bluuebunny/crossref_metadata_2025 huggingface dataset:
The Frequency and Cost of Patient‐Initiated Device Removal in the ICU
Authors: Fraser Gilles L. , Riker Richard R. , Prato Stephen , Wilkins Micheline L.
Published: June 2004

Objective. To determine the frequency and pattern with which patients in the intensive care unit (ICU) remove medical devices on their own, and the costs associated with this problem.

Design. Prospective observational study.

Setting. Two 10‐bed sections of a multidisciplinary ICU in a tertiary care teaching hospital.

Patients. Adults admitted to the ICU for longer than 24 hours during October 1998.

Interventions. None.

Measurements and Main Results. Medical records were reviewed prospectively for the occurrence of patient‐initiated device removal and the responses to those events by health care providers. Associated costs were estimated using hospital databases and Medicare physician reimbursement schedules. Annual cost estimates were calculated using 1997 admission statistics for 1211 adults in an ICU for more than 24 hours. Thirty‐six patients were studied for 199 patient‐days. Ten patients (28%) removed 42 devices: 88% of these events involved gastrointestinal tubes and vascular catheters. Significant agitation was documented within 2 hours before 74% of the events. Estimated cost associated with device removal was $7606, or $181/event. The estimated annual cost in this 42‐bed ICU was more than $250,000.

Conclusions. Patients commonly remove medical devices on their own, and this represents significant consumption of health care resources.
Elevation of Hepatic Transaminases after Enoxaparin Use: Case Report and Review of Unfractionated and Low‐Molecular‐Weight Heparin‐Induced Hepatotoxicity
Authors: Carlson Melissa K. , Gleason Patrick P. , Sen Soma
Published: June 2004

Enoxaparin, a low‐molecular‐weight heparin (LMWH), is widely used for the treatment and prophylaxis of thromboembolic disorders, such as deep vein thrombosis. Low‐molecular‐weight heparin products have smaller and more uniform molecular weights than unfractionated heparin, allowing them to exhibit a much greater affinity for factor Xa than factor IIa. Compared with traditional unfractionated heparin, LMWHs have proved to be equally efficacious and may be safer. The distinctive characteristics of LMWHs have resulted in decreased rates of bleeding and equivalent rates of thrombocytopenia compared with unfractionated heparin. This favorable safety profile has been identified in several studies and may have led clinicians to believe that LMWHs have lower frequencies of all common side effects. A 66‐year‐old woman developed increased hepatic transaminases during treatment with enoxaparin for a deep vein thrombosis; they returned to normal after enoxaparin discontinuation. A causal relationship between unfractionated heparin and asymptomatic, transient increases in hepatic transaminase levels has been documented; these increased levels also appear to be an underrecognized, adverse effect of LMWH therapy.
Phosphorylation of Nucleoside Analog Antiretrovirals: A Review for Clinicians
Authors: Stein Daniel S. , Moore Katy H. P.
Published: June 2004

Nucleoside analogs (zidovudine, didanosine, zalcitabine, stavudine, abacavir, lamivudine) have been administered as antiretroviral agents for more than a decade. They undergo anabolic phosphorylation by intracellular kinases to form triphosphates, which inhibit human immunodeficiency virus replication by competitively inhibiting viral reverse transcriptase. Numerous methods are used to elucidate the intracellular metabolic pathways of these agents. Intracellular and extracellular factors affect intracellular phosphorylation. Lack of standardization and complexity of methods used to study phosphorylation in patients limit interpretation of study results and comparability of findings across studies. However, in vitro and in vivo studies give important insights into mechanisms of action, metabolic feedback mechanisms, antiviral effects, and mechanisms of toxicity, and have influenced dosing regimens of nucleoside analogs.
Visual Disturbance Associated with Celecoxib
Authors: Lund Brian C. , Neiman Richard F.
Published: June 2004

Celecoxib, a specific inhibitor of cyclooxygenase‐2, is used to treat the symptoms of arthritis. A 79‐year‐old woman developed an atypical visual disturbance associated with this agent that resolved on discontinuation of celecoxib. Similar visual disturbances described with the traditional nonsteroidal antiinflammatory drugs are discussed.
Gatifloxacin, an Advanced 8‐Methoxy Fluoroquinolone
Authors: Fish Douglas N. , North Donald S.
Published: June 2004

Gatifloxacin is a new 8—methoxy‐fluoroquinolone antibiotic approved for use in the United States in December 1999. It has a broad spectrum of activity with potent activity against gram‐positive bacteria, including penicillin‐resistant Streptococcus pneumoniae, as well as excellent activity against gram‐negative and atypical organisms. Gatifloxacin is available in both oral and injectable forms and is administered once/day. Bioavailability is 96%, with a plasma half‐life of approximately 8 hours in individuals with normal renal function. Elimination is primarily renal excretion of unchanged drug with no cytochrome P450‐mediated metabolism. The drug is distributed extensively into tissues and fluids and has a favorable pharmacodynamic profile against important pathogens. It had excellent efficacy in clinical studies of acute sinusitis, acute bacterial exacerbations of chronic bronchitis, community‐acquired pneumonia, complicated and uncomplicated urinary tract infections and pyelonephritis, skin and skin structure infections, and uncomplicated gonococcal infections. The agent is well tolerated, with no evidence of hepatic, cardiac, or phototoxicity noted thus far. Drug interactions are uncommon; however, like other fluoroquinolones, coadministration with multivalent cations should be avoided due to significantly decreased absorption. Gatifloxacin should prove to be a safe and effective agent for a wide variety of infections.
Misoprostol Therapeutics Revisited
Authors: Davies Neal M. , Longstreth James , Jamali Fakhreddin
Published: June 2004

Misoprostol, a prostaglandin E1analog, is a racemate of four stereoisomers. On administration it rapidly de‐esterifies to its active form, misoprostolic acid. Misoprostolic acid is 85% albumin bound and has a half‐life of approximately 30 minutes. It is excreted in urine as inactive metabolites. No significant drug interactions have been reported. Besides its gastrointestinal protective and uterotonic activities, misoprostol regulates various immunologic cascades. It inhibits platelet‐activating factor and leukocyte adherence, and modulates adhesion molecule expression. It protects against gut irradiation injury, experimental gastric cancer, enteropathy, and constipation. It improves nutrient absorption in cystic fibrosis. Misoprostol has utility in acetaminophen and ethanol hepatotoxicity, hepatitis, and fibrosis. It is effective in asthmatics and aspirin‐sensitive asthmatic and allergic patients. It lowers cholesterol and severity of peripheral vascular diseases, prolongs survival of cardiac and kidney transplantation, synergizes cyclosporine, and protects against cyclosporine‐induced renal damage. It works against drug‐induced renal damage, interstitial cystitis, lupus nephritis, and hepatorenal syndrome. It is useful in periodontal disease and dental repair. Misoprostol enhances glycosoaminoglycan synthesis in cartilage after injury. It prevents ultraviolet‐induced cataracts and reduces intraocular pressure in glaucoma and ocular hypertension. It synergizes antiinflammatory and analgesic effects of diclofenac or colchicine and has been administered to treat trigeminal neuralgic pain. It reduces chemotherapy‐induced hair loss and recovery time from burn injury, and is effective in treating sepsis, multiple sclerosis, and pancreatitis.
A Multicenter Evaluation of Gentamicin Therapy in the Neonatal Intensive Care Unit
Authors: Glover Mark L. , Shaffer Christopher L. , Rubino Christopher M. , Cuthrell Corey , Schoening Shellie , Cole Erika , Potter David , Ransom J. Laurence , Gal Peter
Published: June 2004

Study Objective. To evaluate traditional nomogram (TN) versus individualized pharmacokinetic gentamicin dosing practices in neonatal intensive care units, focusing on achieving target therapeutic concentrations (peak > 8 μg/ml, trough < 2 μg/ml), number of dosing changes, number of concentrations obtained, and evidence of nephrotoxicity.

Design. Retrospective chart review.

Setting. Three neonatal intensive care units.

Patients. Three hundred nine infants prescribed gentamicin.

Intervention. None.

Measurements and Main Results. Sixty‐seven percent of patients receiving pharmacokinetic dosing had initial peak concentrations of 8 μ/ml or greater compared with 7% of patients receiving TN dosing (p<0.001). Trough concentrations exceeding 2 μ/ml were reported in 23% of patients receiving TN dosing compared with 2% of pharmacokinetic‐dosed patients (p<0.001). Forty‐two percent and 6%, respectively, required dosage adjustments (p<0.01). The mean number of concentrations obtained per patient was 2.8 and 2.1, respectively (p<0.01). Neither group had evidence of gentamicin‐related nephrotoxicity.

Conclusion. Compared with TN dosing, administering gentamicin loading doses and performing initial pharmacokinetic analysis resulted in rapid attainment of desired concentrations and fewer dosage adjustments, and allowed for a decrease in the number of gentamicin concentrations.
Pharmacotherapy of Behavioral and Psychological Symptoms of Dementia: Time for a Different Paradigm?
Authors: Rojas‐Fernandez Carlos H. , Lanctôt Krista L. , Allen David D. , MacKnight Chris
Published: June 2004

Behavioral and psychological symptoms of dementia can occur in 60–80% of patients with Alzheimer's disease or other dementing illnesses, and are important in that they are a source of significant caregiver stress and often precipitate nursing home placement. These symptoms, namely, aggression, delusions, hallucinations, apathy, anxiety, and depression, are clinically managed with a variety of psychotropic drugs such as antipsychotics, antidepressants, antiepileptic drugs, and benzodiazepines. Various advances in the neuropathophysiology and pharmacotherapy must be considered in the optimal design of regimens for patients with these symptoms.
Retrospective Evaluation of the Effect of Valproate Therapy on Transaminase Elevations in Patients with Hepatitis C
Authors: Lott Rex S. , Helmboldt Kristin M. , Madaras‐Kelly Karl J.
Published: June 2004

Study Objective. To evaluate whether a relationship exists between valproate treatment and transaminase elevations in patients who are positive for hepatitis C virus (HCV).

Design. Retrospective medical record review.

Setting. Veterans affairs medical center.

Patients. A total of 214 HCV‐positive patients; 28 were treated with valproate (study group), 186 were not (control group).

Intervention. Demographic characteristics; valproate treatment histories; plasma concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), albumin, and bilirubin; and exposure to other potentially hepatotoxic drugs, including probable alcohol abuse, were evaluated.

Measurements and Main Results. Hepatotoxicity was staged by comparing maximum ALT and AST values against upper limits of normal or patients' mean elevated baseline ALT and AST values. Data were analyzed using a logistic regression model. Control patients and those with longer durations of HCV were more likely (p<0.0171 and p<0.0142, respectively) to exhibit higher stages of hepatotoxicity. More valproate‐treated patients were exposed to other potential hepatotoxins at the time of peak transaminase elevations (50% vs 39%, p<0.005), whereas more control patients received two or more potential hepatotoxins (13% vs 4%, p<0.00005).

Conclusion. Valproate treatment, either alone or in the presence of other potential hepatotoxins, does not appear to be related to increased transaminase elevations in patients with HCV.
Lopinavir‐Ritonavir: A New Protease Inhibitor
Authors: Mangum Eric M. , Graham Kathleen K.
Published: June 2004

Lopinavir is a new protease inhibitor that is structurally related to ritonavir. It recently was approved by the Food and Drug Administration as a coformulation with ritonavir under the brand name Kaletra. Ritonavir substantially increases lopinavir drug exposure by inhibiting cytochrome P450 isoenzyme 3A4. Based on limited data, lopinavir‐ritonavir demonstrates safety and efficacy in both antiretroviral‐naïve and protease inhibitor‐experienced patients. It has the ability to durably suppress human immunodeficiency virus (HIV) RNA for up to 2 years in antiretroviral‐naïve patients. Compared with nelfinavir, it had superior virologic control at 48 weeks in antiretroviral‐naïve patients. Its side effects include diarrhea, abnormal stools, abdominal pain, nausea, vomiting, and asthenia. A number of patients experienced grade 3–4 laboratory abnormalities in liver function tests, cholesterol, and triglycerides while receiving this drug combination. The exact resistance patterns of lopinavir‐ritonavir are unknown, but the Department of Health and Human Services strongly recommends it for the initial treatment of HIV‐infected adults and adolescents.
Prussian Blue for Treatment of Radiocesium Poisoning
Authors: Thompson Dennis F. , Church Chelsea O.
Published: June 2004

Prussian blue is a crystal lattice that exchanges potassium for cesium at the surface of the crystal. When given orally, it binds cesium that is secreted in the gut before it can be reabsorbed. Data suggest that in humans, Prussian blue can reduce cesium's half‐life by approximately 43% and reduce total body burdens. Prussian blue is well tolerated at a dosage of 3 g/day with appropriate monitoring of serum potassium levels and observing for signs of constipation. Clinical data on the efficacy of Prussian blue in the management of radiocesium poisoning were evaluated. Articles published in English describing distribution and elimination of cesium in both humans and animals were reviewed, along with articles describing administration of Prussian blue in clinical toxicology.
Triiodothyronine Supplementation in Patients Undergoing Cardiopulmonary Bypass
Authors: Reichert Marc G. , Verzino Kelly C.
Published: June 2004

Patients undergoing cardiopulmonary bypass may develop clinically significant physiologic alterations in the perioperative period, including alteration of thyroid hormone concentrations. Alterations in the concentration of thyroid hormones are of concern due to the effects of these hormones on cardiac function. Hypothyroidism is associated with a decrease in cardiac performance; therefore, supplementation with the active thyroid hormone triiodothyronine (T3) in patients undergoing cardiopulmonary bypass has been investigated to improve outcomes. In addition, T3 has been studied as an agent to reduce the frequency of atrial fibrillation after cardiopulmonary bypass.
Levetiracetam: A Novel Antiepileptic Drug
Authors: Hovinga Collin A.
Published: June 2004

Levetiracetam is a new antiepileptic drug, structurally and mechanistically dissimilar to other marketed antiepileptic drugs. It is effective in reducing partial seizures in patients with epilepsy, both as adjunctive treatment and as monotherapy. Levetiracetam has many therapeutic advantages for patients with epilepsy. It has favorable pharmacokinetic characteristics (good bioavailability, linear pharmacokinetics, insignificant protein binding, lack of hepatic metabolism, and rapid achievement of steady‐state concentrations) and a low potential for drug interactions. Recommended starting dosages are considered to be clinically effective; therefore, patients can have some protection from seizures soon after they begin levetiracetam. The most common adverse effects observed with levetiracetam are mild and include somnolence, asthenia, and dizziness. Clinical experience and data from meta‐analyses indicate that levetiracetam is well tolerated, with efficacy comparable or slightly better than that observed with other new antiepileptic drugs. Levetiracetam may be particularly useful in patients who are unresponsive to other antiepileptic drugs, patients receiving drugs with increased potential for drug interactions, or those with hepatic impairment.
Three Michaelis‐Menten Pharmacokinetic Dosing Methods Compared with Physician Dosing of Phenytoin in an Outpatient Neurology Practice
Authors: Spruill William J. , Wade William E. , Cobb Henry H. , Akbari Sara
Published: June 2004

We compared predicted phenytoin serum concentrations using three Michaelis‐Menten pharmacokinetic dosing methods with actual concentrations obtained from physician dosing in an outpatient neurology practice. Method 1 used population estimates for the Michaelis‐Menten constant (Km) and maximum velocity (Vmax), method 2 used one dose and serum concentration pair to determine Vmax, and method 3 used two dose‐concentration pairs to determine both Km and Vmax. In addition, physician doses were compared with pharmacokinetically calculated doses. Records of patients who received at least two phenytoin doses followed by two serum concentration determinations were reviewed. Data on age, gender, weight, physician doses, and resultant serum concentrations were collected. Pearson's correlation coefficient was used to compare physician maintenance doses with pharmacokinetically calculated predicted doses, whereas actual and predicted serum concentration data were used to determine precision and bias associated with each of the three methods. Actual serum concentrations fell into therapeutic range more frequently than predicted values in all but one comparison (method 3). Predicted and actual phenytoin doses were significantly correlated only with method 2. Only one of the three Michaelis‐Menten pharmacokinetic dosing methods evaluated (method 3) was more predictive than physician phenytoin dosing.
Treatment Options for a Patient Experiencing Pruritic Rash Associated with Transdermal Testosterone: A Review of the Literature
Authors: McGriff Nayahmka J. , Csako Gyorgy , Kabbani Mahmoud , Diep Long , Chrousos George P. , Pucino Frank
Published: June 2004

A 22‐year‐old man with hypogonadotropic hypogonadism was receiving monthly intramuscular injections of testosterone replacement therapy. The patient refused to self‐administer the injections because of discomfort, so the therapy was switched to testosterone patches. He experienced a pruritic, macular, erythematous rash underneath the reservoir area of two different transdermal formulations, which did not improve after pretreatment with topical corticosteroids. Eventually, he tolerated application of a testosterone gel and his serum testosterone levels returned to normal after 1 month of therapy. Commercially available and investigational testosterone products and therapeutic monitoring guidelines for androgen replacement are reviewed.
Dramatic Worsening of Type 2 Diabetes Mellitus Due to Olanzapine After 3 Years of Therapy
Authors: Bechara Christopher I. , Goldman‐Levine Jennifer D.
Published: June 2004

Olanzapine, a serotonin‐dopamine‐receptor antagonist, is an atypical antipsychotic agent used to treat schizophrenia and other psychotic disorders. It is preferred over older antipsychotics because of its relatively low frequency of sedation, orthostatic hypotension, extrapyramidal symptoms, and anticholinergic side effects. A 45‐year‐old man with well‐controlled type 2 diabetes mellitus experienced an abrupt worsening of his diabetes after 3 years of olanzapine therapy. His hemoglobin A1c (HbA1c) level rose from a baseline of 5.9–6.2% to 12.5%. Discontinuation of olanzapine by means of a 3‐month taper resulted in a reduction in HbA1c to pretreatment levels. Although cases of olanzapine‐induced hyperglycemia have been documented in the literature, this complication has not been reported in a patient maintained on therapy for this duration. Clinicians should be aware of this possible complication in patients receiving long‐term olanzapine therapy.
Establishing and Maintaining System Linearity
Authors: Wood James C. S.
Published: June 2004

A flow cytometer system transforms a received optical signal into an electrical signal whose amplitude ideally is directly proportional to the optical signal but in practice is at least related in a linear fashion. Establishment and maintenance of system linearity are prerequisites to accurate quantitative measurements. The many potential sources of offsets and nonlinearity are found in all parts of the detection, amplification, and data acquisition systems of a flow instrument. This unit aims to help investigators evaluate performance of the hardware in flow cytometer detection systems.
Digital Fluorescence Microscopy
Authors: Tanke Hans J.
Published: June 2004

Fluorescence microscopy is a valuable tool for biomedical research, providing good sensitivity and high multiplicity. Specialized techniques (anisotropy measurement, resonance energy transfer) can provide information on cell spatial arrangement or label microenvironment. The unit discusses camera selection strategy, microscope hardware, and image acquisition and processing.
Shading Correction: Compensation for Illumination and Sensor Inhomogeneities
Authors: Young Ian T.
Published: June 2004

The interaction between objects in real space, the illumination, and the camera frequently leads to a situation in which a microscope image exhibits significant shading across the field of view. In general this shading effect is undesirable and requires elimination, especially for quantitative microscopy. Starting with a simple mathematical model, this unit develops procedures to correct images for the shading thus introduced in the image‐formation process. Two cases are distinguished: the a priori, which assumes the availability of calibration images in addition to the images of interest; and the a posteriori, which assumes that only the recorded images of interest are available.
High‐Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low‐Abundance Membrane Molecules
Authors: Zola Heddy
Published: June 2004

Cell marker identification by traditional phenotyping techniques is now considered straight‐forward and relatively uncomplicated. In immunofluorescence/flow cytometry the sensitivity, or detection limit, depends on the reagents, staining, and instrument parameters. The sensitivity of the most commonly used procedures, based on fluorescein‐conjugated antibodies, is about 2,000 molecules of target antigen per cell, which is adequate for most of the widely used leucocyte markers. However, measuring target antigens of low density has proved very difficult indeed. Flow cytometric immunofluorescence is capable of detecting 100 molecules of target antigen per cell in practical applications, provided every step of the staining and analysis procedure is optimized for sensitivity. This level of sensitivity reveals staining not seen using conventional analytical procedures. This unit discusses the underlying principles of high‐sensitivity immunofluorescence and provides an excellent series of protocols for the practical detection of as few as 100 target antigen molecules per cell.
Whole Blood Analysis of Leukocyte‐Platelet Aggregates
Authors: Barnard Marc R. , Krueger Lori A. , Frelinger A.L. , Furman Mark I. , Michelson Alan D.
Published: June 2004

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. The platelet binding is initiated primarily through platelet surface expression of P‐selectin (CD62P) following activation‐dependent degranulation. The levels of P‐selectin involved can be low enough to make direct measurement difficult, but detection of leukocyte‐platelet aggregates is relatively simply by whole‐blood flow cytometry. Light scatter and at least one leukocyte‐specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet‐specific markers.
Quality Control in Nucleic Acid Analysis
Authors: Hurley Anne A.
Published: June 2004

All flow cytometry systems require instrument quality control procedures such as are described for phenotyping. However, there are additional considerations for DNA analysis beyond those outlined for other types of analyses. Because flow cytometric analysis of cellular DNA content is an established procedure for measurement of DNA ploidy of tumors and of the fraction of cells in the S phase of the cell cycle two parameters that have prognostic utility in clinical oncology careful quality control of this type of analysis is essential. This unit discusses the quality control issues peculiar to quantitative analysis of cellular DNA content and provides information complementary to many of the units in this chapter.
Analysis of DNA Content and BrdU Incorporation
Authors: Darzynkiewicz Zbigniew , Juan Gloria
Published: June 2004

During the past two decades, the techniques of analysis of cell cycle kinetics based upon the flow cytometric detection of BrdU incorporation have replaced the cumbersome autoradiographic methodology utilizing tritiated thymidine. This unit distills the knowledge gained over this time into concise and easy to follow protocols for DNA content analysis following thermal denaturation or acid denaturation, as well as a modified method that combines thermal denaturation with washless immunocytochemical detection of BrdU.
Combination DNA/RNA Fish and Immunophenotyping
Authors: Dirks Roeland W.
Published: June 2004

This unit presents methods for combining immunophenotyping with DNA/RNA FISH. The approach is used in so‐called genotype/phenotype analysis to identify chromosomal aberrations in sub‐populations of cells present in heterogenous populations. Combining RNA and DNA detection with identification of cellular proteins is quite difficult. This series of protocols is provided to enable the successful application of the combination of these techniques.
Assays of Natural Killer (NK) Cell Ligation to Target Cells
Authors: Renò Filippo , Papa Stefano , Vitale Marco , Zamai Loris
Published: June 2004

Assays for natural killer cells have long been established in the flow repertoire, but successful application of a flow‐based protocol requires attention to many details. This unit provides these details in a comprehensive manner. In particular, the assay is designed for simple bench‐top cytometers, rather than for more complex research instruments. Following this protocol, even a novice user should be able to achieve successful completion of an NK assay.
Flow Cytometric Analysis of Cell Division by Dye Dilution
Authors: Lyons A. Bruce , Doherty Kathleen V.
Published: June 2004

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to tag proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division as well as to in vivo division of adoptively transferred cells and can resolve up to eight successive generations. CFSE is long lived, permitting analysis for several months after transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells.
Reporters of Gene Expression: Autofluorescent Proteins
Authors: Rasko John E.J.
Published: June 2004

This unit on GFP provides clear, detailed, easy‐to‐follow directions for successful transduction of GFP into appropriate cell lines, as well as carefully detailed strategy and trouble‐shooting sections. Instructions for sorting and purification of sucessfully transduced cells and for analysis and re‐analysis of cells together with sample data are all included. The discussion provides data on multiple GFP mutants and lists relevant sources and suggested reading material.
Multidimensional Data Analysis in Immunophenotyping
Authors: Loken Michael R.
Published: June 2004

The complexity of cell populations requires careful selection of reagents to detect cells of interest and distinguish them from other types. Additional reagents are frequently used to provide independent criteria for cell identification. Two or three monoclonal antibodies in combination with forward and right‐angle light scatter generate a data set that is difficult to visualize because the data must be represented in four‐ or five‐dimensional space. The separation between cell populations provided by the multiple characteristics is best visualized by multidimensional analysis using all parameters simultaneously to identify populations within the resulting hyperspace. Groups of cells are distinguished based on a combination of characteristics not apparent in any usual two‐dimensional representation of the data.
Antibiotic Susceptibility Testing by Flow Cytometry
Authors: Mason David J. , Mortimer Fiona C. , Gant Vanya A.
Published: June 2004

Flow cytometry can provide a rapid indication of antibiotic susceptibility. This unit describes a number of fluorescent dyes that allow the cytometer to distinguish the heterogeneic nature of populations exposed to antibiotics. No single method can be used for multiple organisms, since it is the interference with the organisms growth and structural integrity that signals the impact of the antibiotic so rapidly. This unit discusses membrane structural changes as well as alterations in nucleic acid and the probes that can be successfully used with flow cytometry.
Functional Assays of Oxidative Stress Using Genetically Engineered Escherichia coli Strains
Authors: Herrera Guadalupe , Martínez Alicia , O'Cornor José‐Enrique , Blanco Manuel
Published: June 2004

Oxidative stress may be induced in bacteria by exogenous biocidal agents and is involved in endogenous metabolism. The oxyR operon is a main sensor of oxidative stress and oxyR‐deficient bacteria show enhanced sensitivity to oxidative stress and increased accumulation of intracellular reactive oxygen species (ROS). Flow cytometric functional assays in bacteria are limited by the impaired penetration of vital dyes trough the cell wall. Escherichia coli B WP2 strains possess an altered cell‐wall lipopolysaccharide that leads to increased membrane permeability. Flow cytometric analysis of WP2 strains is a convenient alternative for cytometric assays of bacterial function. This unit presents protocols for flow cytometric studies of intracellular oxidative stress in two E. coli B WP2 strains, wild‐type or deficient in the oxyR function, using ROS‐sensitive fluorogenic substrates. Support Protocols describe preparation of phage C21 stock for bacterial verification, verification of the WP2 phenotype, and verification of the deficiency in oxyR function.
Assessing and Controlling Microbial Contamination in Cell Cultures
Authors: Coté Rosalie
Published: June 2004

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary.
Preparation of Human Epidermal Keratinocyte Cultures
Authors: Yamada Susan S.
Published: June 2004

Cultured keratinocytes have been used by a number of investigators in studies investigating wound repair and carcinogenesis, and they have also proven useful as a model for differentiation. This unit describes a protocol for establishing human keratinocytes in tissue culture. Human newborn foreskins are proteolytically digested to separate the epidermis and the dermis, and keratinocytes are obtained from the epidermis.
Purification of a Crude Mitochondrial Fraction by Density‐Gradient Centrifugation
Authors: Graham John M.
Published: June 2004

Most mitochondria prepared by differential centrifugation are contaminated to some extent by lysosomes, peroxisomes, tubular Golgi membranes, and small amounts of endoplasmic reticulum. Density gradient centrifugation using a variety of density media‐‐sucrose, Percoll, Nycodenz, Iodixanol‐‐is described here and is used to prepare purer fractions of mitochondria. The resulting gradient can be analyzed for three marker enzymes: succinate dehydrogenase (mitochondria), b‐galactosidase (lysosomes), and catalase (peroxisomes).
Metabolic Labeling of Prenyl and Carboxyl‐Methyl Groups
Authors: Magee Anthony I.
Published: June 2004

This unit provides protocols for prenylation and carboxy‐methylation of proteins in cultured cells. These modifications often accompany fatty acid acylation. Cultured cells can be labeled biosynthetically using radiolabeled mevalonate, a precursor, to label intermediates that are incorporated as prenoids‐‐e.g., farnesyl and geranylgeranyl. Carboxy‐methylation often accompanies prenylation. The methyl group can be labeled using [3H‐methyl]methionine.
Radioiodination of Cellular Proteins
Authors: Caplan Steve , Baniyash Michal
Published: June 2004

Despite the advances of non‐radioactive methods of protein labeling, radioiodination remains an important tool for studying proteins. This unit discusses several commonly used methods for radioiodination of proteins for various purposes. Included are several simple, reliable protocols for radioiodination of cell surface proteins on live cells, radioiodination of purified, solubilized membrane proteins, and radioiodination of purified soluble proteins.
Centrifugal Elutriation to Obtain Synchronous	Populations of Cells
Authors: Wahl Alan F. , Donaldson Karen L.
Published: June 2004

Counterflow centrifugal elutriation is a noninvasive method for separating large numbers of cells on the basis of their size and mass. For mammalian cells, this method is useful for separating mixed populations of cells, in particular cells at different stages of the cell division cycle without perturbing cell metabolism or using synchronizing agents. This unit describes a method for separating 2 x 108 cells using the standard JE‐6B rotor or larger numbers of cells in the JE‐5.0 rotor. To verify the purity and to characterize the cell cycle positions of cells in the elutriated populations, the unit includes protocols for measuring nascent DNA synthesis by [3H]thymidine incorporation and for detecting DNA synthesis and content by propidium iodide flow cytometry alone or in combination with bromodeoxyuridine incorporation.
Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF‐HR9 Endodermal Cells
Authors: Vlodavsky Israel
Published: June 2004

The ECM is an organized complex of collagens, proteoglycans, and glycoproteins, all interacting to produce a highly stable structure upon which cells migrate, proliferate, differentiate, and survive in vivo. Cultured bovine corneal endothelial (BCE) cells and PF‐HR9 endodermal cells produce underlying ECMs that adhere strongly to plastic and closely resemble subendothelial and subepithelial basement membranes in vivo in their morphology, molecular composition, and biological activities. This unit describes the methods for preparation of these ECMs, their properties, and their cellular effects.
In Vitro Analysis of Chloroplast Protein Import
Authors: Smith Matthew D. , Schnell Danny J. , Fitzpatrick Lynda , Keegstra Kenneth
Published: June 2004

This unit describes protocols for isolating chloroplasts from pea (Pisum sativum) and Arabidopsis thaliana for the study of nuclear‐encoded plastid precursor proteins. Chloroplasts from both preparations are competent for the in vitro import of recombinant preproteins synthesized using in vitro translation systems derived from reticulocyte or wheat germ lysates. These assays can be used to test whether a particular protein is targeted to chloroplasts, for analyzing the suborganellar location of newly imported preproteins, or to study the mechanism of import itself.
In VitroRNASplicing in Mammalian Cell Extracts
Authors: Rooke Nanette , Underwood Jason
Published: June 2004

Almost every eukaryotic pre‐mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue‐specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell‐free, system. Cell‐free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre‐mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.
Cell Traction
Authors: Galbraith Catherine G. , Sheetz Michael P.
Published: June 2004

Traction forces are exerted by cells on their substratum as they migrate. These forces under the entire cell or subcellular regions can be measured. This unit describes several protocols for making silicone sheets to measure traction forces under the entire cell, as well as a protocol for developing a micromachined device to measure forces under subcellular regions.
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Authors: Sefton Bartholomew M.
Published: June 2004

Signal transduction pathways may involve protein phosphorylation at one or more residues. Detection of phosphorylation involves labeling with radioactive inorganic phosphate and subsequent immunoprecipitation with appropriate antibodies. This unit describes labeling conditions for adherent and nonadherent cells and preparation of the lysates from these cells for immunoprecipitation. The labeling method is appropriate for labeling other phosphorylated component of cells, but alternate methods are necessary for their detection.
Use of Glycosidases to Study Protein Trafficking
Authors: Freeze Hudson H.
Published: June 2004

As proteins transit through the cell secretory pathway, modification of their substituent sugar chains occurs in a stepwise fashion. In the course of this processing (maturation) of oligosaccharide chains, the chains acquire sensitivity or resistance to highly specific glycosidases. Thus it is possible to identify processing mileposts by analyzing the general structure of the carbohydrate chains. This unit describes reaction conditions for the family of glycosidases and analysis of the results of digestion reactions.
Endocytosis: Biochemical Analyses
Authors: McGraw Timothy E. , Subtil Agathe
Published: June 2004

Many integral membrane proteins synthesized in the endoplasmic reticulum ultimately arrive at the cell surface to contact the cell environment. During transit through the Golgi and trans‐Golgi network, proteins acquire post‐translational modifications that can be used to track the appearance of such modified proteins at the cell surface. Cellular proteins can be treated with enzymes‐‐e.g., sialidase or protease‐‐or antibodies, or biotinylated to identify molecules that have reached the cell surface. Some proteins first enter the endocytic pathway before appearing at the cell surface; this is detected by treating the cells at 4o and 37oC. Analysis of the number of sialic acids on proteins of cells treated at 4oC identifies proteins resident at the cell surface, while cells treated at 37oC internalize the sialidase, which can then act with proteins in the endocytic compartments.
Current Concepts in Cell Death
Authors: Zhivotovsky Boris , Orrenius Sten
Published: June 2004

This unit attempts to define cell toxicity that leads to cellular demise, with a strong emphasis on cell death via both apoptosis and necrosis, by summarizing some of the more recent developments in cellular, molecular, and biochemical studies of the events that govern the induction and execution of cell death. Specific topics pertaining to cell death include structural changes, macromolecular degradation, cellular signaling, the role of mitochondria, and genetic modulation of apoptotic cell death.
Nonisotopic Methods for Determination of Poly(ADP‐Ribose) Levels and Detection of Poly(ADP‐Ribose) Polymerase
Authors: Haince Jean François , Poirier Guy G. , Kirkland Jim B.
Published: June 2004

Poly(ADP‐ribosyl)ation is a post‐translational modification catalyzed mostly by the 116‐kDa enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1), a nuclear enzyme that transfers an ADP‐ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500‐fold increase in net poly(ADP‐ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP‐ribose) synthesis is PARP‐1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD+ depletion. It has also been proposed that the presence of negatively charged poly(ADP‐ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP‐ribose) in cells using nonisotopic reagents and for identifying the poly(ADP‐ribose) polymerase enzymes present in cells.
Compendium of Drugs Commonly Used in Cell Biology Research
Authors: Cole Nelson H.
Published: June 2004

Over 80 drugs that are used for analysis of cell structure and function are listed by function together with recent references. This is followed by an alphabetical listing for each drug that includes a description of the drug's function, solubility information, stock and working concentrations, storage conditions, and incubation periods.
RNA Secondary Structure Analysis Using the Vienna RNA Package
Authors: Hofacker Ivo L.
Published: June 2004

This Current Protocols in Bioinformatics unit documents how to use the Vienna RNA package for RNA secondary structure analysis. Possible tasks include structure prediction for single sequences, prediction of consensus structures, and sequence design.
Sequence File Format Conversion with Command‐Line Readseq
Authors: Gilbert Don
Published: June 2004

One of the major challenges in using bioinformatics software is that there are a wide variety of sequence formats, e.g., GenBank, EMBL, and FASTA. It is often the case that a sequence or a set of sequences is in one format but is needed in another. This unit offers a solution to this problem ‐ Readseq. Readseq is a program that can read and write 18 different formats
Low‐temperature controllable calcination syntheses of potassium dititanate
Authors: Bao Ningzhong , Feng Xin , Lu Xiaohua , Shen Liming , Yanagisawa Kazumichi
Published: June 2004

Thermodynamic calculations were used to estimate the free energy for reactions of anatase and TiO2·nH2O with K2CO3 to generate potassium dititanate at 25–1200°C, and the results showed that amorphous TiO2·nH2O with reaction activity higher than that of anatase can decrease the lowest generation temperature of potassium dititanate. The precise temperatures of 300°C for TiO2·nH2O and 500°C for anatase were determined by experiments. The crystal growth of potassium dititanate was studied experimentally. It was found that potassium dititanate hydrate is first formed from TiO2·nH2O at 300°C, and converts into K2Ti2O5·0.35H2O at 640°C. The dehydration of K2Ti2O5·0.35H2O occurs at 660–820°C, ending with the generation of K2Ti2O5 single crystals at 820°C. Potassium dititanates with a diversity of morphologies, sizes, water contents, and crystallinities, showing various abilities for optical absorption/reflex, were fabricated from TiO2·nH2O under control, whereas only K2Ti2O5 was prepared from anatase at 500–850°C, which indicate that reaction processes and properties of products are determined according to the type of reactants and the reaction temperature. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1568–1577, 2004
Extinction deficits in male rhesus macaques with a history of self‐injurious behavior
Authors: Lutz Corrine , Tiefenbacher Stefan , Meyer Jerrold , Novak Melinda
Published: June 2004

Self‐injurious behavior (SIB) occurs in both human and nonhuman primate populations. Despite the potential for harm, SIB may persist in part because of an inability to inhibit behavior that results in wounding. A lever‐pressing task was used to test the prediction that monkeys with SIB would show greater persistence in lever‐pressing on extinction trials than monkeys without the disorder. The subjects were 15 individually‐housed adult male rhesus macaques, 10 of which (the SIB group) had a veterinary record of self‐inflicted wounding. All of the monkeys were trained to lever‐press for food rewards to a criterion of 400 total responses. The test procedures consisted of five daily 30‐min sessions divided into six 5‐min intervals. On day 1, the subjects received continuous reinforcement. On days 2–4, testing consisted of alternating reinforced/unreinforced 5‐min intervals, beginning with reinforcement. Reinforced intervals were cued with a buzzer. On day 5, the subjects received no reinforcement. The number of lever‐presses and behavioral responses were recorded during each session. Saliva samples were collected for cortisol measurement before and after test sessions on days 1, 2, and 5. As predicted, monkeys with SIB lever‐pressed more than controls during extinction intervals on days 2–4. There was no difference on day 1 or day 5. The frequency of scratching, yawning, and abnormal behavior increased when reinforcement was intermittent (days 2–4) or absent (day 5). Cortisol levels were highest with continuous reinforcement (day 1), and may reflect differential levels of food intake rather than stress. The presence of extinction deficits suggests that SIB may persist in some monkeys because they lack the ability to regulate the intensity of their biting behavior. Am. J. Primatol. 63:41–48, 2004. © 2004 Wiley‐Liss, Inc.
Transmission of STLV in a closed colony of macaques
Authors: Parrish Stephanie W.K. , Brown Arthur E. , Chanbancherd Penprapa , Gettayacamin Montip , Parrish John H.
Published: June 2004

A 3.3% seroprevalence of simian T‐lymphotropic virus (STLV) was found in a closed breeding and research colony of rhesus and cynomolgus macaques in Thailand. Epidemiology of STLV within the colony was assessed by means of a retrospective analysis of banked and freshly collected serum samples, and a review of the animals' medical records. Evidence was found that the virus had been imported into the colony by some of the original animals, and was subsequently transmitted both vertically and horizontally. The cell‐associated nature of STLV was demonstrated by iatrogenic transmission of the virus following a whole blood transfusion, but there was no transmission to animals that received only serum from the same infected donor. Transmission by all routes was infrequent, as indicated by the overall seroprevalence of 3.3% (14 of 420 samples) after the colony had been closed for 11 years. Maternal–infant transmission appeared to be <12%. Am. J. Primatol. 63:103–109, 2004. © 2004 Wiley‐Liss, Inc.