bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single‐Dose Pharmacokinetics of Sodium Ferric Gluconate Complex in Iron‐Deficient Subjects
Authors: Seligman Paul A. , Dahl Naomi V. , Strobos Jur , Kimko Hui C. , Ms. Rhoda B. Schleicher M.S.  , Jones Michael , Ducharme Murray P. 
Published: June 2004

Study Objectives. To determine the single‐dose pharmacokinetics of intravenous sodium ferric gluconate complex in sucrose injection (SFGC) in iron‐deficient human volunteers, and to assess iron transport.Design. Open‐label, randomized study.Setting. Clinical research facility.Subjects. Fourteen iron‐deficient men and women.Interventions. Subjects were randomized to receive a single intravenous dose of either SFGC 62.5 mg administered over 30 minutes or SFGC 125 mg over 60 minutes. Five days later, the same subjects were rerandomized to receive a second intravenous dose of SFGC, either 62.5 mg administered over 4 minutes or 125 mg over 7 minutes.Measurements and Main Results. Blood samples were collected at predefined times before, during, and up to 72 hours after the infusion to determine the single‐dose pharmacokinetics of SFGC. Assays were performed for both total iron and transferrin‐bound iron, from which drug‐bound iron could be calculated. Urine was collected over 24 hours before dosing and for 24 hours after the start of infusion to determine the renal elimination of iron. Clearance of SFGC from serum was rapid and far exceeded rates reported for iron dextran. Pharmacokinetic parameters were unaffected by dose or infusion rate. Serum iron derived from SFGC did not exceed the binding capacity of transferrin. Serum iron from SFGC became rapidly available (< 24 hrs) as transferrin‐bound iron, but only after passage through another compartment, presumably the reticuloendothelial system (RES). At least 80% of the administered iron was transported to bone marrow within 24 hours after infusion.Conclusions. Iron derived from SFGC appears to be rapidly transferred to a bioavailable iron compartment as transferrin‐bound iron after digestion in the RES. At the doses administered in this study, liberation of potentially toxic, free iron was not detectable.
Sex‐Related Differences in the Pharmacokinetics of Once‐Daily Saquinavir Soft‐Gelatin Capsules Boosted with Low‐Dose Ritonavir in Patients Infected with Human Immunodeficiency Virus Type 1
Authors: Pai Manjunath P. , Schriever Christopher A. , Diaz‐Linares Mariela , Novak Richard M. , Rodvold Keith A. 
Published: June 2004

Study Objectives. To compare the steady‐state pharmacokinetics and safety of saquinavir soft‐gelatin capsules (SGC) plus low‐dose ritonavir administered once/day in antiretroviral‐naïve adult patients infected with the human immunodeficiency virus type 1 (HIV‐1) and to evaluate any sex‐related differences.Design. Single‐center, open‐label, pharmacokinetic study.Setting. University‐affiliated outpatient HIV clinic.Patients. Six men and seven women with HIV‐1.Intervention. Each patient received saquinavir SGC 1600 mg and ritonavir 100 mg for a 14‐day course of therapy. Nine serial blood samples during 24 hours were collected on day 14 of therapy.Measurements and Main Results. Plasma saquinavir and ritonavir concentrations were measured by high‐performance liquid chromatography. Standard noncompartmental methods were used to calculate the pharmacokinetic parameters. The unpaired Student t test was used for the statistical comparison of pharmacokinetic parameters between male and female patients. Once‐daily saquinavir SGC plus ritonavir was generally well tolerated. Pharmacokinetic data from five men and five women were evaluable. The median saquinavir area under the concentration–time curve from 0–24 hours (AUC0–24) in the female patients (82,300 ng·hr/ml) was significantly (p=0.036) higher than that in the male patients (47,400 ng·hr/ml). This relationship remained significant for weight‐adjusted saquinavir AUC0–24 values. Ritonavir's apparent oral clearance in the women was significantly (p=0.023) lower than that in the men.Conclusion. Significantly higher plasma concentrations of saquinavir were achieved in female compared with male HIV‐infected patients receiving once‐daily saquinavir SGC 1600 mg plus ritonavir 100 mg.
DNA Microarrays: An Overview of Technologies and Applications to Toxicology
Authors: Zarbl Helmut 
Published: June 2004

AbstractDNA microarrays or chips can be used to simultaneously monitor the expression levels and/or genotypes of thousands of genes. The application of these techniques heralds a new era in toxicology research, where genotypes and toxicant‐induced expression signatures may be used to monitor cellular responses to different doses, to classify toxins on the basis of their mechanisms of action, to monitor exposures, and to predict individual variability in toxicant sensitivity. This unit reviews the current state of microarray technologies and discusses potential applications in toxicology, with emphasis on the strengths and limitations of the technologies.
Determination of Apoptosis and Necrosis
Authors: Zhivotovsky Boris , Samali Afshin , Orrenius Sten 
Published: June 2004

AbstractCell death occurs by one of two mechanisms: apoptosis (programmed cell death) or necrosis due to cellular injury or trauma. Each method has distinct morphological and biochemical characteristics. Distinguishing between apoptosis and necrosis depends selection of appropriate assays and proper timing. This unit provides an extensive series of assays that measure membrane integrity, cell size and DNA content, state of chromatin, DNA fragmentation, and caspase activation. The results of these assays allow one to distinguish between apoptosis and necrosis.
Measurement of Lipid Peroxidation
Authors: Reilly Christopher A. , Aust Steven D. 
Published: June 2004

AbstractThere is currently considerable interest in what is termed “oxidative stress,” or the oxidation of biological macromolecules, with emphasis on its involvement in various diseases and toxicities and methods to limit either its occurrence or effects. This unit describes traditional methods to measure the extent or rate of lipid peroxidations, including assays for conjugated dienes, lipid hydroperoxides, the polyunsaturated lipid breakdown product malondialdehyde, and hemolysis, along with discussion of alternative methods.
Measurement of Expression of the HSP70 Protein Family
Authors: Bargeron Marcelle , Sharp Frank R. 
Published: June 2004

AbstractThe heat shock protein family of proteins include a number of proteins of similar sizes that are inducible by a wide variety of “stress” stimuli including heat shock, ischemia, toxic metals, amino acid analogs, and some chemotherapeutic and anesthetic chemicals. The functions of these proteins are diverse but are generally associated with molecular chaperoning of proteins. This unit contains a wide variety of protocols for analyzing expression of this family at the transcriptional and translational level: northern blot hybridization, in situ hybridization, metabolic labeling and SDS‐PAGE, and immunoblotting.
Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro
Authors: Hotchkiss Sharon A.M. 
Published: June 2004

AbstractNew therapeutic compounds intended for use on the skin or for delivery through application to the skin and agrochemicals, whose use may result in skin exposure, must be tested for bioavailability as the result of absorption. This unit contains a protocol for measuring skin absorption in vitro using the diffusion cell skin absorption method (SAM), which can be used to measure percutaneous absorption after topical application. Usually a radiolabeled compound is used, but if a suitable specific assay is available, nonradioactive compounds may be tested. The procedure is applicable to skin from a variety of species.
Toxicant Transport by P‐Glycoprotein
Authors: Bain Lisa J. 
Published: June 2004

AbstractToxicant Transport by P‐Glycoprotein (Lisa J. Bain, University of Texas at El Paso, El Paso, Texas).This unit describes several different methods of measuring active transport of toxicants by P‐glycoprotein uptake of a radiolabeled toxicant, uptake of an unlabeled toxicant with detection by HPLC or GC, uptake of radiolabeled toxicant in membrane vesicles; movement of radiolabeled toxicant from the basal to apical compartment using polarized epithelial cells grown in Transwell culture. P‐glygoprotein is an important protein for limiting entry into the brain through the blood‐bain barrier. It also alters the rates of uptake and elimination.
Measurement of Glutathione Transferases
Authors: Mannervik Bengt , Jemth Per 
Published: June 2004

AbstractThere are multiple glutathione transferase genes, the proteins for which have different substrate specificities. The various genes are differentially expressed such that species and organs and tissues differ qualitatively and quantitatively for cytosolic and membrane‐bound forms. This unit provides protocols for analysis of transferase activity in a continuous spectrophotometric assay and an assay with dichloromethane as the substrate.
Coenzyme A and Coenzyme A‐Glutathione Mixed Disulfide Measurements by HPLC
Authors: Rogers Lynette K. , Smith Charles V. 
Published: June 2004

AbstractThis unit describes a method for measuring coenzyme A and coenzyme A‐glutathione mixed disulfide in tissue homogenates obtained from tissues frozen in liquid nitrogen, homogenized in the presence of N‐ethylmaleimide, and then acidified. to avoid the oxidation, reduction, and thiol exchange reactions that can occur during tissue processing and isolation of subcellular fractions. This method may provide a way to assess changes in the oxidative state of mitochondria in intact tissues.
Measurement of Glutathione Reductase Activity
Authors: Mannervik Bengt 
Published: June 2004

AbstractReduced glutathione, a thiol, is essential to the survival of the cells of most aerobic organisms. It is present intracellularly and provides protection from hydroperoxides and free radicals. This unit describes a continuous spectrophotometric assay for reductase activity: it follows the reduction of glutathione disulfide to reduced glutathione by monitoring the oxidation of NADPH as visualized by a decrease in absorbance at 340 nm.
Overview of the Pathway and Functions of Nitric Oxide
Authors: Dillman James F. , Dawson Valina L. , Dawson Ted M. 
Published: June 2004

AbstractNitric oxide has been recognized for years as a toxic, reactive, free radical gas, but in recent years it has been identified as having functions in a variety of metabolic and signaling pathways. NO is synthesized, by nitric oxide synthase, on demand and diffuses to the site of action where it forms noncovalent and covalent linkages with target molecules. This overview presents the pathway of NO formation and also discusses the functions of NO in the nervous, immune and vascular systems.
Detection of Nitrosated Proteins
Authors: Zhang Ying‐Yi , Loscalzo Joseph 
Published: June 2004

AbstractThree methods are provided in this unit to detect nitrosated proteins. The Saville assay is the most commonly used and has the advantages that it is less sensitive to interference than the quinine sulfate‐based fluorescent assay, uses simple chemical reagents, and requires a spectrophotometer. The diaminonaphthalene assay (a fluorescence assay) and chemiluminescence assay are used when the experiment requires high sensitivity. Mass spectrometry is employed for precise structural determinations.
Inducible Nitric Oxide Synthase Expression
Authors: Xie Qiao‐wen 
Published: June 2004

AbstractThis unit contains three protocols that can be used to determine iNOS expression in mouse macrophage‐like cells, RAW 264.7, by measuring end product, protein, and mRNA. A bacterial product, lipopolysaccharide (LPS), stimulates iNOS expression in RAW 264.7 cells.
Assessment of Spatial Memory
Authors: Wenk Gary L. 
Published: June 2004

AbstractBehavioral tasks must be evaluated in terms of the cognitive functions they require. The tasks described in this unit are useful for detecting stimulation by drugs or a small electrical current, impairment of normal function by production of lesions or administration of a pharmacologic or toxicologic agent, recording activity during performance of a specific task, or behavioral phenotyping of transgenic or knockout mice. The radial arm maze test is used for basic working memory or working memory versus reference memory; the water maze task is used for spatial memory, spatial probe trials, or working memory; and the T‐maze test is used for spatial memory, working versus reference memory, or spontaneous alternation.
Advanced Behavioral Testing in Rodents: Assessment of Cognitive Function in Animals
Authors: Bushnell Philip J. 
Published: June 2004

AbstractIn risk assessment of a neurotoxic chemical, it may be necessary to assess its effect on cognitive function, the information‐processing capacity of the animal. Such effects may be inferred from behavioral effects only after other explanations have been ruled out. Learning, memory, attention, and performance are elements of cognitive function that can be assessed in a variety of tests described in this unit: autoshaping the lever‐press response, repeated acquisition in the radial maze, delayed matching to position, two‐light visual discrimination, visual signal detection, and multiple fixed interval/fixed ratio operant schedule.
Mouse Models of Global Cerebral Ischemia
Authors: Panahian Nariman 
Published: June 2004

AbstractUse of rodent models of hippocampal neurodegeneration are recommended for use in studies to understand the pathophysiology and molecular mechanisms involved. This unit includes protocols for two‐vessel occlusion, three‐vessel occlusion, and permanent bilateral vertebral artery occlusion plus methods for histological techniques for preparation of brain tissue and analysis of hippocampal injury.
Whole‐Cell Patch‐Clamp Electrophysiology of Voltage‐Sensitive Channels
Authors: Shafer Timothy J. 
Published: June 2004

AbstractIon channels are critical modulators of electrical excitability in neurons and are targets of a wide variety of neurotoxicants, including metals and insecticides. The only way to directly examine effects of toxicants on ion channel function in an individual cell on a physiologically relevant time scale is to utilize patch‐clamp electrophysiological techniques. This unit presents the basics of utilizing whole‐cell patch‐clamp techniques to study effects of toxicants on voltage‐sensitive Ca2+ channels in neurons grown in culture.
Determining the Ability of Xenobiotic Metals to Bind a Specific Protein Domain by Electrophoresis
Authors: Zawia N.H. , Basha Md. Riyaz , Razmiafshari M. 
Published: June 2004

AbstractEnvironmental metals are potentially toxic and can interfere with the metal‐binding motifs of various critical proteins. This unit describes an electrophoretic method that can be used to measure the ability of a xenobiotic metal to bind a zinc‐finger motif. Information gained using this protocol can lead to the identification of protein targets of metals and shed better light on their mechanisms of action.
Rat Embryo Cultures for In Vitro Teratology
Authors: Fantel Alan G. , Bechter Rudolf , Beckman David 
Published: June 2004

AbstractMammalian embryos provide a particular challenge to those who wish to study developmental processes because development takes place inside the mother's body, thus limiting the investigator's ability to directly affect and observe the embryonic stages of development. Fortunately, as this unit illustrates, methods have been developed for in vitro culture of rodent embryos during early postimplantation (gestation days 9 to 11) and early fetal (gestation days 12 to 14) stages, which are the periods during which the major systems of the embryo are established. Embryos at these stages are particularly suitable for screening studies to determine the effects of teratological agents.
Using Chicken Embryos for Teratology Studies
Authors: Henshel Diane S. , DeWitt Jamie , Troutman Andrea 
Published: June 2004

AbstractThis unit describes methods for injecting, incubating, handling and analyzing domestic chicken embryos used in teratology studies. It also includes a discussion of caveats and special handling issues as well as some discussion of statistical analyses that differentiate working with chicken embryos from working with clutches of eggs or litters of pups. As an example of potential data, preliminary data from a study of abnormalities in early embryos and hatchling chicks exposed to chlordane are presented.
In Vivo Assessment of Prenatal Developmental Toxicity in Rodents
Authors: Narotsky Michael G. , Kavlock Robert J. 
Published: June 2004

AbstractThis unit presents two basic protocols for testing prenatal developmental toxicity in rodents. The standard protocol for assessing prenatal developmental toxicity, often referred to as a Segment II study, describes the methods used for performing cesarean sections and examining cesarean‐delivered fetuses for external, skeletal, and visceral alterations. The protocol for the Chernoff/Kavlock assay, a developmental toxicity screen, describes methods for examining parturition, and postnatal growth and viability of prenatally exposed litters.
Overview of Hepatotoxicity
Authors: Meyer Sharon A. 
Published: June 2004

AbstractThis overview presents some of the salient features of liver biology and physiology that form the basis for a variety of experimental tools that are used for the study of chemical histopathology. Prevalence and risk of heptatoxicity are examined among a subset of chemicals from the EPA IRIS database. The goal is to provide an objective assessment of the magnitude of the problem of chemically‐induced hepatotoxicity and contextual background against which the diversity of responses of the liver to toxicants and the analytical methods available for their study can be appreciated.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro 
Published: June 2004

AbstractThe use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. The development of methods to inexpensively synthesize such tagged biological compounds on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells); require amounts on the order of millicuries. In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses storage, handling, and disposal of 35S, 32P, 33P, and 125I.
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R. 
Published: June 2004

AbstractElectrophoresis is used to separate complex mixtures of proteins, (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols for standard Laemmli gels, electrophoresis of peptides and small proteins, continuous SDS‐PAGE ultrathin gels, multiple single‐concentration gels, gradient gels, and multiple gradient gels.
Purification of Glutamate Dehydrogenase from Liver and Brain
Authors: Motherway Martha , Tipton Keith F. , McCarthy Alun D. , Couée Ivan , Irwin Jane 
Published: June 2004

AbstractTwo alternative procedures are described for the purification of the major form of glutamate dehydrogenase (L‐glutamate‐NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3: GDH) from ox liver and brain. The first involves affinity chromatography on a column of the allosteric inhibitor GTP bound to Sepharose, whereas the other uses a bifunctional ligand (bis‐NAD+) composed of two NAD+ molecules linked together by a spacer arm to precipitate the enzyme in the presence of the substrate analogue glutarate. In both procedures the affinity steps are preceded by ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose. Procedures for the synthesis of GTP‐Sepharose and bis‐NAD+ are described and the ancillary procedures, including the assay of GDH activity and the determination of protein concentration, are also presented.
Quantitative Amino Acid Analysis
Authors: Dunn Ben 
Published: June 2004

AbstractColorimetric dye‐binding assays are often used to determine protein concentration, however, many proteins do not bind dyes in the same way as the proteins normally used as standards, making quantitation by those techniques difficult. This unit describes the information that can be derived from quantitative amino acid analysis (including precise determination of protein concentration), presents some details on sample preparation, and gives examples of the calculations that are needed.
In Vitro Radiolabeling of Peptides and Proteins
Authors: Schumacher Ton N.M. , Tsomides Theodore J. 
Published: June 2004

AbstractRadiolabeling of peptides or proteins is often performed to enhance the sensitivity of detection, to quantitate the binding of peptides to other molecules, or for radioimmunoassays. This unit presents a variety of assays for radiolabeling peptides and proteins with 125I, 131I, 14C, and 3H.
Analysis of Selenocysteine‐Containing Proteins
Authors: Gladyshev Vadim N. , Hatfield Dolph L. 
Published: June 2004

AbstractRepresentatives of three primary life domains‐‐bacteria, archaea, and eukaryotes‐‐possess specific selenium‐containing proteins. The majority of naturally occurring selenoproteins contain an amino acid, selenocysteine, that is incorporated into protein in response to the code word UGA. The presence of selenium in natural selenoproteins and in proteins in which this element is introduced by chemical or biological manipulations provides additional opportunities for characterizing structure, function, and mechanism of action. This unit provides an overview of known selenocysteine‐containing proteins, examples of targeted incorporation of selenium into proteins, and methods specific for selenoprotein identification and characterization.
Purification of Organelles from Mammalian Cells
Authors: Castle J. David 
Published: June 2004

AbstractThe protocols in this unit illustrate a range of different procedures that have been used to fractionate tissue homogenates. They emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, other examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells. The basis (or goal) of each separation and the merits and limitations of each procedure are summarized to provide a guide for selecting among the various approaches. The large number of protocols include specific details relevant to a particular sample cell type (and in many cases a particular organelle).