Experientia supplementum (2012)最新文献

Understanding the chemical mechanisms by which drugs and drug metabolites interact with cells of the immune system is pivotal to our knowledge of drug hypersensitivity as a whole.In this chapter, we will discuss the currently accepted mechanisms where there is scientific and clinical evidence to support the ways in which drugs and their metabolites interact with T cells. We will also discuss bioanalytical platforms, such as mass spectrometry, and in vitro test assays such as the lymphocyte transformation test that can be used to study drug hypersensitivity; the combination of such techniques can be used to relate the chemistry of drug antigen formation to immune function. Ab initio T cell priming assays are also discussed with respect to predicting the potential of a drug to cause hypersensitivity reactions in humans in relation to the chemistry of the drug and its ability to form haptens, antigens and immunogens in patients.

This year marks the 30th anniversary of the first description of the cellular distribution of actin within a yeast cell. Since then advances in both molecular genetics and imaging technologies have ensured research within these simple model organisms has blazed a trail in the field of actomyosin research. Many yeast proteins and their functions are functionally conserved in human cells. This, combined with experimental speed, minimal cost and ease of use make the yeasts extremely attractive model organisms for researching diverse cellular processes, including those involving actomyosin. In this chapter, current state-of-the-art fluorescence methodologies being applied to yeast actomyosin research, together with an honest appraisal of their limitations, such as the pitfalls that should be considered when fluorescently labelling proteins interacting within a dynamic cytoskeleton, will be discussed. Papers describing the established techniques developed for yeast localisation studies will be highlighted. This will provide the reader with an informed overview of the arsenal of imaging techniques available to the yeast actomyosin researcher and encourage them to consider novel ways these simple unicellular eukaryotes could be used to address their own research questions.

DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes and accompany RNA molecules throughout their cellular life. Conformational changes in the helicase core of DEAD-box proteins are intimately linked to duplex unwinding. In the absence of ligands, the two RecA domains of the helicase core are separated. ATP and RNA binding induces a closure of the cleft between the RecA domains that is coupled to the distortion of bound RNA, leading to duplex destabilization and dissociation of one RNA strand. Reopening of the helicase core occurs after ATP hydrolysis and is coupled to phosphate release and dissociation of the second RNA strand.Fluorescence spectroscopy provides an array of approaches to study intermolecular interactions, local structural rearrangements, or large conformational changes of biomolecules. The fluorescence intensity of a fluorophore reports on its environment, and fluorescence anisotropy reflects the size of the molecular entity the fluorophore is part of. Fluorescence intensity and anisotropy are therefore sensitive probes to report on binding and dissociation events. Fluorescence resonance energy transfer (FRET) reports on the distance between two fluorophores and thus on conformational changes. Single-molecule FRET experiments reveal the distribution of conformational states and the kinetics of their interconversion. This chapter summarizes fluorescence approaches for monitoring individual aspects of DEAD-box protein activity, from nucleotide and RNA binding and RNA unwinding to protein and RNA conformational changes in the catalytic cycle, and illustrates exemplarily how fluorescence-based methods have contributed to understanding the mechanism of DEAD-box helicase-catalyzed RNA unwinding.

T lymphocytes are instrumental in the prevention of infections. With their antigen-specific T cell receptor (TCR), these cells recognize short peptides in the peptide-binding groove on MHC molecules of antigen-presenting cells. However, conventional T cells can also recognize non-peptide antigens including carbohydrates, phosphate groups, organic chemicals, and metal ions. The molecular basis of the interaction of TCR with these structures in the context of MHC has been partly solved. Organic chemicals and carbohydrates are recognized when bound to MHC-associated peptides, whereas metal ions are recognized due to their ability to form non-covalent coordination bonds with MHC molecules, bound peptides, and TCR. Peptide-independent metal ion recognition has also been described.

Studying the dynamics of the interaction between actin and myosin and how this is modulated by ATP and other nucleotides is fundamental to any understanding of myosin motor protein activity. The fluorescent label pyrene, covalently attached to actin (at Cys 374), has been one of the most useful optical probes to report myosin binding to actin. The unique spectral features of pyrene make it sensitive to changes in the microenvironment of the probe and allow to monitor processes such as conformational changes and protein-protein interactions. Here we describe how to make and use pyrene-labelled actin and describe a set of fluorescence stopped-flow measurements that allow the actin-myosin interaction to be explored at protein concentrations from μM to nM for many of the known myosin motors.

Reagentless biosensors are single molecular species that report the concentration of a specific target analyte, while having minimal impact on the system being studied. This chapter reviews such biosensors with emphasis on the ones that use fluorescence as readout and can be used for real-time assays of concentration changes with reasonably high time resolution and sensitivity. Reagentless biosensors can be designed with different types of recognition elements, particularly specific binding proteins and nucleic acids, including aptamers. Different ways are described in which a fluorescence signal can be used to report the target concentration. These include the use of single, environmentally sensitive fluorophores; FRET pairs, often used in genetically encoded biosensors; and pairs of identical fluorophores that undergo reversible stacking interactions to change fluorescence intensity. The applications of these biosensors in different types of real-time assays with motor proteins are described together with some specific examples. These encompass regulation and mechanism of motor proteins, using both steady-state assays and single-turnover measurements.

Molecular machines are the workhorses of the cell that efficiently convert chemical energy into mechanical motion through conformational changes. They can be considered powerful machines, exerting forces and torque on the molecular level of several piconewtons and piconewton-nanometer, respectively. For studying translocation and conformational changes of these machines, fluorescence methods, like FRET, as well as "mechanical" methods, like optical and magnetic tweezers, have proven well suited over the past decades. One of the current challenges in the field of molecular machines is gaining maximal information from single-molecule experiments by simultaneously measuring translocation, conformational changes, and forces exerted by these machines. In this chapter, we describe the combination of magnetic tweezers with single-molecule FRET for orthogonal simultaneous readout to maximize the information gained in single-molecule experiments.

Motor proteins are multi-potent molecular machines, whose localisation, function and regulation are achieved through tightly controlled processes involving conformational changes and interactions with their tracks, cargos and binding partners. Understanding how these complex machines work requires dissection of these processes both in space and time. Complementing the traditional ensemble measurements, single-molecule assays enable the detection of rare or short-lived intermediates and molecular heterogeneities, and the measurements of subpopulation dynamics. This chapter is focusing on the fluorescence imaging of single motors and their cargo. It discusses what is required in order to achieve single-molecule imaging with high temporal and spatial resolution and how these requirements are met both in vitro and in vivo. It also presents a general overview and applied examples of the major single-molecule imaging techniques and experimental assays which have been used to study motor proteins.

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

Beta-lactam antibiotics (BLs) are the most frequent cause of hypersensitivity reactions mediated by specific immunological mechanisms, with two main types, IgE reactions or T-cell-dependent responses. From a practical point of view, these reactions can be classified into immediate, for those appearing within 1 h after drug intake, and non-immediate, for those appearing at least 1 h after and usually within 24 h of BL administration. The clinical symptoms differ according to this classification. Urticaria and anaphylaxis are the most frequently recorded symptoms in immediate reactions and maculopapular exanthema and delayed urticaria in non-immediate reactions. Although the exact diagnostic approach differs depending on the underlying mechanism, it is based on the performance of skin testing, laboratory tests, and drug provocation tests.T cells are a key factor in all types of hypersensitivity reactions to BLs, regulating both IgE production or acting as effector cells, with a different profile of cytokine production. A Th1 pattern is observed in both CD4(+) and CD8(+) peripheral T cells in non-immediate reactions, whereas a Th2 pattern is expressed in CD4(+) T cells in immediate reactions.