bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling of the Sedative and Airway Obstruction Effects of Propofol in Patients with Parkinson Disease undergoing Stereotactic Surgery
Authors: Fábregas Neus , Rapado Javier , Gambús Pedro L. , Valero Ricard , Carrero Enrique , Salvador Lydia , Nalda-Felipe Miguel A. , Trocóniz Iñaki F.
Published: August 2004

Background

Functional stereotactic surgery requires careful titration of sedation since patients with Parkinson disease need to be rapidly awakened for testing. This study reports a population pharmacodynamic model of propofol sedation and airway obstruction in the Parkinson disease population.

Methods

Twenty-one patients with advanced Parkinson disease undergoing functional stereotactic surgery were included in the study and received propofol target-controlled infusion to achieve an initial steady state concentration of 1 microg/ml. Sedation was measured using the Ramsay Sedation Scale. Airway obstruction was measured using a four-category score. Blood samples were drawn for propofol measurement. Individual pharmacokinetic profiles were constructed nonparametrically using linear interpolation. Time course of sedation and respiratory effects were described with population pharmacodynamic models using NONMEM. The probability (P) of a given level of sedation or airway obstruction was related to the estimated effect-site concentration of propofol (Ce) using a logistic regression model.

Results

The concentrations predicted by the target-controlled infusion system generally exceeded the measured concentrations. The estimates of C(50) for Ramsay scores 3, 4, and 5 were 0.1, 1.02, and 2.28 microg/ml, respectively. For airway obstruction scores 2 and 3, the estimates of C(50) were 0.32 and 2.98 microg/ml, respectively. Estimates of k(e0) were 0.24 and 0.5 1/min for the sedation and respiratory effects, respectively.

Conclusions

The pharmacokinetic behavior of propofol in patients with Parkinson disease differs with respect to the population from which the model used by the target-controlled infusion device was developed. Based on the results from the final models, a typical steady state plasma propofol concentration of 0.35 microg/ml eliciting a sedation score of 3 with only minimal, if any, airway obstruction has been defined as the therapeutic target.
Evaluation of Anesthesia Residents Using Mannequin-based Simulation
Authors: Schwid Howard A. , Rooke G. Alec , Carline Jan , Steadman Randolph H. , Murray W. Bosseau , Olympio Michael , Tarver Stephen , Steckner Karen , Wetstone Susan
Published: August 2004

Background

Anesthesia simulators can generate reproducible, standardized clinical scenarios for instruction and evaluation purposes. Valid and reliable simulated scenarios and grading systems must be developed to use simulation for evaluation of anesthesia residents.

Methods

After obtaining Human Subjects approval at each of the 10 participating institutions, 99 anesthesia residents consented to be videotaped during their management of four simulated scenarios on MedSim or METI mannequin-based anesthesia simulators. Using two different grading forms, two evaluators at each department independently reviewed the videotapes of the subjects from their institution to score the residents' performance. A third evaluator, at an outside institution, reviewed the videotape again. Statistical analysis was performed for construct- and criterion-related validity, internal consistency, interrater reliability, and intersimulator reliability. A single evaluator reviewed all videotapes a fourth time to determine the frequency of certain management errors.

Results

Even advanced anesthesia residents nearing completion of their training made numerous management errors; however, construct-related validity of mannequin-based simulator assessment was supported by an overall improvement in simulator scores from CB and CA-1 to CA-2 and CA-3 levels of training. Subjects rated the simulator scenarios as realistic (3.47 out of possible 4), further supporting construct-related validity. Criterion-related validity was supported by moderate correlation of simulator scores with departmental faculty evaluations (0.37-0.41, P < 0.01), ABA written in-training scores (0.44-0.49, < 0.01), and departmental mock oral board scores (0.44-0.47, P < 0.01). Reliability of the simulator assessment was demonstrated by very good internal consistency (alpha = 0.71-0.76) and excellent interrater reliability (correlation = 0.94-0.96; P < 0.01; kappa = 0.81-0.90). There was no significant difference in METI versus MedSim scores for residents in the same year of training.

Conclusions

Numerous management errors were identified in this study of anesthesia residents from 10 institutions. Further attention to these problems may benefit residency training since advanced residents continued to make these errors. Evaluation of anesthesia residents using mannequin-based simulators shows promise, adding a new dimension to current assessment methods. Further improvements are necessary in the simulation scenarios and grading criteria before mannequin-based simulation is used for accreditation purposes.
Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species
Authors: Tanaka Katsuya , Weihrauch Dorothee , Kehl Franz , Ludwig Lynda M. , LaDisa John F. , Kersten Judy R. , Pagel Paul S. , Warltier David C.
Published: August 2004

Background

Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo.

Methods

Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg. kg(-1).min(-1)), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively.

Results

Isoflurane (P < 0.05) decreased infarct size to 24 +/- 4% (mean +/- SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 +/- 3%; n = 8). NAC (43 +/- 3%; n = 7) and 2-MPG (42 +/- 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 +/- 3%; n = 8 and 46 +/- 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 +/- 12 -6 +/- 9 fluorescence units; P < 0.05).

Conclusions

The results indicate that ROS produced following administration of isoflurane contribute to protection against myocardial infarction in vivo.
Isoflurane-induced Cerebral Hyperemia Is Partially Mediated by Nitric Oxide and Epoxyeicosatrienoic Acids in Mice In Vivo
Authors: Kehl Franz , Shen Hui , Moreno Carol , Farber Neil E. , Roman Richard J. , Kampine John P. , Hudetz Anthony G.
Published: August 2004

Background

Despite intense investigation, the mechanism of isoflurane-induced cerebral hyperemia is unclear. The current study was designed to determine the contributions of neuronal nitric oxide synthase, prostaglandins, and epoxyeicosatrienoic acids to isoflurane-induced cerebral hyperemia.

Methods

Regional cerebral cortical blood flow was measured with laser Doppler flowmetry during stepwise increases of isoflurane from 0.0 to 1.2, 1.8, and 2.4 vol% end-tidal concentration in alpha-chloralose-urethane-anesthetized, C57BL/6 mice before and 45 min after administration of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI, 40 mg/kg, intraperitoneal), the cyclooxygenase inhibitor indomethacin (INDO, 10 mg/kg, intravenous), and the cytochrome P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 20 mg/kg, intravenous).

Results

Isoflurane increased regional cerebral cortical blood flow by 9 +/- 3, 46 +/- 21, and 101 +/- 26% (SD) at 1.2, 1.8, and 2.4 vol%, respectively. The increases in regional cerebral cortical blood flow were significantly (*P < 0.05) smaller after 7-NI (5 +/- 6, 29 +/- 19*, 68 +/- 15%*) or PPOH (4 +/- 8, 27 +/- 17*, 67 +/- 30%*), but not after administration of INDO (4 +/- 4, 33 +/- 18 [NS], 107 +/- 35% [NS]). The effect of combined treatment with 7-NI, PPOH, and INDO was not additive and was equal to that of either 7-NI or PPOH alone (5 +/- 5, 30 +/- 12*, 76 +/- 24%*). Chronic treatment of mice for 5 days with 7-NI (2 x 40 mg/kg, intraperitoneal) produced similar decreases in regional cerebral cortical blood flow as those seen with acute administration. Neither PPOH nor INDO conferred a significant additional block of the hyperemia in these animals.

Conclusions

Nitric oxide and epoxyeicosatrienoic acids contribute to isoflurane-induced hyperemia. However, only approximately one third of the cerebral hyperemic response to isoflurane is mediated by autacoids. The remaining part of this response appears to be mediated by a direct action of isoflurane on smooth muscle by some yet-unknown mechanism.
Propofol Increases Pulmonary Artery Smooth Muscle Myofilament Calcium Sensitivity
Authors: Tanaka Satoru , Kanaya Noriaki , Homma Yasuyuki , Damron Derek S. , Murray Paul A.
Published: August 2004

Background

Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway.

Methods

Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i.

Results

The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with Gö 6976 also attenuated propofol-induced increases in tension.

Conclusion

These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.
Effects of Chronic Alcohol Abuse on Alveolar Epithelial Barrier Function and Glutathione Homeostasis
Authors: Burnham Ellen L. , Brown Lou Ann S. , Halls Lynn , Moss Marc
Published: August 2004

Background: An association between the development and severity of the acute respiratory distress syndrome has been described in individuals who abuse alcohol chronically, possibly through a mechanism involving the deficiency of pulmonary glutathione. In a rodent model of chronic alcohol abuse, this antioxidant contributes to the maintenance of alveolar‐capillary membrane integrity. We postulated that humans who chronically abuse alcohol will have similar alterations in alveolar‐capillary barrier function.

Methods: Bronchoalveolar lavage was performed in 18 healthy chronic alcoholics and 18 control subjects; total protein and glutathione concentrations were measured within the epithelial lining fluid. To examine possible protracted effects of alcohol abuse, a subset of 11 chronic alcoholic subjects underwent a second bronchoalveolar lavage after a week of abstinence.

Results: Chronic alcoholic subjects had significantly elevated protein concentrations compared with controls (8.64 μg protein/ng immunoglobulin A vs. 5.91 μg protein/ng immunoglobulin A, p= 0.01). After a week of abstinence, no significant increase in either the glutathione levels or normalization of the protein concentrations in the epithelial lining fluid was demonstrable.

Conclusions: Increased protein levels in the epithelial lining fluid of individuals who abuse alcohol chronically may signify abnormal alveolar epithelial barrier function that does not appear to readily reverse after a period of abstinence.
Binge Ethanol Treatment Causes Greater Brain Damage in Alcohol‐Preferring P Rats Than in Alcohol‐Nonpreferring NP Rats
Authors: Crews Fulton T. , Braun Christopher J.
Published: August 2004

Background: Genetics is a known risk factor for alcoholism, and human alcoholics are known to suffer from a loss of brain function and mass. A 4 day rat binge drinking model is known to cause brain region‐specific damage. To investigate the role of genetics in binge‐drinking‐induced brain damage, we studied bidirectionally selected rat lines, the alcohol‐preferring P and the alcohol‐nonpreferring NP rat lines.

Method: P and NP rats were treated with a 4 day binge ethanol protocol. Animals were killed, transcardially perfused, and fixed, and their brains were removed, sectioned, and stained by using the amino cupric silver stain of de Olmos or by using immunohistochemistry for phospho‐extracellular signal regulated kinases and other antigens.

Results: Significant brain damage was found in the olfactory bulbs, posterior perirhinal cortex, and entorhinal cortex in both P and NP rats. P rats were found to have significantly greater brain damage, compared with NP rats, in the posterior perirhinal and posterior entorhinal cortexes, 239%± 50% (p < 0.02) and 219%± 46% (p < 0.01), respectively. Phospho‐extracellular signal regulated kinase immunohistochemistry stained prominently in damaged brain areas.

Conclusions: The P rat line, a genetic model of alcoholism, shows greater region‐specific brain damage due to binge ethanol treatment than its genetic counterpart, the NP rat line. These findings suggest that genetics contribute to susceptibility for binge‐induced brain damage.
A Comparative Study on Alcohol‐Preferring Rat Lines: Effects of Deprivation and Stress Phases on Voluntary Alcohol Intake
Authors: Vengeliene Valentina , Siegmund Sören , Singer Manfred V. , Sinclair John David , Li Ting‐Kai , Spanagel Rainer
Published: August 2004

Background: Voluntary alcohol intake in rats can be influenced by alcohol deprivation phases and stress. We investigated the magnitude of the effects of both deprivation and stress (forced swimming in cold water and foot‐shock had been chosen as stressors distinct in their physical and psychological features) on alcohol intake and the influence of these experiences on the time course of alcohol drinking behavior. For the alcohol drinking procedure, a long‐term model of alcohol self‐administration originally developed for heterogeneous Wistar rats was used and was compared with different alcohol‐preferring rat lines.

Methods: Adult male Alko alcohol (AA), alcohol‐preferring (P), high‐alcohol‐drinking (HAD), and unselected Wistar rats were given ad libitum access to water, 5%, and 20% alcohol solutions for 6 months. A deprivation phase of 14 days was performed after 8 weeks of access to alcohol. After 16 weeks and 22 weeks of alcohol access, all animals were subjected to forced swimming and foot‐shock, respectively, for 3 consecutive days, while alcohol intake was still being measured.

Results: Alcohol deprivation led to a significant increase in alcohol intake in Wistar rats and P rats. No alcohol deprivation effect was observed in HAD and AA rats; after deprivation, however, their preference for the 20% alcohol solution increased, immediately in the HAD rats and gradually over time in the AA rats. Repeated swim stress caused an increase in alcohol intake in Wistar rats but no changes in the alcohol‐preferring rat lines. Foot‐shock stress increased alcohol consumption in all lines of rats, but the most pronounced effects were observed in HAD and P rats.

Conclusions: Wistar, HAD, P, and AA rats differentially respond to alcohol deprivation and stress, showing that the genetic background of these different rat lines profoundly affects relapse‐like drinking and stress‐induced drinking.
Alcohol Use and Cognition at Mid‐Life: The Importance of Adjusting for Baseline Cognitive Ability and Educational Attainment
Authors: Krahn Dean , Freese Jeremy , Hauser Robert , Barry Kristen , Goodman Brian
Published: August 2004

Background: The nature of the relationship between cognition and alcohol consumption remains controversial. Studies have reported negative, positive, and nonsignificant effects of alcohol consumption on cognition. Problematic throughout the literature is that baseline cognitive ability has not been adequately controlled in previous studies, and even educational attainment is only sometimes controlled. Because such variables may be associated with both alcohol intake and later‐life cognition, we hypothesize that the observed relationship between alcohol intake and cognition may change when these variables or other conditions in early life have been controlled.

Methods: We examined the relationship of alcohol intake and cognition at age 53 using the Wisconsin Longitudinal Study, which has followed Wisconsin high school graduates from 1957 to 1992. Our measures include cognitive ability test scores from the freshman and junior years of high school, educational attainment, an abstract reasoning test score at age 53, alcohol intake at age 53, and other measures.

Results: When no controls were used, both men and women with low levels of alcohol consumption at 53 (i.e., 0–1 drink per day) had better scores on the abstract reasoning subtest of the Wechsler Adult Intelligence Scale (WAIS‐R) at age 53 than subjects who never drank or currently did not drink. However, after adjusting for adolescent‐measured cognitive ability and educational attainment, men with low levels of consumption no longer had higher abstract reasoning scores than nondrinking men, but they still did have higher abstract reasoning scores than men who drank more than one drink per day. For women, adjusting for cognitive ability and educational attainment eliminated all significant effects of alcohol on cognition, and reversed the nonsignificant result that women with higher consumption had the highest cognition scores. These results demonstrate the importance of adjusting for baseline cognitive ability when attempting to study the effect of long‐term alcohol use patterns on cognition, and that educational attainment cannot be considered a valid substitute for baseline cognition scores.

Conclusions: Much of the apparent benefit of moderate alcohol intake on cognition in our society may well be explained by differential rates of alcohol consumption among subjects with differing baseline cognitive ability scores. Neither is there evidence that moderate alcohol intake reduces cognitive functioning.
Students’ perceptions toward the use of the digital library in weekly web‐based distance learning assignments portion of a hybrid programme
Authors: Koohang Alex
Published: August 2004

This study attempted to investigate students’ perceptions toward the use of the digital library in weekly web‐based distance learning assignments portion of a hybrid instructional programme. Attention was given to three variables—age, gender, and prior experience with the Internet. A Likert‐type instrument consisting of 10 items was designed to collect information about students’ perceptions toward the use of digital library. The subjects of this study were 154 students enrolled in an undergraduate hybrid programme in management at a medium‐sized university in the Midwest, USA. Collected data were analysed using ANOVA. The overall results suggest that students had positive perceptions toward the use of the digital library. Age was found to be an insignificant factor. However, gender and prior experience with the Internet were found to be significant factors. Males had significantly higher positive perceptions toward using the digital library. Students who had more prior experience with the Internet had significantly higher positive perceptions toward using the digital library. Implications for practice are discussed and recommendations are made for future research.
Match Makers and Deal Breakers: Analyses of Assortative Mating in Newlywed Couples
Authors: Watson David , Klohnen Eva C. , Casillas Alex , Nus Simms Ericka , Haig Jeffrey , Berry Diane S.
Published: August 2004

Abstract
We conducted a comprehensive analysis of assortative mating (i.e., the similarity between wives and husbands on a given characteristic) in a newlywed sample. These newlyweds showed (a) strong similarity in age, religiousness, and political orientation; (b) moderate similarity in education and verbal intelligence; (c) modest similarity in values; and (d) little similarity in matrix reasoning, self‐ and spouse‐rated personality, emotional experience and expression, and attachment. Further analyses established that similarity was not simply due to background variables such as age and education and reflected initial assortment (i.e., similarity at the time of marriage) rather than convergence (i.e., increasing similarity with time). Finally, marital satisfaction primarily was a function of the rater's own traits and showed little relation to spousal similarity.
Prevention of erosive oesophagitis relapse with pantoprazole
Authors: Richter J. E. , Fraga P. , Mack M. , Sabesin S. M. , Bochenek W. ,   The Pantoprazole US GERD Study Group
Published: August 2004

Aim : To compare the safety and efficacy of pantoprazole and ranitidine in maintaining erosive oesophagitis healing.

Methods : Gastro‐oesophageal reflux disease patients (349) with endoscopically documented healed erosive oesophagitis (grade 0 or 1) were randomly assigned to receive pantoprazole (10, 20 or 40 mg/q.d.s.) or ranitidine (150 mg/b.d.). Erosive oesophagitis status was assessed endoscopically at months 1, 3, 6 and 12 or when relapse symptoms appeared (relapse = reappearance of erosive oesophagitis grade 2 within 12 months). Symptom‐free days were also assessed.

Results : Pantoprazole 20‐ and 40‐mg were significantly more effective than ranitidine in maintaining healing regardless of initial erosive oesophagitis grade. Response was dose‐related. After 12 months 78, 55, 46 and 21% of patients remained healed (40‐, 20‐, 10‐mg pantoprazole and ranitidine). Pantoprazole 40‐mg produced significantly more symptom‐free days (83%) than ranitidine (58%). Heartburn‐free days/nights were significantly higher with pantoprazole 40‐mg (92 and 93%) than ranitidine (73 and 77%). The most frequent reason for discontinuation, unsatisfactory efficacy, occurred most often with ranitidine (P < 0.001).

Conclusion : Once‐daily pantoprazole therapy prevented relapse of healed erosive oesophagitis more effectively than ranitidine and with fewer heartburn days. Response to pantoprazole was dose‐related. Pantoprazole 40‐mg was the most effective regimen and consistent in maintaining erosive oesophagitis healing with a good safety and tolerability profile.
Isolation from Spleen of a 57‐kDa Protein Substrate of the Tyrosine Kinase Lyn
Authors: Donella‐Deana Arianna , James Peter , Staudenmann Werner , Cesaro Luca , Marin Oriano , Brunati Anna Maria , Ruzzene Maria , Pinna Lorenzo A.
Published: August 2004

A 57‐kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src‐like tyrosine kinase Lyn purified from the same source with a Km of 0.34 μM. The tyrosine kinases c‐Fgr, Fyn, C‐terminal Src kinase and p72syk as well as the Ser/Thr‐specific CAMP‐dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C‐terminal Src kinase, which acts to down‐regulate the Src‐like protein‐tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide‐isomerase which belongs to the superfamily of Cys‐Gly‐His‐Cys‐containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C‐terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti‐Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested ‘processive phosphorylation’ model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).
Phagocytic Activation Induces Formation of Platelet‐Activating Factor in Human Monocyte‐Derived Macrophages and in Macrophage‐Derived foam Cells
Authors: Dentan Christine , Lesnik Philippe , Chapman M. John , Ninio Ewa
Published: August 2004

Monocyte‐derived macrophages and macrophage‐derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet‐activating factor (1‐O‐alkyl‐2–acetyl‐sn‐glycero‐3‐phosphocholine, PAF‐acether, PAF), a proinflammatory phospholipid, in human monocyte‐derived macrophages (macrophages) and in cholesterol‐loaded macrophage foam cells (foam cells). Adherent human monocyte‐derived macrophages were transformed into foam cells upon incubation with acetylated low‐density lipoproteins (Ac‐LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3±1.3 μg/μg DNA and 0.2±0.3 μg/μg DNA, n= 5, respectively). After phagocytic stimulation with serum‐opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5–1.1 pmol PAF/μg DNA, n= 5, corresponding to 4.0–8.8 pmol PAF/106 cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell‐associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, ∼ 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse‐phase liquid chromatography. Addition of exogenous 16:0 lyso‐PAF alone triggered PAF formation (0.9–1.7 pmol PAF/μg DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso‐PAF increased PAF formation in an additive manner.

Acetyltransferase, the enzyme which acetylates the precursor lyso‐PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83–240 pmol PAF formed · min−1· mg DNA−1 (n= 4); such elevated activity was not increased by OPZ‐stimulation. The activity of acetylhydrolase, the PAF‐degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded · min−1· mg DNA−1 (n= 5). Cell‐associated acetylhydrolase activity was increased significantly by 40±15% (P < 0.003, n= 5) after 15–30 min of activation with OPZ compared with non‐stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage‐derived foam cells upon phagocytic‐activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects.
Structural Organization of the Gene Encoding the Neuroendocrine Chaperone 7B2
Authors: Braks Joanna A. M. , Broers Cees A. M. , Danger Jean‐Michel H. A. , Martens Gerard J. M.
Published: August 2004

The neuroendocrine‐specific polypeptide 7B2 is a constituent of the regulated secretory pathway. Recently, 7B2 was found to function as a molecular chaperone for prohormone convertase PC2. This report describes the genomic organization of the 7B2 gene which consists of six exons. Exon 1 corresponds to the 5′‐untranslated mRNA region, while exons 2 and 3 encode the signal peptide and the amino‐terminal half of the 7B2 protein that is distantly related to a subclass of molecular chaperones. The carboxy‐terminal half of 7B2, responsible for its inhibitory action on PC2, is encoded by exons 4–6. Primer‐extension analysis showed that the human 7B2 gene is transcribed from multiple transcription‐initiation sites. The human 7B2 gene promoter contains a cAMP‐responsive element, an AP‐1 site, and several Pit‐1/GHF‐1‐binding domains and heat‐shock‐element‐like sequences but no obvious TATA or CAAT boxes. Of further interest is the finding of two DNA elements which are common to the promoter regions of the 7B2 gene and other genes selectively expressed in neuroendocrine tissues.
Stoichiometry, Organisation and Catalytic Function of Protein X of the Pyruvate Dehydrogenase Complex from Bovine Heart
Authors: Sanderson Sanya J. , Miller Clare , Lindsay J. Gordon
Published: August 2004

Mammalian pyruvate dehydrogenase complex (PDC) contains a subunit, protein X, which mediates high‐affinity binding of dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide acetyltransferase (E2) core. Precise stoichiometric determinations on bovine heart PDC, by means of two approaches, indicate the presence of 12 mol protein X/mol PDC and 60 mol E2/mol PDC.

Studies of the organisation of collagenase‐modified PDC by means of covalent cros‐linking of N,N′‐1,2‐phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross‐linked products have Mr values corresponding to homodimers of protein X. However, significant formation of higher‐Mr aggregates indicates that lipoyl domains of protein X can form an interacting network independent of E2 lipoyl domains. These data suggest that either 12 interacting X monomers or 6 interacting X dimers are involved in the binding of six E3 homodimers to the E2/X core. The presence of 60 E2 subunits/complex also supports proposals for a non‐integrated external position of protein X.

Collagenase‐treated PDC possesses residual activity (15%), indicating that protein‐X‐linked lipoamide groups can substitute for the lipoyl domains of E2 in overall complex catalysis. Protein‐X‐mediated diacetylation of dihydrolipoamide moieties is also performed by the modified complex which raises the possibility of a unique catalytic function for protein X.
The βA4 Amyloid Precursor Protein Gene and Alzheimer's Disease
Authors: Hendriks Lydia , Van Broeckhoven Christine
Published: August 2004

Alzheimer's disease is a senile dementia caused by progressive neurodegeneration of the central nervous system. One of the most prominent pathological characteristics is βA4 amyloid deposition in senile plaques in the brain parenchyma and in cerebral blood vessels. βA4 amyloid is processed from a larger integral membrane protein, the βA4 amyloid precursor protein. Different pathogenic mutations in this protein have been detected in a small number of Alzheimer's disease families. Here functional implications of these mutations on the processing of the precursor protein and the βA4 amyloid deposition will be discussed with respect to the pathogenesis of Alzheimer's disease and related disorders.
The Effects of Sodium Perchlorate on Rabbit Muscle Enolase
Authors: Kornblatt Mary Judith , Al‐Ghanim Adel , Kornblatt Jack A.
Published: August 2004

Incubation of rabbit ββ enolase in NaClO4 (≤0.3 M) results in a loss of enzymatic activity and striking changes in the second‐derivative ultraviolet spectrum of enolase. HPLC gel filtration shows that dissociation of the dimeric enzyme is occurring. We have used molecular modelling, fluorescence and circular dichroic spectroscopy to examine the structural differences between the monomeric and dimeric forms of this protein. In the dimer, the tyrosine residues are in a non‐polar environment; upon dissociation, two of them that were at the dimer interface become exposed. This results in large changes in the second‐derivative spectrum. Both the tryptophan fluorescence emission spectrum and the aromatic region of the CD spectrum indicate that there are also changes in the environment of other aromatic residues. No perturbations in the peptide bond region of the CD spectrum are observed. We propose that the major structural effect of NaClO4, is to increase the flexibility of the loops connecting the helices and strands of the α/β, barrel of enolase. These loops, which contain about half of the aromatic residues, contain some of the residues of the active site and other residues involved in subunit contacts. Increased flexibility of the loops could disrupt both subunit interactions and the structure of the active site.
Cucumber T‐Complex Protein
Authors: Ahnert Volker , May Christian , Gerke Roland , Kindl Helmut
Published: August 2004

T‐complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP‐1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157‐Da protein exhibits only 28% identity to both TCP‐1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat‐degrading non‐dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 230000×g for 4 h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59‐kDa species as constituents of 22‐S particles. From hypocotyls, a preparation of T‐complex was obtained which consisted almost exclusively of proteins in the molecular range of 57–62 kDa. Likewise, the radioactive Cucumis sativus TCP‐1 synthesized from CSTCP‐1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61‐kDa species.
Role of cAMP in the Regulation of Hepatocytic Autophagy
Authors: Holen Ingunn , Gordon Paul B. , Strømhaug Per E. , Seglen Per O.
Published: August 2004

To assess the role of cAMP in the regulation of autophagy, we examined the effects of cAMP analogues and cAMP‐elevating agents on freshly isolated rat hepatocytes, using electroinjected [3H]raffinose as an autophagy probe. Glucagon was found to stimulate, inhibit or have no effect on autophagy, depending on the inclusion of metabolites like pyruvate (which caused ATP depletion and autophagy suppression) and amino acids (a complete mixture that antagonized pyruvate) in the incubation medium. Inhibition was also observed with theophylline, a cAMP‐elevating inhibitor of cyclic nucleotide phosphodiesterases, and with the adenylyl cyclase activator deacetylforskolin. At low concentrations of deacetylforskolin, the inhibition could be abolished by amino acids. N6,2′‐O‐ibutyryladenosine 3′,5′‐monophosphate (Bt2‐cAMP) strongly inhibited both autophagic sequestration of [3H]raffinose and overall autophagic protein degradation; again, amino acids abolished the autophagy‐inhibitory effect of low Bt2‐cAMP concentrations. Several other cAMP analogues {8–thiomethyl‐cAMP, N6‐benzoyl‐cAMP, (S)‐5,6–dichloro‐1‐d‐ribofuranosylbenzimidazole 3′,5′‐[thio]monophosphate, (S)‐8–bromoadenosine 3′,5′‐[thio]monophosphate} inhibited autophagy as well. The effect of Bt2‐cAMP was rapid, dose‐dependent, reversible and did not require concomitant protein synthesis. Neither Bt2‐cAMP nor deacetylforskolin reduced intracellular ATP levels or cell viability, ruling out inhibition of autophagy by non‐specific cytotoxicity. The autophagy‐inhibitory effect of Bt2‐cAMP could be substantially antagonized (40–50%) by KT‐5720, a specific inhibitor of the cAMP‐dependent protein kinase A, and by the nonspecific protein kinase inhibitor K‐252a. Somewhat surprisingly, KN‐62 and KT‐5926, allegedly specific inhibitors of Ca2+/calmodulindependent protein kinase II and myosin light chain kinase, respectively, were also Bt2‐cAMP‐antagonistic. These results suggest that cAMP regulates the early, sequestrational step of hepatocytic autophagy by a highly conditional, dual mechanism, inhibition being predominant under most conditions in freshly isolated hepatocytes, whereas stimulation reportedly predominates in vivo. The effect of cAMP is probably mediated by protein kinase A, but other protein kinases would appear to participate in the regulation of autophagic sequestration as well.
Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser Azotobacter Vinelandii Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy
Authors: Steensma Elles , Heering Hendrik A. , Hagen Wilfred R. , Van Mierlo Carlo P. M.
Published: August 2004

This study deals with the detailed electrochemistry and complete EPR‐monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild‐type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild‐type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild‐type flavodoxin response. EPR‐monitored redox titrations of wild‐type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox‐linked pK. Instead, the presence of at least two redox‐linked protonation sites is suggested: pKred,1= 5.39±0.08, pKox= 7.29±0.14, and pKred,2= 7.84±0.14 with Em,7=−459±4 mV, and a constant redox potential at high pH of −485±4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is −0.5±0.1 mV·K−1, yielding ?H°= 28.6±1.5 kJ·mol−1 and ?S°= ‐50.0±6.2 J·mol−1·K−1. No significant differences in redox properties of wild‐type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II.
Molecular Characterisation of Plant Endoplasmic Reticulum
Authors: Coughlan Sean J. , Hastings Craig , Winfrey Ron J.
Published: August 2004

Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two‐step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components.

A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity‐purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide‐isomerase. Northern and Southern blot analysis showed that the mRNA from a single‐copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide‐isomerase catalytic activity, thus confirming identity of this protein.
Elongation Factor Ts from Thermus thermophilus
Authors: Blank Jutta , Nock Steffen , Kreutzer Roland , Sprinzl Mathias
Published: August 2004

The gene encoding the elongation factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF‐Ts from E. coli with 282 amino acid residues, EF‐Ts from T. thermophilus is considerably shorter, differing by 86 amino acids. EF‐Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins. Purified T. thermophilus EF‐Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys190 by iodoacetamide affects neither the dimerization nor the ability of EF‐Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF‐Ts, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF‐Tu · EF‐Ts)2 complex, a ternary EF‐Tu · EF‐Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF‐Ts, that does not bind to EF‐Tu but is still capable of forming homodimers.
Use of T7 RNA Polymerase in an Optimized Escherichia coli Coupled in vitro Transcription‐Translation System
Authors: Köhrer Caroline , Mayer Christine , Gröbner Peter , Piendl Wolfgang
Published: August 2004

An Escherichia coli coupled in vitro transcription‐translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21(DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio between mRNA and ribosomes has to be regulated in the coupled in vitro system. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The coupled in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the coupled in vitro system.
Acetaldehyde Mediates the Synchronization of Sustained Glycolytic Oscillations in Populations of Yeast Cells
Authors: Richard Peter , Bakker Barbara M. , Teusink Bas , Van Dam Karel , Westerhoff Hans V.
Published: August 2004

In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long‐standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180° out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 μM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 μM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells.
Comparison of Self‐Sustained Sequence‐Replication Reaction Systems
Authors: Gebinoga Michael , Oehlenschläger Frank
Published: August 2004

The 3SR (self‐sustained sequence‐replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA‐dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)‐1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three‐enzyme 3SR and our two‐enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two‐enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three‐enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments.