bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidant‐Induced Regulation of Glutathione Synthesis
Authors: Liu Rui‐Ming , Choi Jinah , Forman Henry J. 
Published: June 2004

AbstractThis unit describes protocols for characterizing the expression of two glutathione biosynthesis enzymes: gamma‐glutamylcysteine synthase (GCS) and gamma‐glutamyl transpeptidase (GGT) in response to oxidants. GCS catalyzes the first and rate‐limiting step of glutathione synthesis, while GGT degrades extracellular glutathione (GSH) to provide the amino acids required for intracellular synthesis of GSH. Northern blot hybridization is used to quantitatively assess the mRNAs for the two enzymes in response to oxidant. Nuclear run‐on is used to determine the rate of transcription.
Measurement of Thioredoxin and Thioredoxin Reductase
Authors: Arnér Elias S.J. , Holmgren Arne 
Published: June 2004

AbstractThe thioredoxin system is ubiquitous, providing reducing equivalents to essential biosynthetic enzymes like ribonucleotide reductase. It is essential for cellular redox regulation, control of oxidative stress, and protection against oxidative damage. This unit includes protocols for measuring thioredoxin or thioredoxin reductase in biological preparations or as purified enzymes.
The Heme Biosynthesis Pathway and Clinical Manifestations of Abnormal Function
Authors: Phillips John D. , Kushner James P. 
Published: June 2004

AbstractBiosynthesis of heme is important for both prokaryotes and eukaryotes. The enzymes for this multistep process are distributed between the cytosol and mitochondria in eukaryotes. In humans there are inherited and acquired disorders characterized by over synthesis of one or more enzymes or absence of an enzyme. This overview discusses each enzyme in the heme biosynthesis pathway.
Measurement of Heme Concentration
Authors: Sinclair Peter R. , Gorman Nadia , Jacobs Judith M. 
Published: June 2004

AbstractHeme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P‐450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed‐phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.
Measurement of δ‐Aminolevulinate Dehydratase Activity
Authors: Fujita Hiroyoshi 
Published: June 2004

AbstractVertebrate ALAD is a cytosolic enzyme that catalyzes the second step of porphyrin formation, and it is expressed abundantly in liver and erythroid tissues as well as in other tissues. This unit describes a colorimetric assay for measuring ALAD activity in mammalian blood or tissues and the extent of reactivation of the enzyme in lead‐inhibited samples.
Measurement of Ferrochelatase Activity
Authors: Taketani Shigeru 
Published: June 2004

AbstractFerrochelatase is the terminal enzyme in the heme biosynthesis pathway. Under anaerobic conditions it catalyzes the insertion of ferrous iron into the protoporphyrin IX ring to form protoheme. In the absence of iron and under aerobic conditions, the enzyme will use zinc or mercury as a substitute. The assay described in this unit uses zinc under aerobic conditions and the artificial substrate mesoporphyrin to analyze the formation of zinc‐mesoporphyrin by HPLC with UV or fluorescence detection.
Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrofluorometry
Authors: Parsons Patrick J. 
Published: June 2004

AbstractQuantification of the level of erythrocyte protoporphyrin can be used as a screening assay for lead exposure. In this method, porphyrins and heme compounds are extracted from whole blood using ethyl acetate, and porphyrins are then separated from heme by back‐extraction with HCl solution. The extracted porphyrins are then quantified using a spectrofluorometer calibrated with protoporphyrin IX standard solutions.
Detection of Heme Oxygenase Activity by Measurement of CO
Authors: Vreman Hendrik J. , Stevenson David K. 
Published: June 2004

AbstractHeme oxygenase (HO) is the first and rate‐limiting step in degradation of heme, and in the presence of NADPH‐cytochrome P‐450 reductase it produces equimolar amounts of biliverdin and CO. CO produced in a closed system can be quantified as described in this unit by gas chromatography as a measure of HO activity in tissue slices, tissue homogenates, and tissue fractions.
Histochemical Analysis of Heme Degradation Enzymes
Authors: Ewing James F. 
Published: June 2004

AbstractHistochemical analysis can be used to study the cellular localization, expression, and regulation of enzymes responsible for catabolism of the heme molecule. This unit describes protocols for assessing the expression and regulation of specific HO‐1 and HO‐2 mRNAs by in situ hybridization using digoxigenin‐tagged probes. Immunohistochemistry is used to visualize specific cellular sites of HO‐1 and HO‐2 protein expression.
Quantitation of Human Heme Oxygenase (HO‐1) Copies by Competitive RT‐PCR
Authors: Abraham N.G. , Shenouda S.K. , Goodman A. 
Published: June 2004

AbstractThe authors have developed a system for quantifying human HO‐1 mRNA in samples limited in cell number and/or mRNA copies. Total RNA from human liver was used to develop the system. The RNA is reverse transcribed and amplified by PCR in a tube also containing an internal standard obtained by deleting 50 base pairs from the original human gene. After amplification the two templates are resolved and quantified. When the internal standard is present in the reaction mixture, the ratio of amplified sample to internal standard is proportional to the amount of sample RNA making it possible to calculate the number of specific mRNA molecules.
Purification and Characterization of Heme Oxygenase
Authors: Wilks Angela 
Published: June 2004

AbstractHigh‐yield expression and purification of human heme oxygenase isozyme 1 provided the breakthrough in characterizing the protein from mechanistic and structural standpoints. This unit provides a protocol for high‐level expression and subsequent purification of HO‐1. The commentary includes a discussion of subsequent biochemical and biophysical characterizations.
Assay of Tissue Activity of Nitric Oxide Synthase
Authors: Schmidt Kurt , Mayer Bernd 
Published: June 2004

AbstractNitric oxide synthase activity can be assayed by a variety of methods. The simplest method to assay activity is the conversion of radiolabeled L‐arginine to L‐citrulline. The assay described in this unit is sensitive, simple to perform, is relatively robust, and can be performed with intact monolayer or suspension cells, or with cell or tissue extracts. The product is easily separated from the reaction by cation‐exchange chromatography.
Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites
Authors: Wink David A. , Kim Sungmee , Miles Allen , Jourd'heuil David , Grisham Matthew B. 
Published: June 2004

AbstractRecently fluorometric techniques have been developed to determine NO and S‐nitrosothiols with improved sensitivity over spectrophotometric techniques based on similar chemistry. This unit contains protocols for detection and quantification of NO from chemical reactions and from cellular systems, and of S‐nitrosothiols in chemical, biochemical, and cellular experiments.
Immunocytochemical Analysis of Cyclic Nucleotides
Authors: deVente J. , Steinbusch H.W.M. 
Published: June 2004

AbstractVisualization of second messenger molecules is a powerful approach to study the role of second messengers in relation to the (sub)cellular localization and/or kinetics of the second messenger response. This unit describes the appropriate fixation procedures for the cyclic nucleotides cAMP and cGMP and the use of specific antibodies to localize these molecules.
Testing for Organophosphate‐Induced Delayed Polyneuropathy
Authors: Moretto Angelo 
Published: June 2004

AbstractOrganophosphorous compounds may cause two distinct types of toxicity: acute cholinergic toxicity and organophosphate‐induced delayed polyneuropathy (OPIDP). The ability of a compound to cause OPIDP is assessed as described by administering the compound to hens and screening the brain, spinal cord, and peripheral nerves for neuropathy target esterase activity to detect OPIDP and acetylcholinesterase activity to rule out the acute toxicity. This assay can also be used as part of a screen for protective agents.
Neurobehavioral Testing in Humans
Authors: Anger W. Kent , Rohlman Diane S. , Storzbach Daniel 
Published: June 2004

AbstractThe evaluation of the effects of chemical exposures on humans is a worldwide concern, and most chemicals have not been evaluated for neurotoxic effect. Human neurobehavioral research or clinical evaluations of populations exposed to chemicals must be carefully planned and structured. This unit describes the steps required to create such a study, select the appropriate measures, and evaluate the results.
Development of an In Vitro Blood‐Brain Barrier
Authors: Janigro Damir , Stanness Kathe A. , Soderland Carl , Grant Gerald A. 
Published: June 2004

AbstractThe blood‐brain barrier represents a significant protective barrier for the passage of pathogens and toxicants, but it is difficult to study in vivo. This unit describes an in vitro system that can be used as a model for the blood‐brain barrier to study its functions in an accessible format.
Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures
Authors: Allen Jeffrey W. , Mutkus Lysette A. , Aschner Michael 
Published: June 2004

AbstractThere is increasing interest in the role of astrocytes as mediators of neurotoxicity. This unit describes a method for preparing astrocyte cultures of greater than 95% purity by enzymatic dissociation from neonatal rat brain. These preparations have both high yield and high viability.
Analytical Cytology: Applications to Neurotoxicology
Authors: Catlin Michelle C. , Guizzetti Marina , Ponce Rafael A. , Costa Lucio G. , Kavanagh Terrance J. 
Published: June 2004

AbstractThis unit describes methods for analyzing the effects of neurotoxicants on cell cycle regulation by dual parameter flow cytometry and on cell signaling by quantifying intracellular calcium concentrations within individual cells by scanning confocal laser microscopy or using the fluorescent calcium probe fluo‐3.
Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator
Authors: Charleston Jay S. 
Published: June 2004

AbstractThis unit describes techniques, based on recent advances in stereological methods, to obtain unbiased estimates of total cell or synapse number in discrete structures of the central nervous system. They combine unbiased counting frames, unbiased systematic random sampling, and unbiased estimates of the structure volume to produce the final estimate of number.
Measurement of Glial Fibrillary Acidic Protein
Authors: O'Callaghan James P. 
Published: June 2004

AbstractToxicant‐induced injury of the adult or developing central nervous system results in hypertrophy of astrocytes at the site of injury. The hallmark of this response is enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acid protein (GFAP). A protocol is provided for assaying GFAP levels in detergent homogenates of brain tissue using a sandwich ELISA.
Aggregating Neural Cell Cultures
Authors: Honegger Paul 
Published: June 2004

AbstractAggregating Neural Cell Cultures (Paul Honegger, University of Lausanne, Lausanne, Switzerland). When freshly dissociated embryonic tissues are kept under gyratory agitation, the cells aggregate to form three‐dimensional spheroids in which the cells can migrate and organize themselves, attaining maximal cellular differentiation after weeks of culture. The three‐dimensional architecture of the aggregates permits direct cell‐to‐cell interactions and the formation of a natural cell matrix, which is fundamental to the acquisition of the histotypic properties of the aggregates. This unit describes protocols for preparing forebrain cells from embryonic rodents for aggregating cultures and maintaining these cultures to the differentiated state.
Coculturing Neurons and Glial Cells
Authors: Viviani Barbara 
Published: June 2004

AbstractThe close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron‐glia and microglia‐astrocyte sandwich cocultures, in which the selected cell populations grow on two different surfaces. This system has the advantage that the two cell populations can be separated at any point in the exposure without disrupting the cells
integrity and organization
Organ Culture of Midfacial Tissue and Secondary Palate
Authors: Abbott Barbara D. 
Published: June 2004

AbstractPalatal organ culture provides an in vitro model for the study of the formation of the secondary palate, which forms the roof of the mouth in the developing fetus. The protocol describes the steps for culture of the mid‐facial region of the fetal mouse or rat. In culture the secondary palatal shelves proceed through stages of growth, elevation and fusion in a manner analogous to that occurring in utero. This model provides a tool for studies of mechanisms of normal and abnormal palatogenesis and has applications for developmental biology and toxicology.
Small Animal Models of Hemorrhagic Shock–Induced Liver Dysfunction
Authors: Bauer Inge , Bauer Michael 
Published: June 2004

AbstractBlood loss and ensuing circulatory failure continues to be the leading cause of death between the ages of 1 and 45 in most western countries. Hepatocellular dysfunction is a key feature of the pathophysiological sequelae of trauma/hemorrhage in patients surviving the first hours and hemorrhagic hypotension has been widely used as a model for indirect liver injury in the critically ill. This unit describes a low‐flow ischemia model of the liver due to hemorrhagic hypotension that can be used to produce a reproducible and graded injury to the pericentral region of the acinus, which is critical for metabolism of xenobiotics. This protocol allows for measurement of cardiac output and regional flow.
Guidelines for Mating Rodents
Authors: Tyl Rochelle W. 
Published: June 2004

AbstractGuidelines for Mating Rodents (Rochelle W. Tyl, Research Triangle Institute, Research Triangle Park, North Carolina). Mating is an important part of assessing the effects of toxicants on the reproductive tract. This unit describes the set up of mating pairs, determining the success of mating, and assessing the various outcomes of a successful mating to evaluate reproductive function.
Histopathology of the Male Reproductive System I: Techniques
Authors: Creasy Dianne M. 
Published: June 2004

AbstractHistopathology of the Male Reproductive System: Techniques (Diane Creasy, Huntingdon Life Sciences, East Millstone, New Jersey). Fixation and sampling of the male reproductive system for routine histopathological examination requires special procedures including the use of nonroutine fixatives and nonroutine stains. High‐resolution light microscopy and electron microscopy require additional specialized techniques including perfusion fixation and plastic embedding. Aspects of comparative anatomy are also important for ensuring that specific structures within the reproductive tract are sampled adequately in various species. This unit provides methodology and guidance on what structures to sample and how to fix and prepare tissues from the male reproductive tract for routine and special investigations.
Histopathology of the Male Reproductive System II: Interpretation
Authors: Creasy Dianne M. 
Published: June 2004

AbstractHistopathology of the Male Reproductive System II: Interpretation (Diane Creasy, Huntingdon Life Sciences, East Millstone, New Jersey) Histopathology is acknowledged as the most sensitive endpoint for detecting testicular toxicity. However, identification and interpretation of chemically induced changes in the testis require fundamental knowledge of spermatogenesis, its dynamics and regulation. Changes in the rest of the reproductive tract are also frequently inter‐related, being the result of or cause of disturbance in testicular spermatogenesis. This unit provides practical guidelines on how to evaluate testicular histopathology and how to utilize staging in a qualitative evaluation. It also provides an overview of the most common chemically induced lesions and their potential significance with respect to etiology and functional consequences.
Epididymal Sperm Count
Authors: Wang Yefan 
Published: June 2004

AbstractMaking Epididymal Sperm Counts (Yefan Wang, TherImmune Research Corporation, Gaithersberg, Maryland). Epididymal sperm counts are a widely used, simple and sensitive method of assessing the effects of male reproductive toxicants on the epididymal and/or testicular site of action. After careful dissection of the tissues and further processing, the sperm suspensions are counted using a hemacytometer and analyzed for effect.
Formation and Functions of Protein Sulfenic Acids
Authors: Poole Leslie B. 
Published: June 2004

AbstractProtein sulfenic acids are generated as reversibly oxidized cysteinyl residues formed upon reaction of thiols with peroxides, nitric oxide, peroxynitrite, and other reactive oxygen or nitrogen species. They can be stabilized within the protein environment, irreversibly oxidized to sulfinic and sulfonic acids by additional oxidant, condensed with protein or exogenous thiol groups to form disulfide bonds, or directly reduced back to thiols. Sulfenic acids in proteins can act as intermediates in redox catalysis or as critical components in cysteine‐dependent redox regulation.
Local Lymph Node Assays
Authors: Dearman Rebecca J. , Kimber Ian 
Published: June 2004

AbstractThe murine local lymph node assay (LLNA) allows identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. In this test, contact allergens are identified as a function of events occurring during the induction phase of skin sensitization; specifically, stimulation of proliferative responses in draining lymph nodes measured as a function of radiolabeled thymidine incorporation in situ. Two methods are presented. In one, lymph nodes are pooled on an experimental group basis, while in the other, lymph nodes of individual animal are pooled, which enables statistical analyses. These methods have been developed for contact sensitization hazard assessment and provide a robust and reliable method as an alternative to guinea pig tests which measure skin sensitizing potential as a function of challenge‐induced skin reactions in previously sensitized animals. It is also possible to use LLNA data for measurement of relative potency in terms of risk assessment.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractMammalian cells in culture are a valuable source of experimental material and/or reagents for many toxicological studies. This unit provides basic information on aseptic technique, preparation of medium, subculturing monolayer cells, passaging suspension cells, freezing and storing monolayer and suspension cells, thawing and recovering cultures, and determining viable cell number.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M. 
Published: June 2004

AbstractThe polymerase chain reaction (PCR) is used to enzymatically amplify small quantities of specific DNA sequences. The reaction must optimized to specifically amplify the sequences and primers of interest; this includes titration of magnesium chloride and selection of enhancing agents, if appropriate, to minimize nonspecific primer target interactions and maximize the specificity, sensitivity, and yield.
Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
Authors: Voytas Daniel , Ke Ning 
Published: June 2004

AbstractThis unit provides procedures for detecting an quantifying radiolabeled proteins in SDS‐polyacrylamide gels or blots. Autoradiography requires fixed, dried gels before exposure to X‐ray film. The results may be enhanced by using intensifying screens and/or preflashing the film. Alternatively fluorography with organic scintillant‐impregnated gels may be used to enhance signal. The autoradiogram may be quantified by densitometry. Phosphorimaging is a useful alternative to film exposure.
Spectrophotometric and Colorimetric Determination of Protein Concentration
Authors: Simonian(spectrophotometric methods) Michael H. , Smith(colorimetric methods) John A. 
Published: June 2004

AbstractSpectrophotometric and Colorimetric Determination of Protein Concentration (Michael H. Simonian, Beckman‐Coulter, Inc., Fullerton, California, and John A. Smith, University of Alabama at Birmingham, Birmingham, Alabama). Measuring absorbance at 280 nm (A280) is one of the oldest methods for determining protein concentration. This method is still widely used because it is simple and does not require incubating the sample with exogenous chromophores. Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A205) is more universally applicable. Furthermore, the absorptivity for a given protein at 205 nm is several‐fold greater than that at 280 nm. Thus, lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm. The aromatic amino acids also exhibit fluorescence emissions when excited by light in the UV range. Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information. However, with a protein standard whose aromatic amino acid content is similar to that of the sample, intrinsic fluorescence can be used for quantitation. The most frequently employed colorimetric methods for determining protein concentration are those of Bradford and Lowry.
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah M. , Titus Julie A. , Zumstein Louis 
Published: June 2004

AbstractConventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small‐molecule) composition of a macromolecule‐containing solution. The solution to be dialyzed is placed in a sealed dialysis membrane and immersed in a selected buffer; small solute molecules then equilibrate between the sample and the dialysate. Concomitant with the movement of small solutes across the membrane, however, is the movement of solvent in the opposite direction. There are several simple and relatively inexpensive methods for concentrating protein solutions. Dialysis against Aquacide 11A (Calbiochem), which removes water through the dialysis tubing, may be used. After concentration, the solution must be redialyzed into the appropriate buffer. Another method is to use Immersible‐CX Ultrafilters (Millipore) which, when connected to a vacuum, remove everything below their molecular weight cutoff (MWCO). Alternatively, centrifugal concentrators, which are operated with the aid of ordinary laboratory centrifuges may be used.