bluuebunny/crossref_metadata_2025 huggingface dataset:
Resources in Emerging Structures and Processes of Change
Authors: Feldman Martha S.
Published: June 2004

In this paper I argue that understanding resources through a social practice perspective enables us to understand more about the role of resources in change. In particular, social practice theory enables us to view resources in context as mutable sources of energy rather than as stable things that are independent of context, and to analyze the reciprocal relationship between actions and resources as they change. This approach to understanding resources requires an elaboration on current social practice theory and provides a new way to understand organizational change. This perspective is used to show how resources transform in unexpected ways as a result of change in organizational routines and how this transformation of resources makes resistance to change difficult to predict.
Active Transport of Nitrofurantoin into Human Milk
Authors: Gerk Phillip M. , Kuhn Robert J. , Desai Nirmala S. , McNamara Patrick J.
Published: June 2004

Study Objective. To determine the extent to which nitrofurantoin is transferred into human milk.

Design. Prospective, single‐dose pharmacokinetic study.

Setting. University‐affiliated clinical research center.

Patients. Four healthy lactating women 8–26 weeks postpartum.

Intervention. All subjects received a single, oral, 100‐mg dose of nitrofurantoin macrocrystals with food. Serial serum and milk samples were obtained and analyzed by high‐performance liquid chromatography.

Measurements and Main Results. Milk pH, milk fat partitioning, and protein binding in serum and milk were determined. Predicted milk:serum ratio (M:S) was compared with the observed M:S. Nitrofurantoin M:S predicted was 0.28 ± 0.05, whereas M:S observed was 6.21 ± 2.71. Average milk concentration was 1.3 mg/L, and estimated suckling infant dosage was 0.2 mg/kg/day or 6% of maternal dose (mg/kg).

Conclusions. Nitrofurantoin is actively transported into human milk, achieving concentrations in milk greatly exceeding those in serum. Concern is warranted for suckling infants younger than 1 month old, or for infants with a high frequency of glucose‐6–phosphate dehydrogenase deficiency or sensitivity to nitrofurantoin.
Economic Impact of Neutropenia and Febrile Neutropenia in Breast Cancer: Estimates from Two National Databases
Authors: Gandhi Sanjay K. , Arguelles Lester , Boyer J. Gregory
Published: June 2004

Study Objective. To estimate the economic burden of neutropenia and febrile neutropenia in female breast cancer hospital admissions in the United States.

Design. Retrospective database analysis.

Patients. Female admissions with a breast cancer diagnosis.

Measurements and Main Results. By reviewing two national databases (Healthcare Costs and Utilization Project, MarketScan), length of stay and charge or payment/admission were estimated from 1994–1996. Neutropenic and febrile neutropenic admissions were longer and incurred higher charges and payments than nonneutropenic and afebrile neutropenic admissions, respectively (p<0.05). The difference in mean charges between neutropenic and nonneutropenic admissions decreased from $13,143 in 1994 to $6913 in 1996, whereas the difference in payment was $4957 (adjusted to 1996 dollars). The difference in mean charges between febrile and afebrile neutropenic admissions decreased from $11,570 in 1994 to $2873 in 1996, whereas the difference in payment was $2390 (adjusted to 1996 dollars).

Conclusion. There was a trend toward decreased charges for inpatient admissions with neutropenia in patients with breast cancer (1994–1996). Interventions that reduce the frequency of neutropenia and febrile neutropenia could reduce hospitalization costs of breast cancer admissions.
Update on Strategies to Improve Thrombolysis for Acute Myocardial Infarction
Authors: Spinler Sarah A. , Inverso Stephanie M.
Published: June 2004

Therapy for acute myocardial infarction involves rapid restoration of blood flow through a coronary artery that has been occluded by a ruptured atherosclerotic plaque. Thrombolytic therapy, the pharmacologic standard to achieve this outcome, significantly improves survival; however, current regimens have limitations: they can fail to achieve complete reperfusion, they can cause significant bleeding events, and they can result in reocclusion. In addition, complex regimens of some agents can cause dosing errors. Accordingly, newer compounds were developed to address some of these issues, and alternative strategies are being implemented. The combination of platelet glycoprotein IIb‐IIIa receptor inhibitors plus thrombolytic agents produced promising results in clinical trials, including faster clot lysis and greater flow rates than either therapy alone. The addition of unfractionated heparin or low‐molecular‐weight heparin to thrombolytic‐antiplatelet therapy is being evaluated, as is administration of thrombolytic‐antiplatelet before percutaneous coronary intervention.
Practice‐Based Research: Lessons from Community Pharmacist Participants
Authors: Simpson Scot H. , Johnson Jeffrey A. , Biggs Catherine , Biggs Rosemarie S. , Kuntz Arlene , Semchuk William , Taylor Jeff G. , Farris Karen B. , Tsuyuki Ross T. ,   Study of Cardiovascular Risk Intervention by Pharmacists Investigators
Published: June 2004

We designed this project to determine community pharmacists' opinions regarding the challenges and motivations of their recent participation in a pharmacy practice‐based research study. At the conclusion of a randomized, multicenter study, 87 community pharmacist‐investigators were sent a questionnaire that explored four areas: motivating factors to participate, barriers to participation, communication tools used by study coordinators, and design issues for future studies. Fifty‐eight (67%) completed questionnaires were returned. Key factors motivating participation in the study were desire to improve the profession and opportunity to learn. Time was the greatest barrier to participation. Pharmacy practice‐based research has two distinct advantages. First, it translates clinical knowledge into direct application in the community. Second, it provides needed data to demonstrate the value of enhanced pharmacy practice. Thorough understanding of pharmacists' opinions is necessary to optimize the design of future studies.
Cerebrospinal Fluid Concentrations of Quinupristin‐Dalfopristin in a Patient with Vancomycin‐Resistant Enterococcus faecalis Ventriculitis
Authors: Garey Kevin W. , Tesoro Eljim , Muggia Victoria , Rodvold Keith A.
Published: June 2004

A 44‐year‐old man was treated successfully for vancomycin‐resistant Enterococcus faecium (VREF) ventriculitis with intrathecal quinupristin‐dalfopristin 1 mg, 2 mg, and 4 mg, and other intravenous antibiotics. Cerebrospinal fluid samples were collected before and after the 1‐mg and 2‐mg doses to determine the concentrations of quinupristin‐dalfopristin and its active metabolites. Concentrations were above the minimum inhibitory concentration for VREF immediately after unclamping the extraventricular drain and were quantifiable for at least 7 hours.
Fatal Hemolysis After High‐Dose Etoposide: Is Benzyl Alcohol to Blame?
Authors: Smith Adrienne L. , Haider Kamal , Schachtner Jill M. , Mathur Sharad , VanOrden Ray , Gentile Teresa C.
Published: June 2004

A 53‐year‐old African‐American man with relapsed non‐Hodgkin's lymphoma developed seizures and respiratory arrest 2 hours after an infusion of high‐dose etoposide in preparation for an autologous bone marrow transplant. Laboratory tests revealed both rapid hemolysis and severe metabolic acidosis. The patient died the following day. Based on toxicities observed, we suspect that our patient possessed an ethnic polymorphism of the enzyme alcohol dehydrogenase. Further research is required to determine the relationship between the benzyl alcohol metabolic rate and toxicity and genetic polymorphisms of alcohol dehydrogenase in African‐Americans.
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.
Argatroban Therapy for Antithrombin Deficiency and Mesenteric Thrombosis: Case Report and Review of the Literature
Authors: Dager William E. , Gosselin Robert C. , Owings John T.
Published: June 2004

Antithrombin deficiency is a hypercoagulable state that can increase the risk for thrombosis, especially in the presence of other procoagulant triggers. Unfractionated heparin and low‐molecular‐weight heparins may not provide effective anticoagulation since they require antithrombin for activity. Direct thrombin inhibitors, however, work independently of antithrombin. A 21‐year‐old man with a history of heavy alcohol consumption had thrombosis of the superior mesenteric vein. Infusion with unfractionated heparin was started, and despite repeated boluses and increases to 21 U/kg/hour, the maximum activated partial thromboplastin time reached was 39 seconds. The unfractionated heparin was discontinued, and the direct thrombin inhibitor argatroban was infused at rates of 0.4–0.5 μg/kg/minute. Over the course of several weeks, the patient had numerous operations to remove and repair necrotic bowel. When no further surgery was anticipated, warfarin therapy was started; the argatroban infusion was discontinued when the patient reached the therapeutic target international normalized ratio with warfarin. No recurrent thrombosis or major bleeding occurred. Direct thrombin inhibitors, such as argatroban, seem to be suitable alternatives for acute anticoagulation in patients with antithrombin deficiency.
Fatal Acute Encephalomyelitis After a Single Dose of Intrathecal Methotrexate
Authors: Brock Stacia , Jennings Heath R.
Published: June 2004

A 40‐year‐old Hispanic man with acute lymphoblastic leukemia was treated with a single dose of intrathecal methotrexate 12 mg for prophylaxis against leptomeningeal spread of tumor. The day after methotrexate administration, the patient complained of severe back pain and urinary retention. The diagnosis of encephalomyelitis was made on day 3 after methotrexate administration, and by day 6 mechanical ventilation was begun secondary to ascending paralysis. By day 8 the patient was comatose, with minimal signs of brain activity and little hope for recovery; on day 12 he died. Although neurotoxicity is a frequent complication of methotrexate therapy, fatal acute neurotoxicity is extremely uncommon, especially in adults. The mechanisms of methotrexate toxicity remain unclear, and no effective treatment exists to prevent its occurrence. This patient rapidly progressed from mild neuro‐toxicity to fatal encephalopathy after one dose of intrathecal methotrexate during his third cycle of chemotherapy. Clinicians should be aware of the signs and symptoms of neurotoxicity during treatment, as well as predisposing factors that put patients receiving methotrexate at risk for neurotoxic effects.
DNA Microarrays: An Overview of Technologies and Applications to Toxicology
Authors: Zarbl Helmut
Published: June 2004

DNA 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

Cell 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

There 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

The 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

New 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

Toxicant 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

There 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

This 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

Reduced 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

Nitric 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

Three 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

This 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

Behavioral 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

In 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

Use 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

Ion 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

Environmental 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

Mammalian 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

This 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

This 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

This 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.
Reporter Genes to Detect Cre Excision in Mice
Authors: Lobe Corrine P. , Guo Caiying
Published: June 2004

Transgenic mouse lines that express Cre recombinase with tissue and temporal specificity are being developed as important new tools to manipulate the mouse genome.
Performing a Testicular Spermatid Head Count
Authors: Seung Han , Wolfe Gary , Rocca Meredith
Published: June 2004

procedure of counting spermatid heads using hemacytometer
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro
Published: June 2004

The 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

Electrophoresis 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

Two 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

Colorimetric 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

Radiolabeling 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

Representatives 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

The 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).
Subcellular Fractionation of Tissue Culture Cells
Authors: Aniento Fernando , Gruenberg Jean
Published: June 2004

Cell fractionation techniques include some of the most important and widely used analytical tools in cell and molecular biology, and are essential for the development of cell‐free assays that reconstitute complicated cellular processes. In addition to simple gradient systems, this unit discusses the immuno‐purification of organelles, in particular endosomes. As antigens, purification can be achieved using endogenous or ectopically expressed proteins, provided that appropriate antibodies are available. Alternatively, tagged proteins can be used, when combined with anti‐tag antibodies. Now that sequencing of the genomes of several organisms has been completed, biochemical strategies, and in particular fractionation and in vitro transport assays, are more necessary than ever to study the numerous protein and protein complexes that are being discovered.
Fermentation and Growth ofEscherichia colifor Optimal Protein Production
Authors: Bernard Alain , Payton Mark
Published: June 2004

Large‐scale production of recombinant proteins inEscherichia colirequires growth of cells in fermentors. This unit listsE. colistrains appropriate for use in fermentors, and also discusses important characteristics of fermentation equipment. Production of recombinant proteins in batch fermentations is described, as are variations of fermentation systems that enable continuous growth and protein production in high‐cell‐density, fed‐batch cultures and that permit labeling of recombinant proteins with heavy atom derivatives such as selenomethionine or with stable isotopes such as2H,13C, and15N. Production of labeled proteins facilitates structural studies by X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The protocols in this unit are designed for expression systems directing intracellular or periplasmic localization of recombinant proteins; however, in the case of extracellular secretion of the desired protein, the culture medium itself, rather than pelleted cells, would be saved, concentrated, and subjected to purification processes. Fermentation experiments require careful monitoring of cell growth and assurance of preinoculation sterility, both which are described here.
Overview of Protein Expression in Saccharomyces cerevisiae
Authors: Strausberg Robert L. , Strausberg Susan L.
Published: June 2004

This overview presents vectors and host strains that are available to direct gene expression in S. cerevisiae, including information on promoters, vector maintenance and copy number, transcription terminators, and selectable markers. Challenges to the expression of foreign proteins are also covered, including attainment of desired production yield, production of protein with appropriate post‐translational modifications, conformation and function, and secretion to the extracellular medium.
Culture of Yeast for the Production of Heterologous Proteins
Authors: Romanos Michael A. , Clare Jeffrey J. , Brown Crawford
Published: June 2004

This unit describes culture of the yeast strainsSaccharomyces cerevisiaeandPichia pastorisfor the production of foreign proteins. The protocols listed here forS. cerevisiaeare for three widely used types of promoter: galactose‐regulated (GAL1, GAL7, GAL10), glucose‐repressible (e.g.,ADH2), and constitutive glycolytic (e.g.,PGKorGAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol‐regulatedAOX1promoter andHIS4selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small‐scaleS. cerevisiaeandP. pastoriscultures and also for high‐density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided forS. cerevisiaeand yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high‐density cultures (e.g., 130 g/liter). Finally, adescribes small‐scale preparation of protein extracts.
Generation of Recombinant Vaccinia Viruses
Authors: Earl Patricia L. , Moss Bernard , Wyatt Linda S. , Carroll Miles W.
Published: June 2004

This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid‐transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
Choice of Cellular Protein Expression System
Authors: Gray David , Subramanian Shyam
Published: June 2004

Recombinant protein expression has become a standard laboratory tool, and a wide variety of systems and techniques are now in use. Because there are so many systems to choose from, the investigator has to be careful to use the combination that will give the best results for the protein being studied. This overview unit discusses expression and production choices, including post‐translational modifications (e.g., glycosylation, acylation, sulfation, and removal of N‐terminal methionine), in vivo and in vitro folding, and influence of downstream elements on expression.
Determining the Fluorescence Spectrum of a Protein
Authors: Pain Roger H.
Published: June 2004

Fluorescence spectra of proteins are determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions. A thorough consideration of fluorescence spectrometers and their calibration is provided along with important information regarding spectrometer cells, buffers and clarification of samples. Protocols are provided for recording fluorescence spectra and for measuring fluorescence quenching to probe the accessibility of tryptophan residues to small molecules (to yield information about the structural environment of the tryptophan). The technique involves quantifying the decrease in protein fluorescence intensity in the presence of increasing concentrations of quencher, followed by analysis of the data to give details of the interaction of the quencher with the tryptophan residue. Finally, agives details on how to interpret fluorescence spectra.
Overview of Conventional Chromatography
Authors: Williams Alan
Published: June 2004

The key to success in purification of any protein is developing the right separation strategy. This involves selecting and correctly applying a combination of separation techniques, chosen on the basis of information about the target protein and contaminants. There is no single separation technique and no single purification scheme that will allow successful purification of all types of proteins. However, there is a single approach to developing a successful purification strategy. This overview discusses the basic steps that must be taken, including selection of source material, and progression from initial “capture” stage of separation to the final polishing stage. Factors that can affect chromatographic resolution are also covered.
Ion‐Exchange Chromatography
Authors: Williams Alan , Frasca Verna
Published: June 2004

This unit outlines the basic steps in planning and carrying out ion‐exchange chromatography to separate proteins. Protocols describe both batch adsorption and column chromatography in conjunction with either step‐ or linear elution gradients. Support protocols describe (1) pilot experiments to determine initial conditions for batch or column chromatography (i.e., pH required for binding, change in pH or salt concentration required for elution, and available capacity of a medium), (2) calculation of the dynamic capacity of an ion‐exchange column, (3) methods for producing continuous gradients of pH and salt concentration to elute proteins from ion‐exchange columns, (4) regeneration of used ion‐exchange media, and (5) storage of ion‐exchange media.
Hydrophobic‐Interaction Chromatography
Authors: Kennedy Robert M.
Published: June 2004

This unit discusses the important parameters in designing and optimizing a separation by hydrophobic interaction chromatography (HIC), including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for packing and testing a column; determining binding and elution conditions; eluting the sample; and cleaning, regenerating, and storing HIC columns.
Dye Affinity Chromatography
Authors: Stellwagen Earle
Published: June 2004

Dye affinity chromatography is a protein purification procedure based on the high affinity of immobilized dyes for the binding sites on many proteins. It is a rapid, inexpensive, and versatile method that is applicable to the purification of crude cellular extracts. This unit presents protocol for the three types of dye affinity chromatography: negative chromatography, positive chromatography, and tandem chromatography. An initial protocol describes a chromatographic procedure in which a small volume of the protein mixture to be purified is applied to a series of miniature columns, each containing a different immobilized dye. Analysis of the flowthrough and bound material allows determination of the optimum dye material for larger‐scale purification. An alternate procedure describes a similar initial selection procedure using centrifugation instead of chromatography. A support protocol describes a simple procedure for immobilization of free dyes.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. , Springer Timothy A.
Published: June 2004

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in‐vitro‐translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear‐cut explanation of the options for employing the technology.
One‐Dimensional Electrophoresis Using Nondenaturing Conditions
Authors: Gallagher Sean R.
Published: June 2004

Nondenaturing or “native“ electrophoresis—i.e., electrophoresis in the absence of denaturants such as detergents and urea—is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Two protocols are presented in this unit: continuous PAGE, which is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH, and discontinuous PAGE, which is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.
Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers
Authors: Righetti Pier Giorgio , Bossi Alessandra , Gelfi Cecilia
Published: June 2004

Capillary electrophoresis in acidic isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and no interaction of macromolecules with the untreated wall ensues. In addition, because of the low conductivity of quasi‐stationary isoelectric buffers, high voltage gradients can be applied (up to 800 V/cm), permitting fast peptide analysis with a high resolving power and minimal diffusional peak spreading.
Digestion of Proteins in Gels for Sequence Analysis
Authors: Stone Kathryn L. , Williams Kenneth R.
Published: June 2004

A high percentage of eukaroytic proteins have blocked amino termini, so it is usually necessary to cleave the protein chemically or enzymatically to obtain the partial sequences needed for cDNA cloning. Because SDS‐PAGE is the current method of choice for the final purification of the >25 pmol amounts of protein that are usually required for internal sequencing, procedures that can be used to digest proteins in situ in SDS‐polyacrylamide gels are often the most useful and have the added benefit of eliminating losses that may occur during blotting. Two strategies have been developed to respond to this need and to deal with the unique problems posed by SDS, which are that SDS inhibits trypsin, one of the enzymes that is most commonly used for internal sequencing studies, and also interferes with reversed‐phase HPLC. In this unit, SDS is removed from the gel prior to enzymatic cleavage by the staining and subsequent washing steps. An acetonitrile wash is performed to remove residual SDS from the protein sample, and Tween 20 is then added back to the sample to maintain the solubility of the denatured protein. In an alternate protocol, the gel slices are washed with ammonium bicarbonate and the protein samples digested in the absence of detergent. Support protocols are provided for determining the amount of protein present in the sample via amino acid analysis, and for reducing and alkylating proteins separated by SDS‐PAGE (which facilitates the identification of cysteine residues during subsequent peptide sequencing reactions).