bluuebunny/crossref_metadata_2025 huggingface dataset:
What's New in Lipid Management?
Authors: Thompson Paul D.
Published: May 2004

Statins are the principal agents for managing hyperlipidemia because of their ability to reduce low‐density lipoprotein cholesterol (LDL) and triglyceride levels, increase high‐density lipoprotein cholesterol (HDL) levels slightly, and decrease clinical events. Nevertheless, not all patients can tolerate the statins or achieve their treatment goals with these drugs. In addition, available statins have only modest effects on HDL, and clinical events still occur despite the best current lipid‐lowering agents. Thus, new lipid‐lowering agents are required to improve clinical outcomes. These recently or soon to be released agents are stanol esters, which can be incorporated into spreads and other foods and reduce LDL levels up to 15%; colesevelam, a new bile acid sequestrant with a higher potency, fewer adverse effects, and less drug interactions than those of older agents in this class; rosuvastatin, a new statin that reduces LDL levels and increases HDL levels at least more effectively than atorvastatin; and a new class of agents that inhibit cholesterol absorption and are likely to be used in combination with statins. Low‐density lipoprotein cholesterol apheresis is another, nonpharmacologic option for patients with severe hyperlipidemia despite maximal medical therapy.
Clinical and Microbiologic Analysis of a Hospital's Extended‐Spectrum β‐Lactamase‐Producing Isolates Over a 2‐Year Period
Authors: Burgess David S. , Hall Ronald G. , Lewis James S. , Jorgensen James H. , Patterson Jan E.
Published: May 2004

Study Objective. To evaluate the microbiologic and clinical outcomes of patients with extended‐spectrum β‐lactamase (ESBL)‐producing isolates over a 2‐year period.

Design. Retrospective analysis.

Setting. Tertiary care teaching hospital.

Patients. Twenty‐one patients with cultures of confirmed ESBL‐producing Escherichia coli, Klebsiella pneumoniae, or Klebsiella oxytoca.

Measurements and Main Results. Antimicrobial susceptibilities of piperacillin‐tazobactam, cefotetan, carbapenems, aminoglycosides, fluoroquinolones, trimethoprim‐sulfamethoxazole, and nitrofurantoin (nitrofurantoin for urinary isolates only) of confirmed ESBL producers at our institution were determined, as well as clinical outcomes of patients with ESBL‐producing isolates. Microbiologic and medical records were reviewed for patient sex and age, antimicrobial susceptibilities, antimicrobial therapy, and clinical and microbiologic outcomes. From January 2000‐December 2001, 31 isolates were confirmed as ESBL producers (6 E. coli, 11 K. pneumoniae, and 14 K. oxytoca). A statistically significant increase occurred over the 2‐year period from 9 (0.6%) of 1414 isolates in 2000 to 22 (1.8%) of 1218 isolates in 2001 (p=0.0055). All isolates were susceptible to carbapenems, and more than 88% were susceptible to amikacin, cefotetan, or nitrofurantoin. Less than 70% of isolates were susceptible to gentamicin, fluoroquinolones, piperacillin‐tazobactam, or trimethoprim‐sulfamethoxazole. All patients treated with a carbapenem experienced clinical cure. Piperacillin‐tazobactam alone and in combination resulted in an overall clinical cure rate of 55%, with a 50% cure rate for isolates susceptible to piperacillin‐tazobactam. All patients in whom antibiotic therapy failed had been treated with piperacillin‐tazobactam or cefepime, either alone or in combination with a fluoroquinolone.

Conclusion. Carbapenems remain the treatment of choice for ESBL‐producing pathogens. Piperacillin‐tazobactam and cefepime should not be routinely administered for the treatment of these organisms.
Comorbidities and Associated Treatment Charges in Patients with Anxiety Disorders
Authors: McLaughlin Trent , Geissler Erika C. , Wan George J.
Published: May 2004

Objective. To describe comorbidities and treatment charges in patients receiving initial anxiolytic therapy for anxiety disorders.

Design. Retrospective data analysis.

Patients. Six thousand six hundred forty‐seven patients with anxiety disorders.

Measurements and Main Results. Analysis was conducted using medical and pharmacy data for patients in 26 United States health plans from the PharMetrics Integrated Outcomes database. Data were collected for 12 months before and after the first anxiolytic prescription date (index date) during the study period (January 1, 1998‐December 31, 2000). The sample comprised patients with a diagnosis of anxiety disorder whose original anxiolytic prescription had been refilled within 120 days. International Classification of Diseases, 9th revision, Clinical Modification (ICD‐9‐CM) codes and medical and prescription data identified the existence of comorbidities, and medical and prescription charges. The most common diagnoses of anxiety disorder were anxiety not otherwise specified (67%), panic disorder (14%), and generalized anxiety disorder (13%). The most commonly prescribed anxiolytics were benzodiazepines, such as alprazolam (43%) and lorazepam (27%). Hypertension was the most common nonpsychiatric comorbidity (22%) and depression the most common psychiatric comorbidity (37%). Other comorbidities observed were lipid disorders (17%), chronic obstructive pulmonary disease (9%), and asthma (7%). Total treatment charges 12 months before and after initial anxiety diagnosis and anxiolytic prescription were $6279 and $9270, respectively (p<0.0001).

Conclusion. Chronic conditions are commonly associated with anxiety disorders. Recognizing these psychiatric and nonpsychiatric comorbidities is important when treating patients with these disorders. Treatment charges are significantly higher when a patient is diagnosed with an anxiety disorder, highlighting the impact of anxiety on the total cost of treatment for these patients.
Pharmacokinetic Study in Women of Three Different Doses of a New Formulation of Oral Testosterone Undecanoate, Andriol Testocaps
Authors: Houwing Natalie S. , Maris Frans , Schnabel Peter G. , Bagchus Wilma M.
Published: May 2004

Study Objective. To assess the pharmacokinetic parameters of testosterone undecanoate after administration of a new oral formulation, Andriol Testocaps.

Design. Randomized, open‐label, group‐comparative, parallel‐design, dose‐proportionality study.

Setting. Clinical pharmacology unit.

Subjects. Forty‐five healthy women without childbearing potential.

Intervention. Two oral doses each of testosterone undecanoate 20, 40, or 80 mg were administered with meals, separated by a 12‐hour dosing interval.

Measurements and Main Results. Serum concentrations of testosterone undecanoate were assayed by liquid chromatography with mass spectrometric detection, and of testosterone and 5α‐dihydrotestosterone (DHT) by gas chromatography with mass spectrometric detection. Pharmacokinetic parameters were calculated using standard methods. Statistical analysis of dose proportionality was performed on the loge‐transforms of dose‐normalized area under the serum concentration‐time curve from 0–12 hours (AUC0–12) and from zero to the sampling time of the last measurable concentration after administration of the second dose (AUC0‐tlast), and maximum serum concentration after the first dose (Cmax1). For testosterone undecanoate, testosterone, and DHT, dose‐related increases in plasma concentrations were found with increasing doses of testosterone undecanoate; maximum concentrations were found 5–7 hours after administration. Using baseline‐corrected testosterone values, dose proportionality for testosterone was found for AUC0–12, AUC0‐tlast, and Cmax1. After higher doses, plasma levels of testosterone undecanoate were higher and plasma levels of DHT lower than could be expected assuming dose proportionality.

Conclusion. Serum testosterone levels are dose proportional after oral administration of two doses of a new formulation of testosterone undecanoate 20, 40, and 80 mg, Andriol Testocaps.
Pharmacologic Treatment of Chronic Obstructive Pulmonary Disease: Past, Present, and Future
Authors: Faulkner Michele A. , Hilleman Daniel E.
Published: May 2004

Pharmacologic treatment of chronic obstructive pulmonary disease (COPD) has evolved considerably during the past several decades. Initial treatment of the disease was accomplished primarily through antibiotics, mucolytic agents, and nonselective sympathomimetic agents. Up‐to‐date treatment guidelines stratified according to strength of evidence are published in the National Heart, Lung, and Blood Institute‐World Health Organization workshop report on the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Current drug therapy for stable COPD focuses primarily on bronchodilation through inhaled β2‐agonists and anticholinergic agents, immunization, and elimination of smoking as a risk factor. Although many pharmacologic agents are available to treat COPD, no drug has demonstrated effectiveness in halting progression of the disease. Rather, the goal of drug therapy at this time is to maintain control of symptoms and prevent COPD exacerbations. Compared with asthma, research into treatment for COPD has been minimal. However, a long‐acting anticholinergic agent, tiotropium, has received approval status by the United States Food and Drug Administration. The drug has been shown to improve spirometric parameters, quality of life, and utilization of health care resources. In addition, several new targets for the treatment of COPD are being studied, and a few agents, including some that theoretically may slow functional decline in patients with COPD, are in development.
New Antiarrhythmic Agents for Atrial Fibrillation and Atrial Flutter: United States Drug Market Response as an Indicator of Acceptance
Authors: LaPointe Nancy M. Allen , Pamer Carol A. , Kramer Judith M.
Published: May 2004

Objective. To determine how well dofetilide and Betapace AF (sotalol, approved solely for atrial fibrillation and atrial flutter), with their detailed dosing and monitoring guidelines for safety, were accepted into clinical practice during the 2 calendar years after their introduction.

Methods and Results. We reviewed the number of new, refill, and total prescriptions of all antiarrhythmic agents in the United States from April 2000‐December 2001 to assess use of dofetilide and Betapace AF in the drug market. Both were prescribed very infrequently throughout the study period. In addition, the infrequent reported use of these drugs for patients with atrial fibrillation and flutter indicated poor acceptance of these agents by prescribing physicians. We speculated that the restricted distribution and required educational program for dofetilide, as well as the availability of generic sotalol products, may have discouraged physicians from prescribing both dofetilide and Betapace AF.

Conclusion. A common goal for both the dofetilide risk‐management program and the creation of a sotalol product indicated solely for atrial fibrillation and atrial flutter was to provide safer treatment for patients with these arrhythmias. Unfortunately, limited penetration of dofetilide and Betapace AF into the U.S. market suggests that drugs without a risk‐management program or detailed dosing guidelines were more likely than dofetilide or Betapace AF to be selected for treatment of atrial fibrillation and atrial flutter.
Peritoneal Dialysis Fluid Concentrations of Linezolid in the Treatment of Vancomycin‐Resistant Enterococcus faecium Peritonitis
Authors: DePestel Daryl D. , Peloquin Charles A. , Carver Peggy L.
Published: May 2004

Objective. To determine linezolid concentrations in peritoneal dialysis fluid after multiple oral doses of the drug in a 46‐year‐old man with vancomycin‐resistant Enterococcus faecium peritonitis who was undergoing peritoneal dialysis.

Methods. After administration of oral linezolid 600 mg twice/day was started, peritoneal dialysis fluid was collected at the end of several 4‐ and 8‐hour dwell times and submitted for analysis of linezolid concentration. Before linezolid therapy was begun, and immediately after several peritoneal dialysis exchanges, 30 ml of expended peritoneal dialysis fluid was collected in a sterile container and immediately frozen at −70°C until analysis by high‐performance liquid chromatography.

Results. Peritoneal dialysis concentrations of linezolid greater than 4 μg/ml were achieved after the first dose of linezolid and maintained after repeated doses. During the course of therapy, mean linezolid concentrations in peritoneal dialysis fluid tended to increase (mean 7.60 μg/ml, range 3.54–16.2 μg/ml). All assayed peritoneal dialysis samples demonstrated linezolid concentrations greater than 4 μg/ml at the end of 4‐ or 8‐hour dwell times, except for one level after a missed dose on linezolid treatment day 3. Duration of dwell times did not appear to correlate with linezolid concentrations.

Conclusion. In this patient, linezolid 600 mg twice/day penetrated into peritoneal dialysis fluid at or above the concentrations necessary to treat common gram‐positive bacteria. Linezolid therapy is likely to have a role in peritoneal dialysis‐associated peritonitis based on its antimicrobial activity, pharmacokinetic properties, ease of administration, and tolerability.
The Role of Multidrug Efflux Pumps in the Antibiotic Resistance of Pseudomonas aeruginosa and Other Gram‐Negative Bacteria
Authors: Aeschlimann Jeffrey R.
Published: May 2004

Gram‐negative bacteria remain clinically important pathogens in both hospital and community settings. Recent research indicates that efflux pumps play a prominent role in the multidrug resistance of Pseudomonas aeruginosa and many other gram‐negative bacteria. Four multidrug efflux pump systems have been well characterized in P. aeruginosa: MexA‐MexB‐OprM, MexC‐MexD‐OprJ, MexE‐MexF‐OprN, and MexX‐MexY‐OprM. These efflux pumps have different substrate specificities, and their production and activity can be increased by many factors commonly present in infections (e.g., high inocula of bacteria, low pH, and stationary‐phase growth). Moreover, fluoroquinolone antibiotics can commonly select mutants that constitutively overproduce Mex‐Opr efflux pump systems. Based on most recent studies, the prevalence of efflux pump overproduction in clinical strains of P. aeruginosa may range from 14–75%. The best treatment for infections caused by bacteria that overproduce efflux pumps is unknown, but pharmacodynamic optimization of antibiotics and the use of antibiotic combinations that are substrates for different pump systems may represent reasonable strategies until more data are available.
Propofol Withdrawal Syndrome in an Adult Patient with Thermal Injury
Authors: Cawley Michael J. , Guse Timothy M. , Laroia Anjali , Haith Linwood R. , Ackerman Bruce H.
Published: May 2004

A 48‐year‐old man with a history of ethanol abuse and bipolar disease fell asleep while smoking in an intoxicated state. The patient received a 30% total body surface area burn involving his face and upper torso that resulted in an inhalation injury. Several attempts at weaning from mechanical ventilation failed due to his extreme agitation, which was unresponsive to benzodiazepines, opiates, and antipsychotic agents. Propofol therapy was begun in combination with valproic acid, fluoxetine, and risperidone to assist in the treatment of his severe agitation associated with the bipolar disease, inhibiting ventilatory weaning. Repeated attempts to discontinue propofol were associated with withdrawal symptoms such as severe agitation, tremors, tachycardia, tachypnea, and hyperpyrexia. His symptoms resolved only after each time the propofol infusion was restarted. The patient received propofol for 95 days for management of his agitation before dying from refractory septic shock and multiple organ failure.
Possible Heart Failure Exacerbation Associated with Rosiglitazone: Case Report and Literature Review
Authors: II Robert Lee Page , Gozansky Wendolyn S. , Ruscin J. Mark
Published: May 2004

Increasing evidence suggests that neurohumoral manifestations of heart failure may lead to insulin resistance, predisposing patients with heart failure to the development of glucose intolerance or worsening of existing diabetes. Theoretically, insulin‐sensitizing thiazolidinediones (TZDs) should be beneficial in this patient population. A 74‐year‐old man with well‐compensated systolic dysfunction and longstanding type 2 diabetes mellitus treated with glyburide began therapy with rosiglitazone 4 mg/day, which was increased to 8 mg/day after 1 month. Two weeks later he was seen with a 5‐kg weight gain, shortness of breath, bibasilar rales, +S3 gallop, and increased jugular venous distention. Twelve days later symptoms worsened, with pulmonary edema on chest radiograph, continued weight gain, and +4 pitting edema resistant to oral diuretics. The patient was admitted to the hospital for exacerbation of heart failure. Five days after discharge he was readmitted for similar symptoms, including an 11.8‐kg weight gain. He reported adherence to drug therapy and diet. Rosiglitazone was immediately discontinued and 11 days later the man's weight stabilized to 79 kg and remained between 79 and 80 kg 2 and 3 months after discharge. This case demonstrates that TZDs may precipitate weight gain and pulmonary and peripheral edema in patients with stable heart failure. Earlier reports documented similar symptoms in patients without a history of heart failure. Although current recommendations state that TZDs should not be administered to patients with New York Heart Association class III or IV disease, practitioners should be aware that these adverse effects also may occur in patients with milder forms heart failure as well as those without heart failure.
The microdistribution of three uncommon freshwater gastropods in the drainage ditches of British grazing marshes
Authors: Watson Alisa M. , Ormerod S.J.
Published: May 2004

The gastropods Segmentina nitida, Anisus vorticulus and Valvata macrostoma occur in drainage ditches on grazing marshes that are now among the most threatened wetland systems in western Europe. Although each of these species is listed in the UK Red Data Book (RDB) and Biodiversity Action Plan, influences on their distribution are poorly understood.

2. To improve management information, the within‐channel distributions of the three snails were examined in 20 ditches in southeast England. Abundance, vegetation cover and other environmental factors were recorded across the ditch profile near the surface (<0.2m) and at depth (<0.15m from the benthos).

3. All three species were significantly more abundant near the surface than at depth. Below 0.6m, ditches had significantly reduced concentrations of dissolved oxygen (<0.6 mg L−1, <5% saturation), possibly sufficient to limit the occurrence of gastropods dependent wholly or partly on aqueous gas exchange.

4. There were no other systematic variations in abundance between the ditch margins and centre channel. However, across all ditches and samples in the survey, each species' abundance varied significantly with vegetation structure. S. nitida was most numerous where there was least open water and hence most vegetation, V. macrostoma among emergent stands and A. vorticulus in ditches with floating vegetation but few submerged plants.

5. These microdistributional data support large‐scale surveys in illustrating the potential importance of vegetation management for these snails; providing that other requirements are satisfied, ditch management could favour each RDB species by optimizing particular vegetation features. Apparent preferences for vegetation structure rather than particular channel locations suggest that refuges left during ditch clearance could be located anywhere in the ditch. Factors that reduce oxygen concentrations below 1mg L −1, such as eutrophication, might be detrimental.

Copyright © 2004 John Wiley & Sons, Ltd.
Development of a mammalian series‐fibered muscle
Authors: Paul Angelika C. , Sheard Philip W. , Duxson Marilyn J.
Published: May 2004

This study examines the processes by which multiply innervated, serially fibered mammalian muscles are constructed during development. We previously reported that primary myotubes of such a muscle, the guinea pig sternomastoid muscle, span from tendon to tendon and are innervated at each of the muscle's four innervation zones. Secondary myotubes form later, in association with each point of innervation (Duxson and Sheard, Dev. Dyn., 1995; 204:391–405). We now describe the further growth and development of the muscle. Secondary myotubes initially insert onto and grow along the primary myotube. However, as they reach a critical length, they encounter other secondary myotubes growing from serially adjacent innervation zones and may transfer their attachment(s) to these serially positioned secondary myotubes. Other secondary myotubes maintain attachment at one or both ends to their primary myotube. Thus, an interconnected network of primary and secondary myotubes is formed. Patterns of reactivity for cell adhesion molecules suggest that early attachment points between myotubes are the embryonic precursors of adult myomyonal junctions, characterized by the expression of α7Bβ1 integrin. Finally, the results show that secondary myotubes positioned near a tendon are generally longer than those lying in the mid belly of the muscle, and we suggest that the environment surrounding the tendinous zone may somehow stimulate myotube growth. Anat Rec Part A 278A:571–578, 2004. © 2004 Wiley‐Liss, Inc.
Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells
Authors: Ignowski Jolene M. , Schaffer David V.
Published: May 2004

Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real‐time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the Km of luciferase in living cells to be ≈ 1 mM, a value much higher than the 10 μM found for pure enzyme in vitro. In addition, a 2‐hour half‐life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior. © 2004 Wiley Periodicals, Inc.
Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration
Authors: Guarino Richard D. , Dike Laura E. , Haq Tariq A. , Rowley Jon A. , Pitner J. Bruce , Timmins Mark R.
Published: May 2004

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen‐sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion‐limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per‐cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol·min−1·cell−1 for bacteria to 0.1–10 fmol·min−1·cell−1 for immortalized mammalian and insect cell lines to >10 fmol·min−1·cell−1 for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture. © 2004 Wiley Periodicals, Inc.
Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures
Authors: Henry Olivier , Dormond Edwige , Perrier Michel , Kamen Amine
Published: May 2004

One of the major limitations in the production of adenoviral vectors is the reduction in cell‐specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed‐batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK‐293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4–3 × 106 cell/mL, while maintaining a similar cell specific productivity (17,900 ± 2400 VP/cell) to that of a batch infected at a low cell density (5 × 105 cell/mL). The highest virus concentrations (4.1 ± 0.6 × 1010 VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on‐line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity. © 2004 Wiley Periodicals, Inc.
Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase
Authors: Rosche Bettina , Breuer Michael , Hauer Bernhard , Rogers Peter L.
Published: May 2004

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)‐phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1‐pentanol, hexadecane or MTBE (methyl tertiary‐butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54–81 mM) in the aqueous phase. 1‐pentanol, 1‐hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed. © 2004 Wiley Periodicals, Inc.
Preparation of single cells from aggregated Taxus suspension cultures for population analysis
Authors: Naill Michael C. , Roberts Susan C.
Published: May 2004

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y‐23, macerozyme, Driselase®, and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y‐23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 μM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism. © 2004 Wiley Periodicals, Inc.
Glutamic acid removal and PHB storage in the activated sludge process under dynamic conditions
Authors: Dionisi Davide , Majone Mauro , Miccheli Alfredo , Puccetti Caterina , Sinisi Carlo
Published: May 2004

Glutamic acid removal in the activated sludge process is studied herein, primarily the formation of storage polymers under dynamic conditions. The activated sludge process was operated by using a sequencing batch reactor (sludge age of 6 d) fed with a synthetic mixture of readily available carbon sources, including glutamic acid. Removal of glutamic acid as the only carbon sources was studied in batch tests, along with oxygen consumption, ammonia uptake – release, and formation of storage polymers. It was found that poly‐3‐hydroxybutyrate (PHB) was stored and that the storage also occurred simultaneously to biomass growth. PHB storage accounted for 16% of the overall solids that were formed from glutamic acid, as the average value of nine batch tests. Neither other Polyhydroxyalkanoates nor polyglutamic acid were detected . Nuclear magnetic resonance analysis, performed on biomass extracts, allowed us to clarify the main metabolic pathways involved in glutamic acid removal and, in particular, the pathways involved in PHB storage. It was found that glutamic acid enters the Krebs cycle as α‐ketoglutaric acid and exits to form pyruvic acid and then acetyl‐CoA, which is the starting point of PHB production pathway. © 2004 Wiley Periodicals, Inc.
Novel chemically synthesized hydroxyl‐containing jasmonates as powerful inducing signals for plant secondary metabolism
Authors: Qian Zhi‐Gang , Zhao Zhen‐Jiang , Xu Yufang , Qian Xuhong , Zhong Jian‐Jiang
Published: May 2004

Novel hydroxyl‐containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 μM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl‐containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 μM 2,3‐dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 ± 0.5 mg/g (at day 21), whereas the Tc content was 29.2 ± 0.6 mg/g (on the same day) with addition of 100 μM MJA. Quantitative structure–activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L‐phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H2O2 production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl‐containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures. © 2004 Wiley Periodicals, Inc.
In situ product removal using a crystallization loop in asymmetric reduction of 4‐oxoisophorone by Saccharomyces cerevisiae
Authors: Buque‐Taboada Evelyn M. , Straathof Adrie J.J. , Heijnen Joseph J. , van der Wielen Luuk A.M.
Published: May 2004

In situ product crystallization was investigated for solid product crystals that were obtained during fermentation. The model reaction was the asymmetric reduction of 4‐oxoisophorone (OIP) using baker's yeast (S. cerevisiae) as a biocatalyst. The target product was 6R‐dihydro‐oxoisophorone (DOIP), also known as levodione, a key intermediate in carotenoid synthesis. DOIP was degraded by baker's yeast mainly to (4S,6R)‐actinol, an unwanted byproduct in the process. Actinol formation reached up to 12.5% of the initial amount of OIP in the reactor during a batch process. However, better results were obtained when the dissolved DOIP concentration was controlled using an integrated fermentation–crystallization process because: (a) actinol formation was reduced to 4%; and (b) DOIP crystal formation in the reactor was avoided. DOIP productivity was improved by 50% and its selectivity was raised from 87% to 96% relative to the batch process. In the integrated process, most of the product was recovered as pure crystals; this may already minimize, if not eliminate, the need for organic solvents in the final purification steps. An almost sixfold reduction in biocatalyst consumption per kilogram product was achieved, which also can contribute to the minimization of waste streams. © 2004 Wiley Periodicals, Inc.
Rapid chromatography for evaluating adsorption characteristics of cellulase binding domain mimetics
Authors: Mosier Nathan S. , Wilker Jonathan J. , Ladisch Michael R.
Published: May 2004

The cost of cellulolytic enzymes is one barrier to the economic production of fermentable sugars from lignocellulosic biomass for the production of fuels and chemicals. One functional characteristic of cellulolytic enzymes that improves reaction kinetics over mineral acids is a cellulose binding domain that concentrates the catalytic domain to the substrate surface. We have identified maleic acid as an attractive catalytic domain with pKa and dicarboxylic acid structure properties that hydrolyze cellulose while producing minimal degradation of the glucose formed. In this study we report results of a rapid chromatographic method to assess the binding characteristics of potential cellulose binding domains for the construction of a synthetic cellulase over a wide range of temperatures (20° to 120°C). Aromatic, planar chemical structures appear to be key indicators of cellulose adsorption. Indole, the side‐chain of the amino acid tryptophan, has been shown to reversibly adsorb to cellulose at temperatures between 30° and 120°C. Trypan blue, a polyaromatic, planar molecule, was shown to be irreversibly adsorbed to cotton cellulose at temperatures of <120°C on the time scale of the experiments. These results confirm the importance of hydrophobic cellulose and the cellulose‐binding component of cellulolytic enzymes and cellulolytic enzyme mimetics. © 2004 Wiley Periodicals, Inc.
Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: Engineering gel structural changes to facilitate cartilaginous tissue production
Authors: Bryant Stephanie J. , Bender Ryan J. , Durand Kevin L. , Anseth Kristi S.
Published: May 2004

A major challenge when designing cell scaffolds for chondrocyte delivery in vivo is creating scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. To address this design challenge, degradable photocrosslinked hydrogels based on poly(ethylene glycol) were investigated. To alter the gel's initial mechanical properties, hydrogels were fabricated by varying the initial macromer concentration from 10% to 15% to 20%. A twofold increase in macromer concentration resulted in an eightfold increase in the initial compressive modulus from 60 to 500 kPa. Gel degradation was tailored by incorporating fast‐degrading crosslinks that enable maximal extracellular matrix (ECM) diffusion with time and a minimal number of nondegrading (or slowly degrading) crosslinks to maintain scaffold integrity and prevent complete gel erosion during tissue formation. Chondrocytes encapsulated in these gels produced cartilaginous tissue rich in glycosaminoglycans and collagen as seen biochemically and histologically. Interestingly, mass loss appeared to more closely match tissue secretion in gels fabricated from a 15% macromer concentration. However, the spatial ECM distribution was grossly similar in all three gels. By tailoring gel degradation and controlling network evolution during degradation, gels with optimal properties can be fabricated to support initially physiologic compressive loads while simultaneously supporting the formation of a neotissue. © 2004 Wiley Periodicals, Inc.
Vascular‐targeting therapies for treatment of malignant disease
Authors: Siemann Dietmar W. , Chaplin David J. , Horsman Michael R.
Published: May 2004

BACKGROUND

Tumor endothelium represents a valuable target for cancer therapy. The vasculature plays a critical role in the survival and continued growth of solid tumor masses; in addition, the inherent differences between tumor blood vessels and blood vessels associated with normal tissue make the tumor vasculature a unique target on which to base the design of novel therapeutics, which may allow highly selective treatment of malignant disease. Therapeutic strategies that target and disrupt the already formed vessel networks of growing tumors are actively being pursued. The goal of these approaches is to induce a rapid and catastrophic shutdown of the vascular function of the tumor so that blood flow is arrested and tumor cell death due to the resulting oxygen and nutrient deprivation and buildup of waste products occurs.

METHODS

Biologic approaches and small‐molecule drugs that can be used to damage tumor vasculature have been identified. Physiologic, histologic/morphologic, and immunohistochemical assessments have demonstrated that profound disruption of the tumor vessel network can be observed minutes to hours after treatment. The small‐molecule agents that have made the greatest advances in the clinical setting (5,6‐dimethylxanthenone‐4‐acetic acid [DMXAA], combretastatin A4 disodium phosphate [CA4DP], and ZD6126) are the focus of the current review.

RESULTS

Loss of patent blood vessels, decreased tumor blood flow, extensive necrosis, and secondary ischemia‐induced tumor cell death have been well documented in a variety of preclinical tumor models treated with agents such as DMXAA, CA4DP, and ZD6126. The use of such agents in conjunction with irradiation and other chemotherapeutic agents has led to improved treatment outcomes.

CONCLUSIONS

The targeting of tumors' supportive blood vessel networks could lead to improvements in cancer cure rates. It is likely that this approach will prove to be most efficacious when used in concert with conventional treatment strategies. Cancer 2004. © 2004 American Cancer Society.
The changing pattern of Kaposi sarcoma in patients with HIV, 1994–2003
Authors: Mocroft Amanda , Kirk Ole , Clumeck Nathan , Gargalianos‐Kakolyris Panaglotis , Trocha Hanna , Chentsova Nelly , Antunes Francisco , Stellbrink Hans‐Jürgen , Phillips Andrew N. , Lundgren Jens D.
Published: May 2004

BACKGROUND

The introduction of highly active antiretroviral therapy (HAART) has radically changed the clinical course of human immunodeficiency virus (HIV) infection. The goals of the current study were to assess the change in the incidence of Kaposi sarcoma (KS) among European patients with HIV since the introduction of HAART and to identify the factors associated with the development of KS among patients receiving HAART.

METHODS

The incidence of KS and the factors associated with the development of this malignancy in patients receiving HAART were evaluated using Poisson regression. Patients examined in the current study were among the 9803 individuals with HIV who were enrolled in the EuroSIDA study, a pan‐European multicenter investigation.

RESULTS

There was an estimated annual reduction of 39% (95% confidence interval [CI], 35–43%; P < 0.0001) in the incidence of KS between 1994 and 2003. The proportion of acquired immunodeficiency syndrome (AIDS) diagnoses made due to KS during prospective follow‐up ranged from 4.1% to 7.5%, and there was no significant change over time in this figure (P = 0.97). Four thousand fourteen patients began receiving HAART during prospective follow‐up; 41 of these 4014 were subsequently diagnosed with KS (1.0%). After adjustment in multivariate analyses, patients with higher current CD4 counts were found to have a decreased incidence of KS (incidence rate ratio [IRR], 0.60; 95% CI, 0.53–0.68; P < 0.0001), as were those for whom more time had elapsed since the initiation of HAART (IRR, 0.77; 95% CI, 0.60–0.98; P = 0.037). In contrast, homosexual men were found to have a significantly increased incidence of KS (IRR, 2.12; 95% CI, 1.00–4.54; P = 0.050)

CONCLUSIONS

The current incidence of KS among patients with HIV is less than 10% of the incidence reported in 1994; the proportion of AIDS diagnoses made on the basis of KS diagnoses remains near 6%. Most individuals who developed KS while receiving HAART began treatment with low CD4 cell counts and developed KS within 6 months of the initiation of HAART. There continues to be an increased incidence of KS among homosexual men and a greatly reduced incidence of KS among patients with higher CD4 counts. Cancer 2004. © 2004 American Cancer Society.
Paramagnetic Chloro‐, Alkoxo‐, or Azidovanadium(IV) Complexes Supported by an [ONNO]‐Type Amine Bis(phenolate) Ligand
Authors: Wolff Fabien , Lorber Christian , Choukroun Robert , Donnadieu Bruno
Published: May 2004

The reactions between V(N3)2(NMe2)2 or V(OiPr)4 and the chelating dianionic bis(phenoxy)amine ligand [ONNO]H2 afford complexes formulated as [V(N3)2{ONNO}] (1) and [V(OiPr)2{ONNO}] (2). When refluxed in toluene with an excess of Me3SiCl, compound 2 leads to mono‐ or dichloro derivatives [V(Cl)(OiPr){ONNO}] (3) and [VCl2{ONNO}] (4) depending on the experimental conditions. The crystal structures of complexes 1−4 were solved and show in all cases an [ONNO] ligand that has phenolate groups in a trans configuration, with both amino‐nitrogen donor atoms of the ligand coordinated to the octahedral vanadium center. Complexes 1−4 were further characterized by EPR spectroscopy and susceptibility measurement studies typical of d1‐paramagnetic VIV species. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Ruthenium(II) Octaphenylporphyrazine Complexes with Mixed Axial Ligands: Peculiarities of Their Formation and Spectral Properties
Authors: Vagin Sergej , Ziener Ulrich , Hanack Michael , Stuzhin Pavel A.
Published: May 2004

A series of new axially bis‐coordinated (octaphenylporphyrazinato)ruthenium(II) complexes {[(Ph8Pz)RuL2], L = cyclohexyl isocyanide, CyNC, (1b); 4,4′‐bipyridine, bpy, (2c); N‐methylimidazole, CH3Im, (3a)} was prepared and characterized. The reaction of the bis‐isocyanide adducts [(Ph8Pz)Ru(tBuNC)2] (1a) or [(Ph8Pz)Ru(CyNC)2] (1b) with monodentate N‐heterocycles gave, under certain conditions, the mixed‐ligand complexes [(Ph8Pz)Ru(Py)(tBuNC)] (4a), [(Ph8Pz)Ru(Py)(CyNC)] (4b) and [(Ph8Pz)Ru(CH3Im)(CyNC)] (3b). The reaction of 1a or 1b with an excess of bidentate 4,4′‐bipyridine under the same conditions resulted in the formation of the corresponding dimeric species [(Ph8Pz)Ru(tBuNC)]2(μ‐bpy) (2a) and [(Ph8Pz)Ru(CyNC)]2(μ‐bpy) (2b) in high yield. The structures of all the complexes were unambiguously proved by 1H NMR spectroscopy, and the possibility of their self‐assembly is discussed. All mixed‐ligand compounds were also characterized by UV/Vis and IR spectroscopy, as well as elemental analysis. The 1H NMR spectra of these compounds are discussed in detail. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Specific cerebellar activation during Braille reading in blind subjects
Authors: Gizewski Elke R. , Timmann Dagmar , Forsting Michael
Published: May 2004

The traditional view that the cerebellum is involved only in the control of movements has been changed recently. It has been suggested that the human cerebellum is involved in cognition and language. Likewise, besides cortical activity in sensorimotor and visual areas, an increased global activation of the cerebellum has been revealed during Braille reading in blind subjects. Our purpose was to investigate whether there is cerebellar activation during Braille reading by blind subjects other than sensorimotor activation related to finger movements. Early blind and normal sighted subjects were studied with functional magnetic resonance imaging (fMRI) during Braille reading, tactile discrimination of nonsense dots, dots forming symbols, and finger tapping. The experiments were done in block design. Echo planar imaging sequences were carried out on a 1.5‐T MR scanner. All blind individuals reading Braille showed robust activation of the posterior and lateral aspects of cerebellar hemispheral lobules Crus I bilaterally but more predominately on the right side. Additionally, activation was present in the medial cerebellum within lobules IV, V, and VIIIA, predominantly on the right. Discriminating nonsense dots did not reveal any activation of Crus I, but did reveal activation within the medial part of lobules IV, V, and VIIIA, predominately on the right. Analysis of sighted subjects during reading of printed text revealed activation of the posterolateral cerebellar hemisphere in Crus I bilaterally, predominantly on the right. Tactile analysis of dots representing symbols revealed an activation in lobules IV and VIII and in right Crus II but not in Crus I. In conclusion, parts of cerebellar activation during Braille reading in blind subjects (i.e., within lobules IV, V, and VIII) overlap with the known hand representation within the cerebellum and are likely related to the sensorimotor part of the task. Cerebellar activation during Braille reading within bilateral Crus I may be due to language processes or inner speech similar to those found during text reading in normal sighted subjects. Object recognition did not account for Crus I activation. Hum. Brain Mapp. 22:229–235, 2004. © 2004 Wiley‐Liss, Inc.