bluuebunny/crossref_metadata_2025 huggingface dataset:
Prepharmacy Predictors of Success in Pharmacy School: Grade Point Averages, Pharmacy College Admissions Test, Communication Abilities, and Critical Thinking Skills
Authors: Allen David D. , Bond C. A.
Published: June 2004

Good admissions decisions are essential for identifying successful students and good practitioners. Various parameters have been shown to have predictive power for academic success. Previous academic performance, the Pharmacy College Admissions Test (PCAT), and specific prepharmacy courses have been suggested as academic performance indicators. However, critical thinking abilities have not been evaluated. We evaluated the connection between academic success and each of the following predictive parameters: the California Critical Thinking Skills Test (CCTST) score, PCAT score, interview score, overall academic performance prior to admission at a pharmacy school, and performance in specific prepharmacy courses. We confirmed previous reports but demonstrated intriguing results in predicting practice‐based skills. Critical thinking skills predict practice‐based course success. Also, the CCTST and PCAT scores (Pearson correlation [pc]=0.448, p<0.001) were closely related in our students. The strongest predictors of practice‐related courses and clerkship success were PCAT (pc=0.237, p<0.001) and CCTST (pc=0.201, p<0.001). These findings and other analyses suggest that PCAT may predict critical thinking skills in pharmacy practice courses and clerkships. Further study is needed to confirm this finding and determine which PCAT components predict critical thinking abilities.
Fenofibrate and Warfarin Interaction
Authors: Aldridge Martha A. , Ito Matthew K.
Published: June 2004

A 79‐year‐old man with atrial fibrillation and coronary heart disease who was taking warfarin (Coumadin) was converted to fenofibrate from gemfibrozil therapy for persistently elevated triglyceride levels. The patient took fenofibrate for 1 month and subsequently experienced rectal bleeding that required a visit to the emergency room. Before starting fenofibrate therapy, his coagulation values were within therapeutic range, but when measured in the emergency room the international normalized ratio (INR) was grossly elevated. The patient denied any changes in diet, alcohol ingestion, compliance with therapy, or use of other new drugs except for fenofibrate. His drug therapy profile consisted of digoxin, fosinopril, and furosemide for chronic heart failure, allopurinol for gout, and potassium supplementation. To minimize the risk of supratherapeutic INR values and/or hemorrhagic events, clinicians should perform serial monitoring of INR when initiating fenofibrate therapy in a patient previously stabilized on a coumarin anticoagulant.
Treatment of Pain Syndromes with Venlafaxine
Authors: Grothe Dale R. , Scheckner Brian , Albano Dominick
Published: June 2004

Major depressive disorder (MDD) and anxiety disorders such as generalized anxiety disorder (GAD) are often accompanied by chronic painful symptoms. Examples of such symptoms are backache, headache, gastrointestinal pain, and joint pain. In addition, pain generally not associated with major depression or an anxiety disorder, such as peripheral neuropathic pain (e.g., diabetic neuropathy and postherpetic neuralgia), cancer pain, and fibromyalgia, can be challenging for primary care providers to treat. Antidepressants that block reuptake of both serotonin and norepinephrine, such as the tricyclic antidepressants (e.g., amitriptyline), have been used to treat pain syndromes in patients with or without comorbid MDD or GAD. Venlafaxine, a serotonin and norepinephrine reuptake inhibitor, has been safe and effective in animal models, healthy human volunteers, and patients for treatment of various pain syndromes. The use of venlafaxine for treatment of pain associated with MDD or GAD, neuropathic pain, headache, fibromyalgia, and postmastectomy pain syndrome is reviewed. Currently, no anti‐depressants, including venlafaxine, are approved for the treatment of chronic pain syndromes. Additional randomized, controlled trials are necessary to fully elucidate the role of venlafaxine in the treatment of chronic pain.
Clopidogrel‐Induced Thrombotic Thrombocytopenic Purpura–Hemolytic Uremic Syndrome After Coronary Artery Stenting
Authors: Manor Shawn M. , Guillory Gregory S. , Jain Suresh P.
Published: June 2004

The antiplatelet drug clopidogrel has largely replaced ticlopidine, due to an association between ticlopidine and thrombotic thrombocytopenic purpura–hemolytic uremic syndrome (TTP‐HUS). Clopidogrel at first was thought to be void of this potentially fatal adverse effect, but recent case reports have called that assumption into question. Even with proper treatment (plasma exchange), TTP‐HUS can persist for weeks. Clinicians should be aware of this possible adverse effect because prompt therapy is imperative for patients' survival. Earlier reports of clopidogrel‐related TTP‐HUS have involved patients who had received at least 72 hours of therapy. We describe a case of TTP‐HUS in a patient who had received only a 300‐mg loading dose of clopidogrel.
Cefazolin Tolerance Does Not Predict Ceftriaxone Hypersensitivity: Unique Side Chains Precipitate Anaphylaxis
Authors: Poston Sara A. , Jennings Heath R. , Poe Kevin L.
Published: June 2004

A 48‐year‐old woman with a questionable history of an unspecified ceftriaxone allergy was treated with cefazolin for surgical antibiotic prophylaxis. After she tolerated cefazolin therapy for 4 days, the medical staff concluded that her allergy history was inaccurate, and she was treated with intravenous ceftriaxone for suspected nosocomial pneumonia. Approximately 10 minutes after the start of the infusion, the patient experienced anaphylaxis. Initial symptoms of oral angioedema and laryngopharyngeal constriction progressed to dyspnea, tachypnea, hypotension, and tachycardia, all of which quickly resolved after immediate treatment with hydrocortisone, diphenhydramine, and epinephrine. Skin testing with cefazolin, cefepime, and ceftriaxone revealed that the likely allergic determinant mediating the patient's hypersensitivity reaction was the unique ceftriaxone R2 side chain and not the β‐lactam ring, which initially was suspected by the physician. Immunoglobulin E‐mediated hypersensitivity reactions to cephalosporins may occur due to antibody complexes with the β‐lactam ring or various cephalosporin side chains. Misconceptions regarding the nature of cephalosporin allergies complicate antibiotic selection for patients with questionable allergy histories and may lead to inappropriate drug reexposure and anaphylaxis. Detailed understanding of the antigenic determinants that mediate hypersensitivity reactions is essential for clinicians to avoid type 1 reactions in patients with a suspected allergy to cephalosporins.
Elevations in Serum Creatinine Concentration: Concerning or Reassuring?
Authors: Epstein Benjamin J.
Published: June 2004

The use of angiotensin‐converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) has significantly reduced morbidity and mortality across the continuum of vascular disease. The utilization of these agents, however, remains suboptimal. The drugs are not prescribed in many patients because of concerns regarding their effects on renal function. Despite overwhelming evidence in favor of renoprotection, it is not uncommon for the glomerular filtration rate (GFR) to decrease shortly after starting treatment with an ACE inhibitor or ARB. This response is functional in nature and should be expected based on renal physiology and its dependence on the renin‐angiotensin system to maintain GFR. Unfortunately, this phenomenon sometimes is viewed as an adverse effect or an indicator of underlying pathology. Although somewhat counterintuitive, early elevations in serum creatinine concentration are associated with improved long‐term renal outcomes in patients with renal insufficiency and thus support, rather than condemn, continued treatment. Clinicians should be aware of the physiologic course associated with blockade of the renin‐angiotensin system so that these agents will not be withheld unnecessarily.
Oral Health of Individuals with Cleft Lip, Cleft Palate, or Both
Authors: Lages Elizabeth Maria Bastos , Marcos Badeia , Pordeus Isabela Almeida
Published: June 2004

Objective

To evaluate the oral health of children with cleft lip and palate.

Design

DMFT (dmft) and DMFS (dmfs) were calculated on the basis of visual examination. Periodontal disease was assessed by classical clinical parameters: volume gain, bleeding, probing depth, loss of insertion, gingival recession. and calculus.

Patients

Seventy-eight individuals aged 1 to 32 years took part in the study.

Results

The dmft was 2.91 (± 3.99) in the 1- to 5-year-old age group and 2.77 (± 3.15) in the 6- to 12-year-old group. The DMFT averages for the age groups of 6 to 12 years, 13 to 18 years, and 19 to 32 years were 1.87 (± 1.78), 6.46 (± 3.11), and 13.62 (± 6.51), respectively. A total of 5.3% of individuals presented a healthy periodontium, and 86.6% presented with gingivitis and 8% presented with periodontitis.

Conclusions

The dental and periodontal condition of individuals with cleft lip and palate was similar to the general population in the region in which this study was conducted. Good preventive measures should result in improved oral health similar to the general population and should be emphasized.
Nonhuman Primates as Animal Models for Toxicology Research
Authors: Burbacher Thomas M. , Grant Kimberly S.
Published: June 2004

There is a long history for the use of nonhuman primates intoxicological research. This unit reviews applications in reproductivetoxicology and teratology, neural toxicology and neurobehavioral toxicology, imunotoxicology, respiratory (lung) toxicology, and chemical carcinogenesis.
The Use of Fish‐Derived Cell Lines for Investigation of Environmental Contaminants
Authors: Dayeh Vivian R. , Bols Niels C. , Schirmer Kristin , Lee Lucy E.J.
Published: June 2004

This unit describes protocols for growing salmonid cell lines and using them in in vitro toxicology studies. Cell viability of cultures is assessed with three indicator dyes: alamar blue for metabolic activity, CFDA‐AM for membrane integrity, and neutral red for lysosomal activity. These protocols are essential tools for investigating environmental toxicity at the cellular level.
Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants
Authors: Buznikov Gennady A. , Slotkin Theodore A. , Lauder Jean M.
Published: June 2004

Sea urchin embryos and larvae provide an inexpensive high‐throughput system for determining developmental actions of neuropharmacologic agents or environmental neurotoxins in both applied and basic biologic contexts. The use of this system for the testing of chlorpyrifos, 1‐nicotine, lipophilic amides of choline, and ritanserin is described in detail.
Zebrafish: An Animal Model for Toxicological Studies
Authors: Zhang Chaojie , Willett Catherine , Fremgen Trisha
Published: June 2004

Zebrafish (Danio rerio) has been extensively studied and well described for environmental toxicity studies. Molecular biology and genetics have recently been used to elucidate the underlying mechanisms of toxicity in zebrafish and to predict effects in mammals. The versatile zebrafish is now incorporated in many areas of toxicological programs for assessing human risk and for preclinical drug discovery and screening.
Preclinical Models of Parkinson's Disease
Authors: Bankiewicz Krys S. , Sanchez‐Pernaute Rosario , Oiwa Yoshitsugu , Kohutnicka Malgorzata , Cummins Alex , Eberling Jamie
Published: June 2004

Parkinson's disease (PD) is a neurodegenerative disorder in which pigmented midbrain neurons progressively die producing a dopamine (DA) deficit in the striatum, which manifests as an akinetic movement disorder. Experimentally induced striatal DA depletion in animals is a valid model of parkinsonism. The capacity of certain substances to damage catecholaminergic neurons has been used extensively to produce DA deficiency in animals. This unit describes methods for inducing parkinsonism in nonhuman primates and rodents using the neurotoxins 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and 6‐hydroxydopamine (6‐OHDA). Additionally, procedures for evaluating the animals are presented.
Measurements of Intracellular Free Calcium Concentration in Biological Systems
Authors: Gunter Karlene K. , Gunter Thomas E.
Published: June 2004

Intracellular calcium plays a role in signaling and as a second messenger in many types of cells and its concentration is closely regulated in cells. Two techniques for measuring intracellular free calcium are using fluorescent ratiometric and nonratiometric probes are described in this unit: fluorescent spectroscopy to measure calcium concentrations in a suspension of cells and fluorescent digital imaging microscopy (FDIM) to measure calcium concentrations in plated cells.
In Vitro Methods for Detecting Cytotoxicity
Authors: Ehrich Marion , Sharova Lioudmilla
Published: June 2004

Toxicant‐induced damage to cells (cytotoxicity) can depress cell growth, compromise intracellular metabolic processes, and/or cause loss of cell viability. Methods that indicate these cytotoxic changes following toxicant exposures are provided, including [3H]thymidine uptake to measure cell growth, MTT dye conversion to detect changes in metabolic activity, and trypan blue uptake to indicate loss of cell viability.
Confocal Microscopy
Authors: Ekström Peter
Published: June 2004

Confocal microscopy allows visualization of optical sections of material labeled with fluorescence or reflecting probes. By excluding light from planes above and below the plane of focus it is possible to obtain sharp images of objects deep within sections. Sections can be combined to construct three‐dimensional images. This unit provides an overview and introduction to confocal microscopy.
Detecting Epigenetic Changes: DNA Methylation
Authors: Broday Limor , Costa Max
Published: June 2004

This unit provides protocols for assessing the state of DNA methylation, including assays for determining the overall DNA methylation levels, measuring DNA methyltranseferase (MTase) activity, determining gene‐specific methylation and overall chromatin condensation patterns.
Measuring the Activity of Arylamine N‐Acetyltransferase (NAT)
Authors: McQueen Charlene A.
Published: June 2004

This unit describes methods for measuring the activity of arylamine N‐acetyltransferases (NAT). Genetic polymorphisms in NAT 1 and NAT 2 have been associated with susceptibility to aromatic amines carcinogens and effects of therapeutic drugs. Evaluation of the activities associated with substrates of NATs is helpful in elucidating the contribution of these enzymes to the pharmacologic and toxicologic effects of arylamines and hydrazines.
Measurement of Carboxylesterase (CES) Activities
Authors: Hosokawa Masakiyo , Satoh Tetsuo
Published: June 2004

Measurement of Carboxylesterase (CES) Activities (Masakiyo Hosokawa, Chiba University, Chiba, Japan and Tetsuo Satoh, Biomedical Research Institute, Chiba, Japan). Mammalian carboxyesterase (CES) efficiently catalyzes the hydrolysis of a variety of ester‐ and amide‐containing chemicals. CES is involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. This unit contains three protocols for measuring CES activities.
Analysis of the Aryl Hydrocarbon Receptor (AhR) Signal Transduction Pathway
Authors: Denison Michael S. , Rogers Jane M. , Rushing S. Renee , Jones Carol L. , Tetangco Selwyna C. , Heath‐Pagliuso Sharon
Published: June 2004

Analysis of the Ah Receptor Signal Transduction Pathway (Michael S. Denison, Jane M. Rohers, S. Renee Rushing. Carol L. Jones, Selwyna C. Tetangico, and Sharon Heath‐Pagliuso, University of California, Davis, California).The protocols in this unit will allow researchers to detect the Ah receptor and characterize its functional activities (i.e., ligand binding, transformation and DNA binding, and gene expression) in their biological test system and to use these methods to detect chemical and biochemical events that affect this signaling system.
Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases
Authors: Anspaugh Douglas D. , Roe R. Michael
Published: June 2004

Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolase and Phosphoric Triester Hydrolase (Douglas D. Anspaugh and Michael Roe, North Carolina State University, Raleigh, North Carolina). This unit describes assays that quantitate two types of esterase the carboxylic ester hydrolases and the phosphoric triester hydrolases. Carboxylic ester hydrolases include the B‐esterases, which are inhibited by organophosphorus compounds. Among the phosphoric triester hydrolases is aryldialkylphosphatase, which has been called A‐esterase or paraoxonase due to its ability to oxidize paraoxon and other organophosphates. These assays are colorimetric and miniaturized for rapid simultaneous testing of multiple, small‐volume samples in a microtiter plate format. There is also a discussion of the history of esterase nomenclature and the reasons why this large group of enzymes is so difficult to classify.
Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole
Authors: Usmani Khawja A. , Tang Jun
Published: June 2004

This unit describes methods for measuring CYP3A4 activity using testosterone as a specific substrate, and for measuring CYP3A4 inhibition using ketoconazole as a selective inhibitor of testosterone oxidation. CYP3A4 is one of the most important and most abundant drug‐metabolizing CYP isoforms in human liver microsomes (∼40% of total CYP), and it has the broadest substrate specificity. It is important to determine whether CYP3A4 is involved in its metabolism.
Overview of Glutathione Function and Metabolism
Authors: Will Yvonne
Published: June 2004

Glutathione is a key player in reduction processes in the cell. It is responsible for maintaining thiol groups of intracellular proteins by providing reducing power for cysteine, dihydolipoate, CoA, ascorbate, and vitamin E. It also plays a role in reduction of NTPs to dNTPs. Glutathione is also involved in detoxification of endogenous and exogenous compounds, participates in the synthesis of leukotrienes and prostaglandins, serves as a cofactor for various enzymes, stores and transports cysteine, and may be involved in cell cycle regulation and thermotolerance.
Measurement of Glutathione Conjugates
Authors: Shultz Michael A. , Morin Dexter , Chan Katherine Watt , Buckpitt Alan R.
Published: June 2004

The involvement of reactive metabolites in cancer and cellular necrosis has been well established. The nucleophile, glutathione, provides a major mechanism of intracellular protection from electrophilic metabolites. Conjugation with glutathione to generate stable, water‐soluble metabolites has been utilized to determine the nature and rates of formation of precursor reactive metabolites. In addition, because activities of the glutathione transferases may play a key role in tissue/cellular susceptibilities to electrophilic compounds, measurement of catalytic activities of these proteins can play an important role in discerning the underlying mechanisms of cell‐selective toxicities. This unit outlines HPLC methods found to provide good separation of glutathione conjugates and includes two additional procedures that can be utilized in experiments where high throughput assays are needed for measuring transferase activities.
Analysis of Superoxide Dismutase Activity
Authors: McCord Joe M.
Published: June 2004

Measuring the activity of superoxide dismutases (SODs), the enzymes responsible for maintaining the steady state level of hydrogen peroxide, is challenging because the substrate is unstable at physiological pH and it reacts with itself. Fortunately the rate of reaction with dismutase is far greater than the rate of self reaction. As described in this unit, this activity can be measured indirectly based on competition between SOD and an indicator molecule that reacts avidly with superoxide to produce a measurable change in absorption, thus it is possible to measure total SOD activity or that of CuZn‐SOD and MnSOD. The activity can also be measured by an activity stain applied to thin‐film agarose or native polyacrylamide gels.
Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates
Authors: Spitz Douglas R. , Oberley Larry W.
Published: June 2004

Three basic forms of mammalian SODs exist and they are distinguished by their sizes and locations. SOD enzyme activity is not easily monitored by direct measurement because the substrate disappearance is very rapid at physiological pH. Activity can be measured as described in this unit by a number of indirect competitive inhibition assays based on the principle that the superoxide anion radical will reduce an inhibitory substrate [such as nitroblue tetrazolium (NBT) or cytochrome c] and SOD activity will reduce the rate of reduction in a competitive fashion. The SOD‐mediated inhibition of the indicator substrate reduction can then be quantitated and plotted as a function of the quantity of protein added to the reaction to construct an inhibition curve.
Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples
Authors: Lykkesfeldt Jens
Published: June 2004

Ascorbic acid and dehydroascorbic acid are commonly used biomarkers of oxidative stress in a variety of experimental models. However, the accurate measurement of these labile compounds remains a challenge both in terms of sample collection and analysis. Determination of dehydroascorbic acid most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a method referred to as the subtraction method. Consequently, successful determination of dehydroascorbic acid is dependent upon proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. The unit presents a detailed introduction to ascorbate analysis in biological samples and discusses common problems and pitfalls. The analytical method described is based on reversed‐phase HPLC with coloumetric detection. This method includes co‐analysis of isoascorbic acid and uric acid. Where applicable, uric acid can conveniently be used as an endogenous intrasample standard that significantly improves the accuracy of the subsequent dehydroascorbic acid calculation.
Measurement of ALA Synthase Activity
Authors: Sinclair Peter R. , Gorman Nadia , Cornell Neal W.
Published: June 2004

In most cells, δ‐aminolevulinate (ALA) synthase is the rate‐limiting enzyme in heme synthesis. It is inducible by drugs and toxins and is feedback regulated by heme. This unit describes a radiometric assay using [14C]succinate as a substrate and a colorimetric assay based on the conversion of ALA to a pyrrole.
Measurement of Uroporphyrinogen Decarboxylase Activity
Authors: Phillips John D. , Kushner James P.
Published: June 2004

Uroporphyrinogen 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

Protoporphyrinogen 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

Changes 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

The 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

Basal 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

The 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

NO‐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

Neurobehavioral 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

As 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

Cultured 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

Cerebellar 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.
Overview of Teratology
Authors: Kimmel Carole A.
Published: June 2004

Teratology is the study of developmental processes to understand the causes and mechanisms of abnormal developmental outcomes. This unit provides an explanation of the basic principles of the field as a context for the more specialized protocol units to follow in the chapter.
Micromass Cultures in Teratology
Authors: Ponce Rafael A.
Published: June 2004

This 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

This 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

A 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.
Genotyping Embryonic Stem (ES) Cells
Authors: Schmidt Jennifer V.
Published: June 2004

This unit describes a means for isolating DNA from clones cultured in 96‐well plates. The extracted DNA can be used for Southern blotting or PCR to verify the genotype of the ES clone.
Aggregation Chimeras (ES Cell–Embryo)
Authors: Lobe Corrinne G. , Guo Caiying
Published: June 2004

One 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

In 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

Monitoring 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

This 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

Protein 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

This 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

Abnormalities 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

This 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.
Transgenic and Gene‐Targeted Mouse Lines for Toxicology Studies
Authors: Sanders George , Ware Carol , Ladiges Warren
Published: June 2004

This unit contains an extensive tabular listing of transgenic and gene‐targeted mice useful in toxicological studies. Each listing contains a brief description of the phenotype, relevant references, and where applicable, commercial sources.
Overview of Protein Purification and Characterization
Authors: Scopes R.K.
Published: June 2004

This 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

This 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

Protein 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

This 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

The 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

Protein 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

The 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

This 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 the Baculovirus Expression System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen
Published: June 2004

Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This overview unit describes the baculovirus life cycle and expression system, and also provides information on vectors and protocols for using the baculovirus expression system.
Overview of Protein Expression by Mammalian Cells
Authors: Gray David
Published: June 2004

This 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

This 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

After 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.