double-stranded terminus inaccessible to nucleases or recombination enzymes as well as ensuring that natural chromosome ends do not activate cell cycle checkpoints which monitor DNA damage (see Chapter 2). Other telomere functions which may well reflect the activity of telomere binding proteins include physical interactions with the nuclear envelope and telomere-telomere interactions. Although almost nothing is known of the molecular nature of the interactions with the nuclear envelope (discussed in Chapter 2) the role of telomere binding proteins in the interactions between ciliate telomeres has been studied in some detail.
Lysosomes and endocytosis
Authors: Gjøen Tor , Berg Trond Olav , Berg Trond 
Published: November 2023

Abstract
Endocytosis is a multistep process whereby molecules are internalized into cells and subsequently transported to one of four destinations: lysosomes, the opposite side of the (epithelial) cell, storage granules, or back to the plasma membrane from which internalization took place. The various steps in the endocytic process are mediated by specialized organelles. Internalization may take place inside or outside coated pits and therefore involves two different areas of the plasma membrane (1). Clathrin-dependent internalization is a well-known process whereas the structure of the plasma membrane that mediates clathrin-independent internalization is not known. Invagination of coated pits leads to coated vesicles from which ‘primary endosomes’ are formed. Clathrin-independent endocytosis leads directly to formation of smooth-surfaced primary endosomes. Primary endosomes carry their cargo to early endosomes that consist of tubules and luminal parts. In some cells the early endosomes may gradually develop into multivesicular bodies. This ‘maturation’ involves formation of internal vesicles and loss of tubular extensions (2). The early endosomes serve as a sorting station. Some endocytic receptors are returned to the plasma membrane in ‘receptosomes’ whereas others follow a transcytotic route to the opposite side of the cells. Some receptors follow the ligand to the lysosomes. In other cells the transport from early to late endosomes may not primarily be due to a maturation process. Instead, specific carrier vesicles may bud off from early endosomes and these are subsequently moved along microtubules to late endosomes (3). The mechanism whereby receptor-ligand complexes are transported from endosomes to lysosomes is not known. Moreover, lysosomes may exist in different states of activity, and functions previously ascribed to lysosomes (e.g. proteolysis), may certainly also take place in endosomes (4). In some cells, such as macrophages, the dense lysosomes may primarily serve as storage granules while the main degradation takes place in a prelysosomal, late endosomal compartment. Transport to lysosomes may be mediated by vesicles budding off from the prelysosomal compartment. In other cells, late endosomes may fuse with lysosomes.
The membranes of the secretory and exocytic pathways
Authors: Graham John M 
Published: November 2023

Abstract
This chapter is concerned primarily with isolation of plasma membrane, Golgi membranes, and endoplasmic reticulum from rat liver. The first sections concentrate on the preparative isolation of these three major membrane systems using routine centrifugation techniques, and by immunoaffinity and density perturbation techniques. The later sections are concerned with the use of centrifugation to analyse the events associated with exocytic or secretory pathways in other animal cells and in yeast, which in addition to the endoplasmic reticulum, Golgi membranes, and plasma membrane, involve a number of compartments including the trans-Golgi network and a variety of vesicle populations which shuttle between membranes and membrane domains.
Isolation and purification of functionally intact mitochondria and chloroplasts from plant cells
Authors: Moore Anthony L , Whitehouse David G 
Published: November 2023

Abstract
The photosynthetic higher plant cell is compartmentalized into organelles such as chloroplasts, mitochondria, peroxisomes, and vacuoles and is unique amongst higher plant cells in containing two bioenergetic organelles. The success and the survival of the plant cell depends upon the co-ordination and integration of the differing metabolic activities of the different organelles. Clearly the metabolic activities and roles of the organelles will vary not only with the developmental state of that cell but also with the type of cell, e.g. mesophyll, epidermal, or bundle sheath. Photosynthetic higher plant cell systems offer the opportunity to isolate both mitochondria and chloroplasts from the same cell at a similar stage of development, a factor of some importance when attempting to present a complete picture of the bioenergetics of that cell (1, 2). There is a large diversity of plant cells each with differing characteristics and developmental patterns and a complete description of the life cycle of a plant cell will only emerge when the integrated pattern of both metabolism and development is known. An answer to this question necessitates the isolation and purification of functionally intact organelles.
RNA-protein interactions in ribosotnes
Authors: Draper David E 
Published: November 2023

Abstract
In recent years, the functional role of ribosomal RNAs in protein synthesis has received a great deal of attention, as domains of highly conserved structure have been identified with the decoding site, the peptidyl transferase active centre, binding sites for the elongation factors, and other functional centres of the ribosome (1). Now that a large database of ribosomal protein sequences is available, it is also apparent that many of the ribosomal proteins are as highly conserved as the rRNAs, including many of the proteins which bind directly and independently to the RNA (2-4). The basic ribosomal machinery is probably an RNA-protein complex which traces its ancestry to the beginnings of biotic evolution.
RNA-protein interactions in the splicing snRNPs
Authors: Nagai Kiyoshi , Mattaj Iain W 
Published: November 2023

Abstract
In 1977, several groups made the striking discovery that some protein-coding eukaryotic genes are discontinuous, being interrupted by non-coding intervening sequences now known as intrans (1-3). The entire length of these genes, including the intrans, is transcribed into pre-mRNA. The intrans are subsequently excised within the nucleus to produce continuous protein-coding mRNA sequences prior to transport to the cytoplasm. This process is called pre-mRNA splicing and its molecular mechanism has been subject to intensive research. The establishment of in vitro splicing-assay systems using either nuclear extracts of HeLa cells (a trans formed human epithelial cell line) or yeast cell extracts has allowed detailed investigation of the catalytic mechanism as well as biochemical characterization of cellular components involved in this process (4-8). Yeast genetics has also facilitated identification of protein and RNA-splicing factors and characterization of inter actions between them (6-9).
Using peptides to study RNA-protein recognition
Authors: Frankel Alan D 
Published: November 2023

Abstract
In biology, as in other sciences, the study of a particular system often is made simpler if the system is first reduced to small components. In structural biology, studies of large macromolecules can be hampered if, for example a molecule has too many flexible segments that prevent it from crystallizing or if it exceeds the practical size limit for nuclear magnetic resonance (NMR), currently -20-30 kDa. To overcome these problems, structural biologists often try to identify small, stable subdomains that retain the essential characteristics of the larger system but behave well under NMR conditions or crystallize. Some types of proteins, such as transcription factors, seem particularly well-suited to this approach because many are constructed from small structural and functional domains that can be studied as isolated modules (1). The structures of several DNA-binding domains were solved only after the domains were localized to short polypeptides, typically &lt;100 amino acids, that bound to DNA with the same specificity as the intact proteins.
Footprinting and modification–interference analysis of binding sites on RNA
Authors: Merryman Chuck , F. Noller Harry 
Published: November 2023

Abstract
In its intracellular state RNA is usually complexed with proteins, other RNAs, and various small ligands. Chemical probing is a convenient and rapid approach that can be med to identify nucleotides that are involved in ligand binding. The two most common strategies employed in chemical probing are footprinting and modification-interference. In footprinting experiments, nucleotides that are protected from chemical attack in the presence of a bound ligand are identified. In modification-interference experiments, nucleotides whose modification prevents ligand binding are identified.
Purification Of Proteolytic Enzymes
Authors: Wilk Sherwin 
Published: November 2023

Abstract
Proteolytic enzymes are purified according to well established and extensively reviewed procedures for purification of proteins in general. However, there are aspects of the purification of some proteolytic enzymes which may require special attention. Many intracellular proteinases are under tight regulation to prevent uncontrolled cellular proteolysis. Tissue disruption may release potent inhibitors, which, if tightly bound, can pose serious problems in purification. In addition. unlike other proteins, proteinases are susceptible to autolysis. The first section of this chapter discusses issues that must be dealt with prior to initiating purification, and also discusses potential problems and pitfalls that are associated with the purification of this class of enzymes in particular. The second section of this chapter deals with the purification procedure in general. Particular attention is paid to affinity chromatography as it relates to proteolytic enzymes. The third section deals with optimizing the purification protocol and judging homogeneity.
Finding, Purification And Characterization Of Natural Protease Inhibitors
Authors: Nagase Hideaki , Salvesen Guy S 
Published: November 2023

Abstract
The activity of a proteolytic enzyme in a living organism is precisely regulated by synthesis and secretion of the enzyme, by zymogen activation and frequently by inhibition. Although some proteinase activities may be limited by very restricted substrate selectivity, or by specific cellular location {e.g. on the cell surface), most proteases are regulated by endogenous inhibitors. Thus, to understand the biological regulation of proteolysis, one must understand the role of endogenous inhibitors. In this chapter, we describe general approaches of how to discover, purify, and characterize endogenous protease inhibitors.
Using Proteinases For Edman Sequence Analysis And Peptide Mapping
Authors: Shannon John 
Published: November 2023

Abstract
When identifying or comparing proteins, it is usually necessary to analyze the component peptides, despite the availability of analytical techniques for intact proteins. One and two-dimensional electrophoresis, mass spectrometry, chromatography, N-and C-terminal sequencing and amino acid analysis all characterize proteins but if differences between proteins are small, the differences may be lost when analyzing a whole protein. However a difference of one amino acid between two peptides from a protein can be detected.
The DNA mobility shift assay
Authors: Dent C L , Smith, M D , Latchman D S 
Published: November 2023

Abstract
A knowledge of the DNA binding protein content of a cell or tissue type can be an important aid towards a more thorough understanding of the functional role of that tissue. Important processes, such as the development of an adult organism from the single-celled zygote and the maintenance of the developed structure and biochemical characteristics of tissues, are increasingly being demonstrated to be dependent on DNA binding transcription factors. The spectrum of transcription factors present determines what genes may be transcribed in a cell type, including genes that encode further transcription factors. The expression of a single gene may be determined by a number of different transcription factors, and the ratio of their concentrations may be vital, particularly if competing positive and negative factors are involved. Factors other than those that bind the DNA may be involved, such as protein cofactors, metal ions, and ligand molecules, and a further important factor is the chromatin structure of the DNA. The gene must be available for binding of factors and subsequent transcription.
Analysis of transcriptional control in RNA virus infection
Authors: Makino S 
Published: November 2023

Abstract
This chapter will illustrate several commonly used methods for the analysis of mRNAs and their transcriptional control by RNA viruses. I will first describe the conventional methods for characterizing mRNAs in virus-infected cells. Subsequently, methods utilizing genetically engineered virus RNA or cDNA with or without reporter genes will be described. These methods enable the analysis of cis- and trans-acting RNA sequences to be determined.
Analysis of RNA virus quasispecies
Authors: Ball Jonathan 
Published: November 2023

Abstract
A virus quasispecies can be thought of as a complex population of genetically related yet distinct variants (1). The term has been used to describe populations of a number of RNA viruses, e.g. human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). Although this definition of a quasispecies appears simple in theory, in practice it can be rather ambiguous (2). For example, the term has been applied to describe unrelated viruses present in different individuals, unrelated viruses arising from multiple infections within the same individual, and to genetically related but distinct variants co-circulating within the same individual and arising from a single infection. It is probably less confusing to use the term to describe the virus population co-existing within a single individual.
Reporter gene systems for the study of G protein-coupled receptor signal transduction in mammalian cells
Authors: Rees Stephen , Brown Susan , Stables Jenny 
Published: November 2023

Abstract
Traditional protocols for the measurement of G protein-coupled receptor (GPCR) signal transduction in mammalian cells have relied upon a number of biochemical techniques. These include the measurement of the rate of guanine nucleotide exchange at the activated G-protein alpha subunit (1), the measurement of the changes in the level of one of a plethora of intracellular second messenger metabolites, such as cAMP, calcium, or inositol phosphates (2), or the activation or inhibition of an ion channel (3). In recent years these assays have been supplemented by the development of reporter gene systems for the study of GPCR signal transduction (4-5). In this chapter we shall describe a number of such reporter gene systems, we shall outline the advantages of reporter genes over standard biochemical analyses, and we shall describe a number of protocols developed for the study of GPCR signal transduction using reporter gene systems.