bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of Uroporphyrinogen Decarboxylase Activity
Authors: Phillips John D. , Kushner James P. 
Published: June 2004

AbstractUroporphyrinogen decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.
Measurement of Protoporphyrinogen Oxidase Activity
Authors: Jacobs Judith M. , Jacobs Nicholas J. 
Published: June 2004

AbstractProtoporphyrinogen oxidase catalyzes the oxidation of protoporphyrinogen to protophyrin. It is a membrane‐bound mitochondrial enzyme and it is the target of photobleaching herbicides. The basic assay presented in this unit for measuring oxidase activity is based on oxidation of the colorless, nonfluorescent substrate, protoporphyrinogen, to the colored, fluorescent protophyrin. Alternate protocols are provided for the measuring the accumulation of protoporphyrinogen resulting from a decrease in oxidase activity due to treatment with diphenylether herbicides or oxidase inhibitor.
HPLC Methods for Analysis of Porphyrins in Biological Media
Authors: Woods James S. , Simmonds P. Lynne 
Published: June 2004

AbstractChanges in porphyrin concentrations in biological media may serve as biological indicators of exposure and toxicity from a wide variety of drugs and chemical agents. This unit describes procedures for quantitative extraction of porphyrins from urine, feces, blood, and biological tissues as well as their separation and analysis by HAPLY spectrofluorometrc techniques.
Detection of Biliverdin Reductase Activity
Authors: Huang Tian‐Jun 
Published: June 2004

AbstractThe conversion of biliverdin to the bile pigment bilirubin is catalyzed by biliverdin reductase, a cytosolic enzyme with two pH optima with different cofactors. The enzyme is assayed at pH 8.7 with NADPH as a cofactor and at 6.75 with NADH. The production of bilirubin is detected as described in this unit with a spectrophotometric assay. The enzyme can be qualitatively assayed by immunoblotting and changes in reductase isoform expression can be detected by two‐dimensional electrophoresis.
Functional Analysis of the Heme Oxygenase‐1 Gene Promoter
Authors: Alam Jawed 
Published: June 2004

AbstractBasal and inducer‐dependent transcription activity of the heme oxygenase‐1 gene promoter can be measured using the luciferase reporter gene in transfection assays. Methods are provided in this unit for purification of plasmid DNA, transient and stable transfection of DNA into cells, and measurement of luciferase and beta‐galactosidase enzyme activities using chemiluminescent‐based assays. These protocols can be used to identify inducer‐activated cis elements.
Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining
Authors: Gonzalez‐Zulueta Mirella , Dawson Valina L. , Dawson Ted M. 
Published: June 2004

AbstractThe presence of NOS coincides with that of NADPH diaphorase activity; therefore, NADPH diaphorase staining is widely used to detect NOS‐containing cells in neural and non‐neural tissues. With the appropriate fixation procedure, this method is capable of detecting cells containing any of the NOS isoforms.
Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide
Authors: Wink David A. , Kim Sungmee , Miles Allen , Jourd'heuil David , Grisham Matthew B. 
Published: June 2004

AbstractNO‐derived intermediates formed under aerobic conditions may engage in complex chemical reactions with biologically important molecules. The outcomes of these reactions and their ultimate effect on biological systems depend on the selectivity of the species and the concentrations of different substances present and whether the reaction takes place in the gas or aqueous phase. In this unit conversion of two different compounds to fluorescent products is used to distinguish between oxidative and nitrosative chemistry of different reactive nitrogen oxide species.
Neurobehavioral Screening in Rodents
Authors: Moser Virginia C. 
Published: June 2004

AbstractNeurobehavioral evaluations are an important component of screening for neurotoxicological effects. A neurobehavioral test battery is chosen to assess a variety of endpoints, including autonomic, neuromuscular, sensory, and excitability functions. Such a battery includes observation of gait and posture, reactivity and arousal, gait and postural characteristics, involuntary/abnormal motor movements, clinical signs and manipulation of neurological reflexes and reactions and neuromuscular activity and postural reactions. Finally, the battery includes assessment of sensory responses.
Mouse Models of Focal Cerebral Ischemia
Authors: Panahian Nariman 
Published: June 2004

AbstractAs with models of global ischemia (UNIT here), models of focal ischemia in the mouse are useful for understanding the pathophysiology of this condition. The models are also useful for assessing the neuroprotective effects of particular genes and pharmacologic agents. This unit describes two methods for inducing focal ischemia: one by proximal middle cerebral artery occlusion and one by permanent endovascular occlusion. Alternatives of transient endovascular occlusion and subarachnoid hemorrhage are also given. A number of protocols are described for determining the effects of ischemia induction: assessing physiological features, mapping of the circulation by perfusing the blood vessels with carbon black, determining stroke volume by staining with 2,3,5‐triphenyltetrazolium chloride, evaluating post‐treatment behavior, assessing stroke volume and cerebral edema, assessing the size of ischemic penumbra, and analyzing the statistics and presenting the data.
Culturing Rat Hippocampal Neurons
Authors: Audesirk Gerald , Audesirk Teresa , Ferguson Charles 
Published: June 2004

AbstractCultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.
Isolation of Cerebellar Granule Cells from Neonatal Rats
Authors: Oberdoerster Jan 
Published: June 2004

AbstractCerebellar Granule Cells in Neurotoxicology (Jan Oberdoerster, Aventis Corporation, Research Triangle Park, North Carolina). Cultured neurons allow the researcher to investigate mechanisms of toxicity on a relatively uniform population of cells. Primary cultures of cerebellar granule cells are post‐mitotic neurons that are readily isolated and may be used for experimental procedures including electrophysiology, neuronal maturation, and various biochemical and molecular analyses.
Micromass Cultures in Teratology
Authors: Ponce Rafael A. 
Published: June 2004

AbstractThis unit describes methods for culture of undifferentiated midbrain (mesencephalon) and limb bud cells from gestation day 12 rat embryos. When grown over 5 days in vitro, these mixed cell populations express many morphological, biochemical, molecular, and immunophenotypic characteristics observed during in vivo differentiation. These cultures can be used in a wide variety of studies designed to investigate normal cellular ontogeny, the teratogenic potential of test agents, or the mechanisms underlying the cellular response to environmental stress.
Preparation of Hepatocytes
Authors: Mudra Daniel R. , Parkinson Andrew 
Published: June 2004

AbstractThis unit describes a process for isolation of intact, viable hepatocytes for use in integrated drug metabolism studies. Isolated hepatocytes are increasingly being used as a biological system for studying the in vitro metabolism of xenobiotics. The isolation of hepatocytes from laboratory animals and nontransplantable human livers donated for research activities involves a two‐step enzymatic digestion of the liver tissue. Two methods for the perfusion of liver tissue are included: in situ perfusion and isolation of hepatocytes and perfusion of excised tissue. The basic protocol also includes suggestions for designing drug metabolism experiments using hepatocytes. The final protocol describes cryopreservation and long‐term storage of isolated hepatocytes.
Measurement of Hepatobiliary Transport
Authors: Ballatori Nazzareno 
Published: June 2004

AbstractA major hepatic function involves the removal of xenobiotics from portal blood and their subsequent storage, metabolism, and excretion into bile, or delivery of various products back into the bloodstream. Transport across the liver cell basolateral (or sinusoidal) plasma membrane is a key step in the overall metabolism of many compounds, whereas transport across the apical (or canalicular) plasma membrane into bile is often the principal pathway for their elimination. Although significant progress has been made in the molecular identification of the transport proteins that mediate these processes, much remains to be learned about regulation and physiological integration. The critical limiting factor in studying biliary excretion is that the bile canaliculus is very small (∼1 µm in diameter) and is generally inaccessible to sampling by conventional approaches. The bile canalicular membrane is a specialized part of the hepatocellular plasma membrane, such that bile is separated from blood plasma only by the tight junctions that encircle each hepatocyte. Because hepatocyte polarity is rapidly lost during cell isolation, most cell culture models provide only limited information on mechanisms of biliary excretion. Thus, biliary secretion has been studied using four major experimental models: canalicular plasma membrane vesicles, cultured hepatocyte couplets, perfused liver, and in vivo (bile duct‐cannulated animals). This unit describes basic methods for collecting bile from anesthetized mice and rats, for carrying out isolated perfused rat liver studies, and for the simultaneous isolation of plasma membrane vesicles derived from the basolateral and canalicular domains of rat liver.
Aggregation Chimeras (ES Cell–Embryo)
Authors: Lobe Corrinne G. , Guo Caiying 
Published: June 2004

AbstractOne approach for generating transgenic mice from ES cell lines is to aggregate ES cells with morula‐stage embryos to generate chimeric mice. The chimeras are then bred to generate transgenic offspring. This method offers a simpler and less expensive alternative to the method of ES cell injection into blastocyst embryos.
In Vivo Models for Male Reproductive Toxicology
Authors: Tyl Rochelle W. 
Published: June 2004

AbstractIn Vivo Models for Male Reproductive Toxicology (Rochelle W. Tyl, Research Triangle Institute, Research Triangle Park, North Carolina). Assessment of male reproductive function requires a specific set of evaluations of the various steps in successful mating
from sperm production to copulation to fertilization to production of a viable litter. This unit outlines the measurements that are standard for determining the effects of treatment with toxicant on the reproductive capacity of male mice and rats.
Monitoring Endocrine Function in Males: Using Intra‐Atrial Cannulas to Monitor Plasma Hormonal Dynamics in Toxicology Experiments
Authors: Fail Patricia A. , Anderson Stephanie A. 
Published: June 2004

AbstractMonitoring Endocrine Function in Males: Using an Intra‐Atrial Cannula to Monitor Plasma Hormonal Dynamics in Toxicology Experiments (Patricia A. Fail and Stephanie A. Anderson, Research Triangle Institute, Research Triangle Park, North Carolina). Intra‐atrial cannulation provides assessment of endocrine change within animals. Protocols for permanent tethers used in short‐term 5‐ to 10‐day experiments and permanent vascular access used in long‐term (>10 days) experiments are presented. A protocol for blood processing is also included. Data from a longitudinal endocrine baseline assessment (LEBA) and an endocrine challenge test (ECT) a presented as well.
Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats
Authors: Ostby Joseph S. , Gray L. Earl 
Published: June 2004

AbstractThis protocol is designed to evaluate the effects of endocrine disrupting compounds (EDCs) through fetal (transplacental) and/or neonatal (via the dam's milk) exposure during the critical periods of reproductive organogenesis in the rat. Continued direct exposure to the F1 pups after weaning is an option that can also be included. Reproductive indices, growth, and viability are monitored throughout the life of the F1 generation and an extensive necropsy is conducted after sexual maturity is attained. The endpoints evaluated are designed to detect compounds such as androgen receptor antagonists or steroid hormone synthesis inhibitors which inhibit normal development of the male reproductive system and growth stimulating hormones which masculinize females and facilitate development of male reproductive tissues. Data collected from these studies can be used to determine the no and lowest observed effects levels and predict potential adverse consequences of EDCs in human and wildlife populations. The information also provides a foundation for future studies designed to identify mechanism(s) of action, specific genes and or proteins involved, and susceptible populations these compounds might target.
Measurement of Protein Sulfenic Acid Content
Authors: Poole Leslie B. 
Published: June 2004

AbstractProtein sulfenic acids are reactive, reversibly oxidized, cysteinyl residues with roles in redox catalysis and regulation. Detection and quantitation of these species in proteins is accomplished through chemical modification by reagents such as 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD chloride), 2‐nitro‐5‐thiobenzoate (TNB) or dimedone, followed by UV‐visible spectral or mass spectrometric analyses.
Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation
Authors: Zhang Jin‐Gang , Fariss Marc W. 
Published: June 2004

AbstractThis unit describes a method for the measurement of cellular membrane antioxidant capacity or susceptibility of tissue samples to lipid peroxidation using a fluorescence microplate reader. The assay is simple and has the advantage of monitoring susceptibility to lipid peroxidation in a large number of samples in real time.
In Situ Localization of Nonenzymatic Peroxidase‐Like Activity of Tissue‐Bound Transition Metals
Authors: Sayre Lawrence M. , Harris Peggy L.R. , Perry George , Smith Mark A. 
Published: June 2004

AbstractAbnormalities in redox and/or transition metal homeostasis is characteristic of many pathological states. Here, we present protocols that can be used for the detection of both transition metals and redox state.
Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment
Authors: Luster Michael I. , Germolec Dori R. , Parks Christine G. , Blaciforti Laura , Kashon Michael , Luebke Robert 
Published: June 2004

AbstractThis overview unit discusses the relationship between immunosuppression, a potential consequence of immunotoxicity, and disease progression. It also discusses other factors, such as stress and age, that affect disease susceptibility. These factors play an important role in risk assessment for exposures to environmental factors.
Overview of Protein Purification and Characterization
Authors: Scopes R.K. 
Published: June 2004

AbstractThis insightful overview by one of the most respected names in protein research discusses a broad array of the aspects involved with protein purification, including historical background, determining the purpose for purifying a particular protein, and actual methods and recommendations for purification procedures. Also covered are methods of analysis and detection, and techniques for characterizing a purified protein. Finally, a useful discussion of the equipment needed in a protein purification lab is provided.
Strategies for Protein Purification
Authors: Scopes R.K. 
Published: June 2004

AbstractThis discussion unit presents information on classification of proteins according to shape, oligomeric structure, and function. In addition, strategies for purifying different types of proteins are discussed based on their location and associations within the cell. Specifically, critical issues are detailed for extracellular, cytoplasmic, and membrane‐associated proteins, as well as for insoluble proteins. Finally, strategies for the purification of both soluble and insoluble (inclusion bodies) recombinantly expressed proteins are provided.
Protein Purification Flow Charts
Authors: Scopes R.K. 
Published: June 2004

AbstractProtein purification flow charts are presented to give a broad outline of the methods used for different types of proteins. They cannot give any detail, as the process appropriate for each protein will have its own variations at each stage. In most cases, the first stage is to obtain a solution containing the desired protein, after which it can be fractionated by various separation techniques. This unit provides charts tailored for purification of soluble and insoluble recombinant and non‐recombinant proteins, as well as membrane‐associated proteins.
Protein Secondary Structure Prediction
Authors: Krystek Stanley R. , Metzler William J. , Novotny Jiri 
Published: June 2004

AbstractThis unit describes procedures developed for predicting protein structure from the amino acid sequence. The first of the four sections is an overview and brief history of structure prediction schemes. The second section describes four distinct prediction schemes, with emphasis on their differences. In the third part each prediction scheme is used to evaluate three proteins that have different folding patterns. The final section is a comparison of the prediction results and suggestions for secondary structure prediction.
Sequence Similarity Searching Using the BLAST Family of Programs
Authors: Wolfsberg Tyra G. , Madden Thomas L. 
Published: June 2004

AbstractThe BLAST (Basic Local Alignment Search Tool) family of sequence similarity search programs allows users to input either a nucleotide or amino acid query sequence, and search a nucleotide or amino acid sequence database. The program returns a list of the sequence “hits”, alignments to the query sequence, and statistical values. This unit describes how to choose an appropriate BLAST program and database, perform the search, and interpret the results.
Protein Databases on the Internet
Authors: Xu Dong , Xu Ying 
Published: June 2004

AbstractProtein databases have become a crucial part of modern biology. Huge amounts of data for protein structures, functions, and particularly sequences are being generated. Searching databases is often the first step in the study of a new protein. Comparison between proteins and between protein families in databases provides information about the relationship between proteins within a genome or across different species, and hence offers much more information than can be obtained by studying only an isolated protein. In addition, secondary databases derived from experimental databases are also widely available. These databases reorganize and annotate the data or provide predictions. The use of multiple databases often helps researchers understand the structure and function of proteins. Although some protein databases are widely known, they are far from being fully utilized in the protein science community. This unit provides a starting point for readers to explore the potential of protein databases on the Internet.
Kinetic Assay Methods
Authors: Allison R. Donald 
Published: June 2004

AbstractThe purpose of this unit is to provide a brief review of issues important in the design of initial‐rate assay methods. General aspects of kinetic assay design are discussed, including enzyme and substrate purity, concentration and stability. Also covered are issues such as continuous versus stop‐time assay formats, coupled enzyme assays, binding studies, and presentation of initial‐rate data.
Selection of Escherichia coli Expression Systems
Authors: Bernard Alain , Payton Mark 
Published: June 2004

AbstractThis unit lists the most useful expression strains of E. coli for fermentation processes. Standard procedures are provided for several expression systems, namely, temperature induction via the pL promoter and chemical induction via the trp promoter, lac or tac promoters, and the T7 promoter. These protocols require that the gene encoding the protein of interest has been identified and cloned into an appropriate expression vector using standard molecular biology techniques. Transformation of a suitable host strain (e.g., by electroporation) is also described and is a prerequisite. Protocols for the analysis of plasmid stability and subsequent storage are provided. Support protocols describe how to prepare samples for electrophoresis, how to analyze the solubility of the expressed proteins, and how to make samples of periplasmic extracts and extracellular media (using TCA precipitation). Many of the support protocols are small‐scale analysis procedures that are used to guide subsequent purification strategies and determine the suitability of the expression system for further development and scale‐up.
Overview of Protein Expression by Mammalian Cells
Authors: Gray David 
Published: June 2004

AbstractThis unit reviews the stages involved in protein production in mammalian cells using a stable‐expression approach. Choice of cell type is discussed, as is transfection of the host cells, methods for selection and amplification of transformants, and growth of cells at appropriate scale for protein production. Since post‐transcriptional modification and intracellular protein transportation are important features of recombinant‐protein production in mammalian cells, some description of these mechanisms is included.
Preparation of Cell Cultures and Vaccinia Virus Stocks
Authors: Earl Patricia L. , Cooper Norman , Wyatt Linda S. , Moss Bernard , Carroll Miles W. 
Published: June 2004

AbstractThis unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range‐restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.
Characterization of Recombinant Vaccinia Viruses and Their Products
Authors: Earl Patricia L. , Moss Bernard 
Published: June 2004

AbstractAfter a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dot‐blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.