Models and Methods in Call Signalling and Gene Expression: Application to Oxidative Stress ResearchMODELS AND METHODS IN CALL SIGNALLING AND GENE EXPRESSION: APPLICATION TO OXIDATIVE STRESS RESEARCH

Edited by Tammy Bray and Norberta Schoene
Published 2000, Hardback/Cloth, pages 200
OICA International: London
ISBN 1 903063 03 5

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Cat. No. 2003
Chapter 1: Assessment of oxidative stress: concepts and approaches
Chapter 2:

Air-liquid interface culture systems for exposure of differentiated cells to oxidative stress

Chapter 3: In vitro culture of differentiated human cells as models for , differentiated human cells as models of oxidant injury
Chapter 4: In vivo Fenton reaction model and application of immunohistochemistry to visualisation of oxidative damage
Chapter 5: Measurement of oxidative lesions in specific mitochondrial and nuclear DNA sequences
Chapter 6: Use of laser scanning confocal fluorescence microscopy for the detection of reactive oxygen species: ROS and viral gene regulation.
Chapter 7: Determination of nuclear factor ?B activity
Chapter 8: Application of the electrophilic mobility shift assay to study gene regulation and genomic disease
Chapter 9: Techniques for the detection of apoptosis as an endpoint of oxidant exposure
Chapter 10: Detecting ROS in cells using flow cytometry

Readership: Research and Development in Academia and Industry, Pharmacologists, Oncologists, Biochemists, Molecular Biologists, Physiologists. Final year undergraduate and graduate students in molecular biology will find this book invaluable.


The recent growth in knowledge of free radicals and reactive oxygen species (ROS) in biology is producing a medical revolution that promises a new age of health. In recent years, as our understanding of the involvement of ROS in the etiology of chronic disease has expanded, ROS research has led to a new paradigm of human health. Today there is a much greater emphasis placed on the early detection of oxidative stress and, through the use of antioxidants, development of preventive strategies to combat ROS-mediated diseases. This has created new interdisciplinary scientific fronts concerned not only with identifying the role of ROS in degenerative disease, but also with identifying factors that may regulate and/or combat ROS mediated tissue damage. This book is presented as an authoritative laboratory tool for those wishing to embark on research in free radical biology and medicine.

The airway epithelium is the body's first physiologic barrier to deleterious air-borne substances such as allergens, industrial particles, pollutants, and infectious agents. Air liquid interface (ALI) culture systems, developed to meet this goal for airway epithelial cells, allow differentiated cells to be continually exposed to air as they are in vivo. When airway epithelial cells are grown in ALI cultures, they continue to respond to oxidant stress and retain their antioxidant capabilities. Thus, ALI cultures are ideal for the study of cellular mechanisms involving reactive oxygen and nitrogen species (ROS/RNS) in airway epithelial cells. The book then discusses the techniques for the isolation and culture of human bronchial epithelial and human pleural or peritoneal mesothelial cells are described. These techniques utilize media developed specifically for each cell type and describe the initial isolation of cells from bronchi, pleural or peritoneal exudates.

Intraperitoneal injection of an iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage associated with lipid peroxidation and oxidative DNA damage that eventually leads to a high incidence (approximately 60 to 90 %) of renal cell carcinoma in rodents after repeated administration. This Fenton-chemistry model is extensively discussed. The alterations in the cellular genome introduced by these DNA-damaging agents can lead to errors in transcription, mutations, recombinations and rearrangements, or even gross chromosomal abnormalities. Resistance to these alterations in DNA transactions depends in large part upon a variety of enzymatic pathways for repairing lesions in DNA. The book discusses the advantages and limitations of three different methodologies (are qualitative Southern blot analysis, quantitative extended length PCR and ligation-mediated PCR) that have been utilized to evaluate the formation and repair of oxidative damage in specific nuclear and mitochondrial DNA sequences.

The book turns to the use of laser scanning confocal fluorescence microscopy, applications of the electrophoretic mobility shift assay to study gene regulation and genomic disease. Landmark scientific initiatives such as the Human Genome Project and recent successes in sequencing the full genome of other species have opened new doors to functional genomics. Although it is well established that genomic transcriptional activity is regulated by complex interactions between proteins known as Transcription Factors (TFs) and DNA sequences in promoter regions of genes, it is now becoming essential to understand how genes are expressed during complex biological processes and their precise mechanism of regulation. This understanding is essential as the loss of control of TF- mediated gene expression is associated with many genomic diseases. New insights arising from the characterization of the DNA binding activity of TFs within the chromatin structure and the regulatory regions of specific genes is driving the further study of TFs by both academia and private industry. By using appropriate techniques and developing new technologies to elucidate their precise function, the potential role of TFs in the correction and control of genomic diseases may be determined and subsequently applied towards future therapeutic approaches.

Questions related to apoptosis are likely to be raised for every system of oxidative stress in which cell death is seen. The first question is whether the oxidative stress is leading to death by apoptosis or necrosis. If the mode of death is apoptosis, the second question will likely be how much apoptosis is occurring. For example, an investigator may want to determine whether particular agents decrease the amount of apoptosis seen following an oxidant exposure. Finally, there is, perhaps, the most interesting question: What molecules in the cell sense the oxidative stress and how do they trigger the suicide program? This book provides an overview of techniques for addressing these questions.

In 1983, the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), was successfully used in tandem with flow cytometry to monitor the hydrogen peroxide generated in neutrophils. Information from this seminal report has been effectively extended and applied by many researchers to investigate intracellular ROS generation in many types of cells under differing conditions.

The approaches described in Models and Methods in Cell Signaling and Gene Expression Applications to Oxidative Stress Research can be easily modified to suit the specific research needs of the individual investigator. Pertinent references are supplied to facilitate integration of these techniques into experimental protocols to study the role of ROS signals within cells.

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