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11099363
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Electrophysiologic findings of a patient with inappropriate sinus tachycardia cured by selective radiofrequency catheter ablation.
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Radiofrequency catheter ablation (RFCA) for inappropriate sinus tachycardia (IST) is associated with a high recurrence rate and sometimes requires pacemaker implantation, especially after extensive ablation. We report a patient with drug-refractory IST who was successfully treated by selective RFCA to the 2 earliest activation sites. During tachycardia, the earliest atrial activation preceded the surface P wave by 50 ms or more, whereas it was only 27 ms for the rest of the right atrium after ablation. Our patient had the longest activation period during tachycardia among the reported patients. In IST patients, a longer activation time at the site of the earliest atrial activation may imply that the abnormality is confined to a small area within the sinus node and may predict the efficacy of selective RFCA.
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11099364
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Right bundle branch block-induced Q waves simulating anterior myocardial infarction extension.
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A concept generally accepted in clinical electrocardiography is the assumption that a right bundle branch block (RBBB) does not alter significantly the initial portion of the plex and because the left bundle branch is intact, the septum is activated normally in a left-to-right direction. We report a woman with an acute anterior myocardial infarction (MI) in which a small R wave was present in leads V1 and V4, but with the development of RBBB associated with PR-interval prolongation, these R waves were replaced by Q waves. Subsequently, the electrocardiographic features of anterior MI disappeared itantly with the loss of RBBB. The clinical and electrophysiologic implications of these findings are discussed.
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11099365
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Intravenous versus oral rehydration during a brief period: stress hormone responses to subsequent exhaustive exercise in the heat.
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The purpose of this study was to determine if intravenous fluid rehydration, versus oral rehydration, during a brief period (20 min) differentially affects plasma ACTH, cortisol, and norepinephrine concentrations during subsequent exhaustive exercise in the heat. Following dehydration (DHY) to Eth 4% of body weight, 8 nonacclimated highly trained males (age = 23.5 +/- 1.2 years, VáO2peak = 61.4 +/- 0.8 ml á kg á min-1, % body fat = 13.5 +/- 0. 6%) cycled to exhaustion at 74% VáO2peak in 36.8 C on three different occasions. These included: (a) no fluid (NF), where no fluid was provided during the rehydration period; (b) DRINK, where oral rehydration (0.45% NaCl) was provided equal to 50% of the prior DHY; and (c) IV, where intravenous infusion (0.45% NaCl) was provided equal to 50% of the prior DHY. Exercise time to exhaustion was not different (p =.07) between the DRINK (34.86 +/- 4.01) and IV (29.48 +/- 3.50) trials, but both were significantly (p <.05) longer than the NF (18.95 +/- 2.73) trial. No differences (p >.05) were found for any of the hormone measures among trials. The endocrine responses at exhaustion were similar regardless of hydration state and mode of rehydration, but rehydration prolonged the exercise time to exhaustion.
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11099366
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Effects of ingesting a large volume of carbohydrate-electrolyte solution on rehydration during recovery and subsequent exercise capacity.
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This randomized, double-blind study examined the effects of rehydration per se and rehydration plus carbohydrate (CHO) ingestion during recovery (REC) on subsequent endurance running capacity. Nine men ran at 70% VáO2max on a level treadmill for 90 min (T1) on two occasions, followed by a 4 hour REC and a further exhaustive run at the same speed (T2). During the first 3 hours of REC, subjects drank either a 6.9% CHO-electrolyte solution (CE) or a CHO- and electrolyte-free sweetened placebo (PL) every 30 min. Volumes prescribed were 200% of the fluid lost after T1, but the actual volume of fluid ingested during the REC ranged from 113-200% and 88. 5-200% of the body mass lost for the CE and PL trials (NS). However, positive fluid balance was found in both trials after REC. During T2, run time was 24.3 +/- 4.4 min longer in the CE trial (69.3 +/- 5.5 vs. 45.0 +/- 4.2 min; p <.05). Higher blood glucose concentrations were observed throughout REC in the CE trial. These results suggest that ingesting a CHO-electrolyte solution is more effective in restoring endurance pared to the same large volume of placebo, even plete rehydration was achieved in both trials.
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11099367
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Active recovery counteracts the post-exercise rise in plasma-free fatty acids.
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The present study investigated the effect of active recovery (AR) pared to rest recovery (RR) upon FFA concentrations following moderate- (MI) or high-intensity (HI) running. Fourteen well-trained males (23.7 +/- 6 years, VáO2max = 69.5 +/- 1.8ml á min-1 kg-1) were randomly assigned into two trials (HI = 30 min at 82% of VáO2max; MI = 60 min at 75% of VáO2max). Within each group, the pleted two sets of experiments of running followed by either AR (15 min running at 50% of VáO2max) or RR (complete rest in the supine position). Plasma volume changes after the exercise did not deviate between the AR or RR trials. In both the HI and MI trials, AR resulted in lower FFA peaks and lower overall FFA concentrations while performing AR (p <.05). However, upon discontinuing AR, there was a rise in the FFA concentration. At 120-min post-exercise, the FFA concentrations after AR and RR were not significantly different. The changes in the FFA/albumin ratio were similar to the FFA responses. It is concluded that AR may counteract the rise in FFA 5-15 minutes after exercise.
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11099368
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Effects of meal form and composition on plasma testosterone, cortisol, and insulin following resistance exercise.
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The purpose of this study was to examine the effects of postexercise feeding on plasma levels of insulin, testosterone, cortisol, and testosterone:cortisol (T:C). Ten experienced, resistance trained males (20.7 +/- 0.95 years) were given whole food (WF: protein 38 g; carbohydrate 70 g; fat 7 g), a supplemental drink (SD; isocaloric and isonitrogenous to WF), an isocaloric carbohydrate beverage (C), or a placebo beverage (P) immediately, 2 and 4 hours after a standardized weight training protocol on 4 days, each separated by 1 week, in a repeated measures design. Subjects also received a standardized meal at 7 and 12 hours postexercise. Insulin, testosterone, and cortisol were measured pre-exercise and during 24 hours of recovery (at 0.5, 2.5, 4.5, 8, and 24 hours) using venous blood samples. Significant (condition 3 time) interactions were found for insulin, testosterone, and T:C, but not for cortisol (p <. 05). The SD yielded a greater response for insulin than all other conditions. Conversely, P demonstrated the greatest values for testosterone and T:C at 2.5 and 4.5 hours postexercise. Cortisol did not vary between conditions and there were no condition effects for insulin, testosterone, cortisol, and T:C at 8 or 24 hours. In conclusion, the efficacy of postexercise feeding for optimizing T:C and muscle growth is unclear; however, consumption of SD appears to maximize circulating insulin for several hours following resistance exercise.
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11099370
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Effect of iron supplementation on thyroid hormone levels and resting metabolic rate in two college female athletes: a case study.
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Iron plays an important role in thyroid hormone metabolism; thus, iron deficiency anemia may lead to alterations in resting metabolic rate (RMR). Based on this premise, two iron-deficient-anemic female athletes, 18 (A1) and 21 (A2) years of age, were supplemented with 23 mg/day of elemental iron to assess its effects on iron and thyroid hormone status and RMR at 0, 8, and 16 weeks. Anemia was clinically corrected in both subjects (hemoglobin: A1 = 11.0 to 13.0 to 12.6 g/dL and A2 = 11.5 to 13.9 to 12.6 g/dL, 0 to 8 to 16 weeks, respectively). Serum ferritin (SF) concentration also improved in both subjects (A1: 5.0 to 11.0 to 15.0 ng/dL and A2: 5.0 to 16.0 to 20.0 ng/dL; 0 to 8 to 16 weeks, respectively); however, 16 weeks of iron supplementation did not fully replete iron stores. A2 increased dietary iron and ascorbic acid intakes from 8 to 16 weeks, possibly accounting for her higher SF concentrations. RMR and total thyroxine changed over time: A1 increased while A2 decreased in these variables. Although clinical correction of iron deficiency anemia occurred after 16 weeks of low-level iron supplementation, RMR and thyroid hormone metabolism were oppositely affected in the two subjects.
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11099369
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Iron status and resting immune function in female collegiate swimmers.
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Iron deficiency may lead to anemia and may result promised endurance exercise performance. Iron deficiency has also been reported to adversely affect the immune system and has been associated with attenuation of natural killer cell (NK) activity. This study was conducted to examine the relationship between iron status and NK activity in highly conditioned female athletes. Ten collegiate female swimmers (SWM) and 9 inactive females (SED) participated in this investigation. Resting blood samples were obtained and analyzed for serum iron and ferritin. NK activity (% lysis) was determined using a whole blood method (51Cr release assay). No significant relationship was found between iron and NK activity (r = 0.55, p =.09), nor between serum ferritin and NK activity (r = 0.33, p =.35) for SWM. ANOVA revealed significantly greater NK activity for SWM (51.63 +/- 15.79%) versus SED (30.34 +/- 13.67%). Serum ferritin levels were not significantly different between SWM (20.38 +/- 8.62 hg á ml-1) and SED (16.79 +/- 10.53 hg á ml-1), nor were iron values different between groups (16.54 +/- 2. 17 mmol á L-1 SWM; 11.92 +/- 2.61 mmol á L-1 SED). A significant relationship between iron status and resting immune function could not be established. Exercise training may affect NK activity; however, the influence of iron status on immune function requires further evaluation.
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11099371
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The effect of siberian ginseng (Eleutherococcus senticosus) on substrate utilization and performance.
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It has been suggested that Eleutherococcus senticosus (ES), also known as Siberian ginseng or ciwuija, increases fat utilization in humans. The purpose of this study was to examine the physiological responses to supplementation with ES in endurance cyclists. Using a randomized, double-blind crossover design, 9 highly-trained men (28 +/- 2 years, VáO2max 57.3 +/- 2.0 ml á kg-1 á min-1) cycled for 120 min at '60% VáO2max followed by a simulated 10-km time trial. Diet was controlled, and ES (1,200 mg á day-1) or a placebo (P) were administered for 7 days prior to each of the two trials. Oxygen consumption, respiratory exchange ratio, and heart rate were recorded every 30 min, and rating of perceived exertion, plasma [lactate], and plasma [glucose] were recorded every 20 min during the 120 min of steady state cycling. There were no significant differences (p >.05) between the ES and P groups at any steady-state time interval or during the cycling time trial (ES = 18.10 +/- 0.42, P = 17.83 +/- 0.47 min). In contrast with previous reports, the results of this study suggest that ES supplementation does not alter steady-state substrate utilization or 10-km cycling performance time.
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11099372
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Creatine monohydrate supplementation enhances high-intensity exercise performance in males and females.
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Creatine monohydrate supplementation has been shown to enhance high-intensity exercise performance in some but not all studies. Part of the controversy surrounding the ergogenic effect(s) of creatine monohydrate supplementation may relate to design issues that result in low statistical power. A further question that remains unresolved in the creatine literature is whether or not males and females respond in a similar manner to supplementation. We studied the effect of creatine supplementation upon high intensity exercise performance in 24 subjects (n = 12 males, n = 12 females). Creatine monohydrate (Cr; 5g, 4x/d 3 4d) and placebo (Pl; glucose polymer 3 4d) were provided using a randomized, double-blind crossover design (7 week washout). e measures included: 2 3 30-s anaerobic cycle test, with plasma lactate pre- and post-test; dorsi-flexor: maximal voluntary contraction (MVC), 2-min fatigue test, and electrically stimulated peak and tetanic torque; isokinetic knee extension torque and 1-min ischemic handgrip strength. Significant main effects of Cr treatment included: increased peak and relative peak anaerobic cycling power ( 3.7%; p <. 05), dorsi-flexion MVC torque ( 6.6%; p <.05), and increased lactate ( 20.8%; p <.05) with no gender specific responses. We concluded that short-term Cr supplementation can increase indices of high-intensity exercise performance for both males and females.
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11099373
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Improved 2000-meter rowing performance in competitive oarswomen after caffeine ingestion.
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petitive oarswomen (age, 22 +/- 3 years; mass, 64.4 +/- 3.8 kg) performed three simulated 2,000-m time trials on a rowing ergometer. The trials, which were preceded by a 24-hour dietary and training control and 72 hours of caffeine abstinence, were conducted 1 hour after ingesting caffeine (6 or 9 mg á kg-1 body mass) or placebo. Plasma free fatty acid concentrations before exercise were higher with caffeine than placebo (0.67 +/- 0.34 vs. 0.72 +/- 0.36 vs. 0.30 +/- 0.10 mM for 6 and 9 mg á kg-1 caffeine and placebo, respectively; p <.05). Performance time improved 0.7% (95% confidence interval [CI] 0 to 1.5%) with 6 mg á kg-1 caffeine and 1. 3% (95% CI 0.5 to 2.0%) with 9 mg á kg-1 caffeine. The first 500 m of the 2,000 m was faster with the higher caffeine pared with placebo or the lower dose (1.53 +/- 0.52 vs.1.55 +/- 0.62 and 1. 56 +/- 0.43 min; p =.02). We concluded that caffeine produces a worthwhile enhancement of performance in a controlled laboratory setting, primarily by improving the first 500 m of a 2,000-m row.
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11099374
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Effects of carbohydrate and chromium ingestion during intermittent high-intensity exercise to fatigue.
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This study was designed to test the hypothesis that addition of chromium (Cr) to a carbohydrate-electrolyte drink would enhance the reported benefits of carbohydrate on exercise capacity during intermittent high-intensity shuttle running.
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11099375
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Ligand-induced strain in hydrogen bonds of the c-Src SH3 domain detected by NMR.
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Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biological activity. A new solution NMR method has been applied to monitor ligand-induced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. The structural response of this domain to ligand binding has been investigated by measuring trans-hydrogen bond (15)N-(13)C' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2, containing residues RALPPLPRY. parison between hydrogen bonds in high resolution X-ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabilization of loop structure. Reproducible ligand-induced changes in trans-hydrogen bond scalar couplings are observed across the domain that translate into changes in hydrogen bond length ranging between 0.02 to 0.12 A. The observed changes can be rationalized by an induced fit mechanism in which hydrogen bonds across the protein participate in pensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mutual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bonded peptide planes. The unprecedented resolution obtained demonstrates the ability to characterize subtle structural rearrangements within a protein upon perturbation, and represents a new step in the endeavor to understand how hydrogen bonds contribute to the stabilization and function of biological macromolecules.
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11099376
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Movement of the decoding region of the 16 S ribosomal RNA accompanies tRNA translocation.
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The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the plex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of plexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the plex during translocation.
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11099377
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Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II.
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Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC 2.4.2.26, XT-I) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine residues of the core protein. Based on the partial amino acid sequence of the purified enzyme from human JAR choriocarcinoma cell culture supernatant we isolated a cDNA encoding XT-I using the degenerate reverse transcriptase-polymerase chain reaction method. This enzyme, which is involved in chondroitin sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, belongs to a novel family of glycosyltransferases with no homology to proteins known so far. 5' and 3'-RACE were performed to isolate a novel cDNA fragment of 3726 bp with a single open reading frame encoding at least 827 amino acid residues with a molecular mass of 91 kDa. The human XT-I gene was located on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibited marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 865 amino acid residues was also isolated by PCR using degenerate primers based on the amino acid sequence of human XT-I. The amino acid sequence of this XT-II isoform displayed 55% identity to the human XT-I. The XT-II gene was located on chromosome 17q21.3-17q22, and the exon/intron structure of the 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from various tissues confirmed that both XT-I and XT-II transcripts are ubiquitously expressed in the human tissues, although with different levels of transcription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were cloned. The deduced amino acid sequences of rat xylosyltransferases displayed 94% identity to the corresponding human enzyme.
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11099379
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BII nucleotides in the B and C forms of natural-sequence polymeric DNA: A new model for the C form of DNA.
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bination of solid-state (31)P and (13)C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10(1) double helix involving BI nucleotides and a 9(1) double helix involving BII nucleotides. The 10(1) BI model is similar to a previous model of B-form DNA, while the 9(1) BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.
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11099378
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Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins.
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Oct-4 is a transcription factor that is specifically expressed in mouse embryonic stem cells and in cell lines derived thereof. In these cells, Oct-4 activates transcription from remote binding sites due to as of yet unknown co-activators. Expression of Oct-4 in differentiated cells is not sufficient to activate transcription from a distance, rather it requires the co-expression of co-activators such as the adenoviral oncoprotein E1A. In this paper, we used phage display to identify Oct-4-interacting proteins. We first analyzed the interaction between Oct-4 and E1A in order to optimize the biochemical conditions that enable Oct-4-specific interactions with other interacting proteins. A panning approach was used to enrich Oct-4 interacting phages from a pool of excess unspecific phages. The biochemical conditions established in our interaction assays were then used to screen a P19 EC cell cDNA expression library in M13 filamentous phage. A number of phage clones displaying portions of unknown and known transcription factors were obtained, from which the HMG-1 transcription factor was identified. HMG-1, and the closely related factor HMG-2, interact with Oct-4 when co-expressed in mammalian cells. In addition, HMG-1 was found to cooperate with Oct-4 in P19 EC cells. These results provide the first evidence of a non-viral factor that enhances Oct-4 distance-dependent transactivation in stem cells.
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11099380
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Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study.
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Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A parison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to mon view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.
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11099381
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Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation.
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N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the plex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.
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11099382
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Crystal structures of a Rab protein in its inactive and active conformations.
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We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural mon to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.
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11099383
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Searching the protein structure databank with weak sequence patterns and structural constraints.
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A method is described in which proteins that match PROSITE patterns are filtered by the root-mean-square deviation of the local 3D structures of the probe and target over the ponents. This was found to increase the discrimination between true and false members of the protein family but was dependent on how unique the structural features in the pattern pared to equivalent fragments extracted from the structure databank (for example; if the pattern fell in an alpha-helix, then discrimination was poor.) We then generalised the sequence patterns (by widening the range of amino acid residues allowed at each position) and monitored how well the structural information helped retain specificity. While the discrimination of the pure sequence pattern had generally disappeared at information content values less than ten bits, the discrimination of bined sequence structure probe remained high at this point before following a similar decay. The displacement between these curves indicates that the ponent is, on average, equivalent to about ten bits. The sequence patterns were also filtered using the parison program SAP, giving a global, rather than local "view" of the proteins. This allowed the information content of the sequence patterns to e even less specific but raised problems of whether some proteins encountered with the same fold but no PROSITE pattern should constitute family members.
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11099384
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The TolA-recognition site of colicin N. ITC, SPR and stopped-flow fluorescence define a crucial 27-residue segment.
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Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region. Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown. We present, for the first time, a fully mapped TolA binding site for a colicin. It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/fluorescence binding studies. The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein. Stopped-flow kinetic studies show that the binding to TolA follows slow association kinetics. The role of other E. coli Tol proteins in colicin translocation was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB. ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis.
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11099385
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Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants.
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Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered pared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.
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11099386
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Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation.
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Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit plementation. When certain mutant versions of alkaline phosphatase bined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline plementation mutants were re-examined in this work in order to determine a molecular explanation of plementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phosphate K(m) were observed for binations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.
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11099388
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Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans.
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Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new plex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of plex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.
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11099389
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Splanchnic hyposensitivity to glypressin in a haemorrhage/transfused rat model of portal hypertension: role of nitric oxide and bradykinin.
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Hyposensitivity to vasopressin is a well documented phenomenon in animals with portal hypertension and patients with cirrhosis subject to haemorrhage. Haemorrhage is associated with the endogenous release of bradykinin, which may subsequently stimulate the formation of nitric oxide (NO). The present study investigated the relative contribution of NO synthase (NOS) isoforms and the role of bradykinin in the pathogenesis of splanchnic hyposensitivity to a long-acting vasopressin analogue, glypressin, in rats with portal hypertension induced by partial portal vein ligation (PVL). At 14 days after the operation, systemic and portal haemodynamics were measured in stable or bleeding PVL rats receiving an intravenous infusion of glypressin (0.07 mg/kg). In the treatment groups, N(G)-nitro-L-arginine methyl ester (L-NAME; a non-selective NOS inhibitor), L-canavanine (a specific inhibitor of inducible NOS) or HOE 140 (a bradykinin B(2) receptor antagonist) was administered 45 min before the infusion of glypressin. In rats with a hypotensive haemorrhage, 4.5 ml of blood was withdrawn and 50% of the withdrawn blood was re-infused before the administration of glypressin or various inhibitors. Splanchnic hyposensitivity to glypressin was demonstrated in the haemorrhage/transfused PVL rats. The infusion of L-NAME elevated the mean arterial pressure in the bleeding PVL rats without the modulation of portal pressure. The addition of L-NAME or HOE 140, but not L-canavanine, significantly and similarly potentiated the portal-hypotensive effects of glypressin. It is concluded that constitutive NOS and bradykinin are responsible, at least partly, for the splanchnic hyposensitivity to glypressin observed in the early stages of the haemorrhage/transfused rat model of portal hypertension.
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11099390
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Acute effects of oxygen and carbon dioxide on retinal vascular network geometry in hypertensive and normotensive subjects.
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The optimal design of vascular networks maximizes circulatory efficiency while minimizing power costs. We investigated the effects of acute changes in vascular tone on retinal arteriolar network geometry. Six hypertensive and six normotensive subjects each breathed air, 5% CO(2) (with 12% O(2)), and 100% O(2) for 5 min periods in random order. Retinal photographs were taken at the end of each test period. Bifurcation angles and arteriolar diameters were measured using operator-directed image analysis, and junction exponents were calculated. Arteriolar diameters narrowed on breathing O(2). The magnitude of this change was significantly greater in normotensive than in hypertensive subjects. Angles narrowed in normotensive subjects, but not significantly in hypertensive subjects. Arteriolar diameters increased significantly on breathing CO(2) in normotensive but not in hypertensive subjects, but there were no changes in angles. Despite changes in diameter, junction exponents did not change under any conditions. Vascular reactivity in the retinal arteriolar bed appears to be diminished in hypertensive subjects. The failure of junction exponents to change, despite alterations in diameter, suggests that arteriolar diameters at retinal bifurcations adhere to optimality principles when exposed to acute vasoactive stress. As vasoconstriction is associated with the narrowing of bifurcation angles, previous observations showing narrowed angles in hypertensive subjects could be explained by increased tone in the retinal arteriolar bed.
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11099391
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Induction of the stress response increases interleukin-6 production in the intestinal mucosa of endotoxaemic mice.
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Previous studies suggest that production of interleukin-6 (IL-6) is increased in the intestinal mucosa during sepsis and endotoxaemia. We tested the hypothesis that mucosal IL-6 production during endotoxaemia is increased further by the heat-shock (stress) response. The stress response was induced in mice by hyperthermia (rectal temperature of 42 degrees C for 3 min) or by intraperitoneal injection of sodium arsenite (10 mg/kg). At 2 h after induction of the stress response, groups of mice were injected subcutaneously with endotoxin (10 mg/kg) or sterile saline. IL-6 mRNA and protein levels in the jejunal mucosa were determined by an RNase protection assay and an ELISA respectively, and levels of hsp72 (heat-shock protein of 72 kDa) were determined by Western blot analysis. Hyperthermia and sodium arsenite increased hsp72 levels in the intestinal mucosa. IL-6 concentrations were increased in the jejunal mucosa of endotoxaemic mice, and this effect of endotoxaemia was potentiated by the stress response. Mucosal IL-6 mRNA levels were increased in endotoxaemic mice, and were increased further by the stress response. Thus it is concluded that mucosal IL-6 production during endotoxaemia may be further stimulated by the stress response. Increased IL-6 levels in the intestinal mucosa may be a potential mechanism by which the stress response exerts a protective effect during sepsis and endotoxaemia.
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11099392
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Role of the L-arginine/nitric oxide pathway in ischaemic/reoxygenation injury of the human myocardium.
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The role of the L-arginine/nitric oxide (NO) pathway in myocardial ischaemic/reperfusion injury remains controversial in experimental animal models. The aim of the present studies was to investigate the role of this pathway in the human myocardium. Myocardial specimens from right atrial appendages of patients undergoing elective coronary bypass graft surgery were incubated in crystalloid buffer at 37 degrees C and subjected to 120 min of simulated ischaemia followed by 120 min of reoxygenation. Tested drugs were added 15 min before ischaemia, and maintained during ischaemia and throughout reoxygenation. Ischaemia resulted in severe myocardial damage, as assessed by the leakage of lactate dehydrogenase (LDH) into the incubation medium and by the capacity of the tissue to reduce 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan product. L-Arginine (10 mM), a precursor of NO, significantly decreased LDH leakage (from 9.0+/-0.6 to 5.3+/-0.3 units/g wet wt; P<0.05), but had no effect on MTT reduction or oxygen consumption. D-Arginine (10 mM), N(G)-nitro-L-arginine methyl ester (L-NAME; 0.5 mM), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (at 1, 100, 500 and 1000 microM), an NO donor, had no significant effects on the measured indices, and L-NAME did not reverse the protection afforded by L-arginine against LDH leakage. In addition, the formation of nitrotyrosine was not influenced by ischaemia/reoxygenation alone or by the agents investigated. In conclusion, these data suggest that L-arginine affords modest protection against ischaemic/reoxygenation injury of the human myocardium, an action that is NO-independent, and that NO metabolism does not play a significant role in this model.
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11099393
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Resting peripheral blood flow in normal pregnancy and in pre-eclampsia.
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Multiple organ dysfunction followed by end organ failure occurs in pre-eclampsia. While one would intuitively reason that one of the factors contributing to the end organ failure is poor nutritional blood flow, this has yet to be demonstrated. The aim of the present study was to determine whether changes in resting nutritional blood flow occur in pre-eclampsia. We used strain-gauge plethysmography to study calf blood flow in 19 women with pre-eclampsia, 13 normal pregnant women and 17 non-pregnant controls. We reasoned that, since the prises mostly skeletal muscle, without anastomotic channels, blood flowing through this region would primarily reflect nutritive flow. Calf blood flow was significantly reduced in women with pre-eclampsia (1.95+/-0.9 ml.min(-1).100 pared with normal pregnant (3.9+/-1.4 ml.min(-1).100 ml(-1)) and non-pregnant (3.8+/-1.0 ml.min(-1).100 ml(-1)) women (P=0.0004 and P=0.0005 respectively; ANOVA). Blood flow in pre-eclampsia was also correlated significantly with platelet count as an index of disease severity. In addition, there was a significant negative correlation between blood flow and systolic blood pressure (r=-0.69, P=0.004) in the women with pre-eclampsia. These findings support the hypothesis that nutritional blood flow is reduced in pre-eclampsia. We suggest that measurement of resting calf blood flow could give a non-invasive index of deterioration of nutritive blood flow to vital organs in pre-eclampsia.
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11099395
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Non-invasive quantification of liver perfusion with dynamic computed tomography and a dual-input one-compartmental model.
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Various liver diseases lead to significant alterations of the hepatic microcirculation. Therefore, quantification of hepatic perfusion has the potential to improve the assessment and management of liver diseases. Most methods used to quantify liver perfusion are invasive or controversial. This paper describes and validates a non-invasive method for the quantification of liver perfusion puted tomography (CT). Dynamic single-section CT of the liver was performed after intravenous bolus administration of a low-molecular-mass iodinated contrast agent. Hepatic, aortic and portal-venous time-density curves were fitted with a dual-input partmental model to calculate liver perfusion. Validation studies consisted of simultaneous measurements of hepatic perfusion with CT and with radiolabelled microspheres in rabbits at rest and after adenosine infusion. The feasibility and reproducibility of the CT method in humans was assessed by three observers in 10 patients without liver disease. In rabbits, significant correlations were observed between perfusion measurements obtained with CT and with microspheres (r=0.92 for total liver perfusion, r=0.81 for arterial perfusion and r=0.85 for portal perfusion). In patients, total liver plasma perfusion measured with CT was 112+/-28 ml.min(-1).100 ml(-1), arterial plasma perfusion was 18+/-12 ml.min(-1).100 ml(-1) and portal plasma perfusion was 93+/-31 ml.min(-1).100 ml(-1). The measurements obtained by the three observers were not significantly different from each other (P>0.1). Our results indicate that dynamic bined with a dual-input partmental model provides a valid and reliable method for the non-invasive quantification of perfusion in the normal liver.
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11099394
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Conjugated linoleic acid induces lipid peroxidation in men with abdominal obesity.
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Conjugated linoleic acid (CLA) has been shown in experimental studies to have chemoprotective properties, and may decrease the deposition of body fat. CLA is prone to oxidation, and it has been suggested that increased lipid oxidation may contribute to the anti-tumorigenic effects of this agent. The present study investigates the urinary levels of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a major isoprostane, and of 15-oxo-dihydro-PGF(2alpha), a major metabolite of PGF(2alpha), as indicators of non-enzymic and enzymic arachidonic acid oxidation respectively after dietary supplementation with CLA in middle-aged men (mean age 53 years) with abdominal obesity for 1 month in a randomized controlled trial. Significant increases in the levels of both 8-iso-PGF(2alpha) and 15-oxo-dihydro-PGF(2alpha) in urine (P<0. 0001 and P=0.0013 respectively) were observed after 1 month of daily CLA intake (4.2 g/day) pared with the control group. The lipid peroxidation parameters had returned to their basal levels at 2 weeks after the cessation of CLA intake, and remained at the same levels for a further 2 weeks until the end of the study. CLA had no effect on serum alpha-tocopherol and gamma-tocopherol levels, or on the urinary levels of 2,3-dinor-thromboxane B(2). Thus CLA may induce both non-enzymic and enzymic lipid peroxidation in vivo in middle-aged men with abdominal obesity, without any side effects. The consequences of the increased lipid peroxidation after CLA supplementation are unknown.
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11099396
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Elevated circulating leptin levels in arterial hypertension: relationship to arteriovenous overflow and extraction of leptin.
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Leptin, a peptide hormone produced mainly in fat cells, appears to be important for the regulation of metabolism, insulin secretion/sensitivity and body weight. Recently, elevated plasma leptin levels have been reported in patients with arterial hypertension. Because a change in circulating leptin concentrations in such patients could be caused by altered rates of production or disposal, or both, the aim of the present study was to identify regions of leptin overflow into the bloodstream and of leptin extraction. Patients with arterial hypertension (n=12) and normotensive controls (n=20) were studied during catheterization with elective blood sampling from different vascular beds (artery, and renal, hepatic, iliac and cubital veins). Plasma leptin was determined by a radioimmunoassay. Patients with hypertension had significantly elevated levels of circulating leptin (12.8 pared with 4.1 ng/l in the controls; P<0.001), and this was also the case when adjusted for body mass index (BMI) [0.435 and 0.167 ng/l per unit BMI (kg/m(2)) respectively; P<0.001]. Circulating leptin was directly related to arterial blood pressure (r=0.38-0.62, P</=0.05-0.005) and immunoreactive insulin (r=0.51, P<0.62), but not to plasma renin activity. A significant renal extraction ratio for leptin was seen in the hypertensive patients, but this was not significantly lower than that in the controls pared with 0. 16; P=0.1). The hypertensive patients had a significantly higher hepatic venous/arterial leptin ratio than the controls pared with 0.93; P<0.02), and this ratio was correlated directly with the BMI (r=0.38, P=0.05) and immunoreactive insulin (r=0.43, P<0.05). In both hypertensive patients and controls there was a significant spillover of leptin into the iliac vein, but not into the cubital vein. In conclusion, the high concentration of circulating leptin in patients with arterial hypertension is probably caused by increased release of leptin from abdominal (especially mesenteric and omental) and gluteal adipose tissue stores, and renal extraction is slightly reduced. Leptin kinetics in arterial hypertension require further investigation.
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11099397
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Improved neoangiogenesis in transmyocardial laser revascularization combined with angiogenic adjunct in a pig model.
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Recent research has revealed neoangiogenesis as a basic phenomenon in transmyocardial laser revascularization (TMLR). Theoretically, myocardial neoangiogenesis could be further enhanced by the addition of angiogenic growth factors. Triads of TMLR channels were created in the lateral wall of the left ventricles of 12 pigs (mean body weight 73+/-5 kg), using a holmium:yttrium-aluminium garnet (YAG) laser. The animals were allocated randomly either to receive an injection of 100 microg of a bovine bone-derived growth factor mixture within the triads (n=6), or to a control group (n=6). Animals were killed 1 month later. Capillary and arteriolar densities were determined puted morphometric analysis of histological sections of the triads. The capillary density of myocardial areas within the triads was significantly greater in the group receiving the bovine bone-derived growth factor mixture than in the control group (14.3+/-3.5/mm(2) and 5.7+/-1.4/mm(2) respectively; P<0.001). The difference was also significant when considering arteriolar density (0.7+/-0.4/mm(2) and 0.2+/-0.1/mm(2) respectively; P<0.001). parison, capillary and arteriolar densities of the TMLR channel scars were 48.7+/-9.7 and 1.9+/-0. 5/mm(2) respectively in the angiogenic group, and 46.3+/-13.7 and 2. 3+/-1.3/mm(2) respectively in the control group (no significant differences). These results demonstrate that the addition of angiogenic factors to TMLR stimulates neoangiogenesis significantly in the areas adjacent to the channels, but not within the channel scars. The latter are themselves strongly vascularized. Hence bined approach, potentiating the effect of TMLR by establishing vascular connections between the neovessels of the channel scars, has the potential for improved clinical e.
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11099399
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Effects of the Ca2+ sensitizer EMD 57033 on intracellular Ca2+ in rat ventricular myocytes: relevance to arrhythmogenesis during positive inotropy.
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We have investigated the effects of the calcium-sensitizing inotropic agent EMD 57033 on Ca(2+) handling in intact and skinned rat ventricular myocytes. Intracellular Ca(2+) was monitored using fura 2. Myocytes were saponin-skinned, allowing study of sarcoplasmic reticulum (SR) function. In intact myocytes EMD 57033 (1-10 micromol/l) produced a concentration-dependent decrease in the amplitude of the Ca(2+) transient and prolonged its declining phase, but had no effect on the rise time. In skinned myocytes, the amplitude of spontaneous Ca(2+) release from the SR was decreased by EMD 57033 (5 and 10 micromol/l), although this agent had no significant effect on the frequency of spontaneous Ca(2+) release. In the presence of the cross-bridge inhibitor 2,3-butanedione monoxime (5 mmol/l), or in a low bathing Ca(2+) concentration (1 mmol/l), EMD 57033 (10 micromol/l) had smaller effects on both the amplitude and time course of the Ca(2+) transient in intact cells than in the absence of 2,3-butanedione monoxime or in the presence of 2 and 5 mmol/l Ca(2+) respectively. These data suggest that the effects of EMD 57033 on Ca(2+) are due to changes in Ca(2+) binding to troponin C, secondary to cross-bridge formation. Thus, during positive inotropy, EMD 57033 is unlikely to provoke arrhythmias due to effects on SR Ca(2+) handling. In intact cells, its effects on Ca(2+) handling would be expected to protect against arrhythmias.
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11099398
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Increased gene expression of adrenomedullin and adrenomedullin-receptor complexes, receptor-activity modifying protein (RAMP)2 and calcitonin-receptor-like receptor (CRLR) in the hearts of rats with congestive heart failure.
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Adrenomedullin is a vasodilator peptide produced in various organs, including heart and kidney. A novel adrenomedullin plex has recently been identified, namely the calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein (RAMP) 2. In the present study, we have examined gene expression of RAMP2, CRLR and adrenomedullin in hearts and kidneys of rats with congestive heart failure caused by coronary artery ligation. Partial cloning was performed to determine the rat RAMP2 nucleotide sequence. Messenger RNA levels were then determined petitive, quantitative reverse transcription-PCR techniques. Significantly increased expression levels (means+/-S.E.) of RAMP2, CRLR and adrenomedullin mRNA were found in the atrium (1.8+/-0.2-fold, 1. 8+/-0.2-fold and 2.1+/-0.1-fold, pared with sham operated rats) and in the ventricle (1.4+/-0.1-fold, 1.3+/-0.03-fold and 3.0+/-0.5-fold respectively). On the other hand, expression levels of RAMP2, CRLR and adrenomedullin mRNAs were not significantly changed in the kidney. These findings suggest potential roles of locally-produced and locally-acting adrenomedullin in the failing heart.
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11099400
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Effect of inhibition of nitric oxide synthase on dynamic cerebral autoregulation in humans.
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Cerebral blood flow is maintained constant over a range of cerebral perfusion pressures by cerebral autoregulation. Impaired cerebral autoregulation may be important in the pathogenesis of cerebral ischaemia. The mechanisms mediating normal cerebral autoregulation in humans are poorly understood. We used a recently described transcranial Doppler technique, which allows non-invasive measurement of dynamic cerebral autoregulation, to test the hypothesis that nitric oxide mediates cerebral autoregulation. The rate of rise of middle cerebral artery blood flow pared with that of arterial blood pressure, was determined following a stepwise fall in arterial blood pressure, in order to calculate an autoregulatory index. The effect of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on dynamic autoregulation pared with that of noradrenaline titrated to result in a similar rise in blood pressure. Six healthy subjects were studied in each group. The mean (S.D.) change in autoregulatory index following noradrenaline at a similar pressor dose was significantly greater than the change following the L-NMMA bolus: 1. 1 pared with -0.8 (0.8) for the left middle cerebral artery (P=0.002), and 1.1 pared with -0.8 (0.8) for the right middle cerebral artery (P=0.002). There was no difference in the mean (S.D.) blood pressure increase resulting from the two agents: L-NMMA, 19.7 (7.4) mmHg; noradrenaline, 15.5 (4.8) mmHg (P=0.281). These results suggest that nitric oxide mediates at least part of the dynamic phase of cerebral autoregulation in humans. Reduced nitric oxide release may play a role in the impaired cerebral autoregulation seen in patients with, or at risk of, cerebral ischaemia.
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11099402
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A compound heterozygote for a novel missense mutation (G105R) in exon 3 and a missense mutation (D204E) in exon 5 of the lipoprotein lipase gene in a Japanese infant with hyperchylomicronaemia.
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We systematically investigated the molecular defects resulting in primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (hereafter called 'the patient') with severe fasting hypertriglyceridaemia (type I hyperlipoproteinaemia). The primary LPL deficiency was diagnosed on the basis of the findings that no LPL activity was detected in post-heparin plasma (PHP) and that the immunoreactive LPL mass in PHP was less than 2% of the control level. The patient was pound heterozygote for a novel missense mutation (G(568)GA-->AGA/Gly(105)-->Arg; G105R) in exon 3 and a missense mutation (GAC(867)-->GAG/Asp(204)-->Glu; D204E) in exon 5 of the LPL gene. The biological significance of both missense mutations was examined by an in vitro study of the expression of the mutant G105R LPL cDNA and D204E LPL cDNA in COS-1 cells. Both mutant LPLs were catalytically inactive and were barely released by heparin from the expressing COS-1 cells. These findings explain the failure to detect LPL activity and immunoreactive LPL mass in the patient's PHP. The G105R allele could be detected by digestion with the BsmAI restriction enzyme, and the D204E allele by digestion with HincII. The patient inherited the G105R allele from his mother and the D204E allele from his father. His parents were heterozygotes for the corresponding mutant allele, but normolipidaemic. The novel G105R missense mutation could not be detected by conventional analysis of single-strand conformation polymorphism, but it was identified by extensive sequencing of the entire exons and their flanking regions in the LPL gene.
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11099401
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Mitogenic action of lysophosphatidic acid in proximal tubular epithelial cells obtained from voided human urine.
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Focal tubular cell multiplication at sites on an injured nephron is a critical event in the recovery phase following acute tubular necrosis. During this process, numerous viable tubular cells exfoliate and are shed into the urine. Lysophosphatidic acid (LPA) is generated in the plasma membrane of injured cells and acts as an intercellular mediator of various biological processes, including inflammation, proliferation and repair. In the present study, exfoliated proximal tubule (PT) cells were isolated from human urine and the mitogenic effects of LPA were investigated as a model of repair and proliferation following renal injury. LPA stimulated a 23. 5% increase in DNA synthesis, a 29.4% increase in cell number and an 86.6% decrease in cAMP content. All of these responses were pertussis toxin sensitive, indicating the involvement of G(i)-type G-proteins in LPA signalling. Conversely, the LPA-induced DNA synthesis and the decrease in intracellular cAMP content were insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), suggesting a mitogenic response via PI3K-independent mechanisms. Furthermore, we detected specific mRNA transcripts for the recently cloned human LPA-receptors, endothelial differentiation gene (Edg)-2 and Edg-4 (Edg-2>>Edg-4) by reverse transcription-PCR in PT cells. Our data suggest that LPA may behave as a local growth factor in PT cells following tubular injury.
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11099406
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Self-mode-locking of quantum cascade lasers with giant ultrafast optical nonlinearities.
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We report on the generation of picosecond self-mode-locked pulses from midinfrared quantum cascade lasers, at wavelengths within the important molecular fingerprint region. These devices are based on intersubband electron transitions in semiconductor nanostructures, which are characterized by some of the largest optical nonlinearities observed in nature and by picosecond relaxation lifetimes. Our results are interpreted with a model in which one of these nonlinearities, the intensity-dependent refractive index of the lasing transition, creates a nonlinear waveguide where the optical losses decrease with increasing intensity. This favors the generation of ultrashort pulses, because of their larger instantaneous intensity relative to continuous-wave emission.
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11099405
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How cells handle cholesterol.
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Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Recent advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts in a functional state. How rafts contribute to cholesterol metabolism and transport in the cell is still an open issue. It has long been known that cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells, and influx of lipoprotein cholesterol into cells. The regulation of cholesterol homeostasis is now receiving a new focus, and this changed perspective may throw light on diseases caused by cholesterol excess, the prime example being atherosclerosis.
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11099407
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Tunable resistance of a carbon nanotube-graphite interface.
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The transfer of electrons from one material to another is usually described in terms of energy conservation, with no attention being paid to momentum conservation. Here we present results on the junction resistance between a carbon nanotube and a graphite substrate and show that details of momentum conservation also can change the contact resistance. By changing the angular alignment of the atomic lattices, we found that contact resistance varied by more than an order of magnitude in a controlled and reproducible fashion, indicating that momentum conservation, in addition to energy conservation, can dictate the junction resistance in graphene systems such as carbon nanotube junctions and devices.
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11099404
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Autophagy as a regulated pathway of cellular degradation.
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Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a plex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.
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11099408
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Formation of sphalerite (ZnS) deposits in natural biofilms of sulfate-reducing bacteria.
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Abundant, micrometer-scale, spherical aggregates of 2- to 5-nanometer-diameter sphalerite (ZnS) particles formed within natural biofilms dominated by relatively aerotolerant sulfate-reducing bacteria of the family Desulfobacteriaceae. The biofilm zinc concentration is about 10(6) times that of associated groundwater (0.09 to 1.1 parts per million zinc). Sphalerite also concentrates arsenic (0.01 weight %) and selenium (0.004 weight %). The almost monomineralic product results from buffering of sulfide concentrations at low values by sphalerite precipitation. These results show how microbes control metal concentrations in groundwater- and wetland-based remediation systems and suggest biological routes for formation of some low-temperature ZnS deposits.
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11099409
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Tropical climate at the last glacial maximum inferred from glacier mass-balance modeling.
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Model-derived equilibrium line altitudes (ELAs) of former tropical glaciers support arguments, based on other paleoclimate data, for both the magnitude and spatial pattern of terrestrial cooling in the tropics at the last glacial maximum (LGM). Relative to the present, LGM ELAs were maintained by air temperatures that were 3.5 degrees to 6.6 degrees C lower and precipitation that ranged from 63% wetter in Hawaii to 25% drier on Mt. Kenya, Africa. Our results imply the need for a approximately 3 degrees C cooling of LGM sea surface temperatures in the western Pacific warm pool. Sensitivity tests suggest that LGM ELAs could have persisted until 16,000 years before the present in the Peruvian Andes and on Papua, New Guinea.
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11099410
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The formation of chondrules at high gas pressures in the solar nebula.
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High-precision magnesium isotope measurements of whole chondrules from the Allende carbonaceous chondrite meteorite show that some aluminum-rich Allende chondrules formed at or near the time of formation of calcium-aluminum-rich inclusions and that some others formed later and incorporated precursors previously enriched in magnesium-26. Chondrule magnesium-25/magnesium-24 correlates with [magnesium]/[aluminum] and size, the aluminum-rich, smaller chondrules being the most enriched in the heavy isotopes of magnesium. These relations imply that high gas pressures prevailed during chondrule formation in the solar nebula.
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11099411
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Support for the lunar cataclysm hypothesis from lunar meteorite impact melt ages.
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Lunar meteorites represent a more random sampling of lunar material than the Apollo or Luna collections and, as such, lunar meteorite impact melt ages are the most important data in nearly 30 years with which to reexamine the lunar cataclysm hypothesis. Within the lunar meteorite breccias MAC 88105, QUE 93069, DaG 262, and DaG 400, seven to nine different impact events are represented with 40Ar-39Ar ages between 2.76 and 3.92 billion years ago (Ga). The lack of impact melt older than 3.92 Ga supports the concept of a short, intense period of bombardment in the Earth-moon system at approximately 3.9 Ga. This was an anomalous spike of impact activity on the otherwise declining impact- frequency curve.
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11099412
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Nitric acid trihydrate (NAT) in polar stratospheric clouds.
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prehensive investigation of polar stratospheric clouds was performed on 25 January 2000 with instruments onboard a balloon gondola flown from Kiruna, Sweden. Cloud layers were repeatedly encountered at altitudes between 20 and 24 kilometers over a wide range of atmospheric temperatures (185 to 197 kelvin). position analysis showed that a large fraction of the cloud layers posed of nitric acid trihydrate (NAT) particles, containing water and nitric acid at a molar ratio of 3:1; this confirmed that these long-sought solid crystals exist well above ice formation temperatures. The presence of NAT particles enhances the potential for chlorine activation with subsequent ozone destruction in polar regions, particularly in early and late winter.
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11099413
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Reinterpreting space, time lags, and functional responses in ecological models.
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Natural enemy-victim interactions are of major applied importance and of fundamental interest to ecologists. A key question is what stabilizes these interactions, allowing the long-term coexistence of the two species. Three main theoretical explanations have been proposed: behavioral responses, time-dependent factors such as delayed density dependence, and spatial heterogeneity. Here, using the powerful moment-closure technique, we show a fundamental equivalence between these three elements. Limited movement by organisms is a ubiquitous feature of ecological systems, allowing spatial structure to develop; we show that the effects of this can be naturally described in terms of time lags or within-generation functional responses.
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11099414
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Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis.
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Many apoptotic molecules relocate subcellularly in cells undergoing apoptosis. The pro-apoptotic protein BID underwent posttranslational (rather than classic cotranslational) N-myristoylation when cleavage by caspase 8 caused exposure of a glycine residue. N-myristoylation enabled the targeting of plex of p7 and myristoylated p15 fragments of BID to artificial membranes bearing the position of mitochondria, as well as to intact mitochondria. This post-proteolytic N-myristoylation serves as an activating switch, enhancing BID-induced release of cytochrome c and cell death.
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11099415
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Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis.
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The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.
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11099416
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Down-regulation of the macrophage lineage through interaction with OX2 (CD200).
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OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.
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11099417
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Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.
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In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family (six mutations in ABCG8 and one in ABCG5) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.
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11099418
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From marrow to brain: expression of neuronal phenotypes in adult mice.
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After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.
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11099419
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Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.
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Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.
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11099420
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Coding the location of the arm by sight.
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Area 5 in the parietal lobe of the primate brain is thought to be involved in monitoring the posture and movement of the body. In this study, neurons in monkey area 5 were found to encode the position of the monkey's arm while it was covered from view. The same neurons also responded to the position of a visible, realistic false arm. The neurons were not sensitive to the sight of unrealistic substitutes for the arm and were able to distinguish a right from a left arm. These neurons appear bine visual and somatosensory signals in order to monitor the configuration of the limbs. They could form the basis of plex body schema that we constantly use to adjust posture and guide movement.
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11099421
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Neurons in monkey prefrontal cortex that track past or predict future performance.
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Although frontal cortex is thought to be important in controlling behavior across long periods of time, most studies of this area concentrate on neuronal responses instantaneously relevant to the current task. In order to investigate the relationship of frontal activity to behavior over longer time periods, we trained rhesus monkeys on a difficult oculomotor task. Their performance fluctuated during the day, and the activity of prefrontal neurons, even measured while the monkeys waited for the targets to appear at the beginning of each set of trials, correlated with performance in a probabilistic rather than a determinist manner: neurons reflected past or predicted future performance, much more than they reflected current performance. We suggest that this activity is related to processes such as arousal or motivation that set the tone for behavior rather than controlling it on a millisecond basis, and could result from ascending pathways that utilize slow, second-messenger synaptic processes.
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11099422
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Rapid induction of mild cerebral hypothermia by cold aortic flush achieves normal recovery in a dog outcome model with 20-minute exsanguination cardiac arrest.
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Resuscitation attempts in trauma victims who suffer cardiac arrest (CA) from exsanguination almost always fail. The authors hypothesized that an aortic arch flush with cold normal saline solution (NSS) at the start of exsanguination CA can preserve cerebral viability during 20-minute no-flow.
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11099423
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Histopathologic effects of cutaneous tape stripping in pigs.
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The stratum corneum (SC) is the major barrier to topical absorption of medications. Skin tape stripping (TS) removes the stratum corneum, allowing more rapid absorption of drugs such as local anesthetics. Prior to evaluating TS in human clinical trials, this study was performed to evaluate its immediate and delayed histopathologic effects in swine.
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11099426
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A comparison of succinylcholine and rocuronium for rapid-sequence intubation of emergency department patients.
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pare rocuronium and succinylcholine for rapid-sequence intubation (RSI) in the emergency department (ED).
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11099425
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Critical care in the emergency department: A physiologic assessment and outcome evaluation.
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The changing landscape of health care in this country has seen an increase in the delivery of care to critically ill patients in the emergency department (ED). However, methodologies to assess care and es similar to those used in the intensive care unit (ICU) are currently lacking in this setting. This study examined the impact of ED intervention on morbidity and mortality using the Acute Physiology and Chronic Health Evaluation (APACHE II), the Simplified Acute Physiology Score (SAPS II), and the Multiple Organ Dysfunction Score (MODS).
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11099427
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Comparison of intravenous midazolam with pentobarbital for sedation for head computed tomography imaging.
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pare the efficacy of intravenous (IV) midazolam with that of IV pentobarbital when used for sedation for puted tomography (CT) imaging in emergency department (ED) pediatric patients.
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11099428
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Sports-related injuries in children.
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To describe the demographics and types of sports-related injuries (SRIs) in children.
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11099429
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Acceptability of emergency department-based screening and brief intervention for alcohol problems.
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To adapt screening and brief intervention for alcohol problems (SBI) to a high-volume emergency department (ED) setting and evaluate its acceptability to patients.
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11099430
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Breast cancer knowledge and preventive behaviors: An urban emergency department-based survey.
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To assess general knowledge and preventive behaviors regarding breast cancer among women who present to an urban emergency department.
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11099432
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Resident perception of academic skills training and impact on academic career choice.
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1) To evaluate residents' perceptions of the quality of training in basic academic skills and the availability and quality of research resources during residency; 2) to evaluate the association between these attitudes and choice of an academic career; and 3) to assess residents' attitudes toward the importance of postgraduate fellowship training for success in an academic career.
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11099431
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Emergency medicine residency applicant educational debt: relationship with attitude toward training and moonlighting.
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Heated debate persists regarding the role of resident moonlighting in emergency medicine (EM). The attitudes of EM residency applicants have not been assessed. The objectives of this study were to assess: 1) the level of educational debt among EM residency applicants, 2) their perception of increased risk potential to patients from unsupervised EM resident practice, and 3) their opposition to laws restricting moonlighting. The authors then report the relationship between the degree of indebtedness and these stated positions.
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11099433
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Emergency department use by adult medicaid patients after implementation of managed care.
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To determine whether the advent of a mandatory Medicaid managed care (MMC) plan had any effect on emergency department (ED) utilization by adult Medicaid patients at an urban teaching hospital.
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11099436
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The rationale for developing public health surveillance systems based on emergency department data.
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Emergency departments (EDs) are well positioned to provide national data on several aspects of public health. The large volume of patients seen annually, improving medical record technology, and emergency uniform data sets make the development of public health surveillance systems a realistic opportunity for emergency medicine. Such data could identify public health concerns and suggest interventions to improve the health of the nation. This article describes current concepts and status of ED surveillance systems, their advantages and disadvantages, the rationale for their existence, and mendations to allow their continued consideration and development.
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11099437
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Teaching patient empathy: the ED visit program.
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Residency programs only are not challenged with petent emergency clinicians, but should strive to develop caring, empathetic, munity-minded physicians. An exercise was designed to help residents experience emergency department (ED) visits from the patient's perspective.
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11099438
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Follow-up program for emergency department patients with gonorrhea or chlamydia.
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To study the performance of a centralized regional follow-up program organized by a municipal department of health (DH) for female patients presenting to the emergency department (ED) with Neisseria gonorrhoeae and/or Chlamydia trachomatis, who are not diagnosed or treated at the time of presentation.
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11099442
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The neurological basis of developmental dyslexia: an overview and working hypothesis.
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Five to ten per cent of school-age children fail to learn to read in spite of normal intelligence, adequate environment and educational opportunities. Thus defined, developmental dyslexia (hereafter referred to as dyslexia) is usually considered of constitutional origin, but its actual mechanisms are still mysterious and currently remain the subject of intense research endeavour in various neuroscientific areas and along several theoretical frameworks. This article reviews evidence accumulated to date that favours a dysfunction of neural systems known to participate in the normal acquisition and achievement of reading and other related cognitive functions. Historically, the first arguments for a neurological basis of dyslexia came from neuropathological studies of brains from dyslexic individuals. These early studies, although open to criticism, for the first time drew attention towards a possible abnormality in specific stages of prenatal maturation of the cerebral cortex and suggested a role of atypical development of brain asymmetries. This has prompted a large amount of subsequent work using in vivo imaging methods in the same vein. These latter studies, however, have yielded less clear-cut results than expected, but have globally confirmed some subtle differences in brain anatomy whose exact significance is still under investigation. Neuropsychological studies have provided considerable evidence that the main mechanism leading to these children's learning difficulties is phonological in nature, namely a basic defect in segmenting and manipulating the phoneme constituents of speech. A case has also been made for impairment in brain visual mechanisms of reading as a possible contributing factor. This approach has led to an important conceptual advance with the suggestion of a specific involvement of one subsystem of vision pathways (the so-called magnosystem hypothesis). Both phonological and visual hypotheses have received valuable contribution from modern functional imaging techniques. Results of recent PET and functional MRI studies are reported here in some detail. Finally, one attractive interpretation of available evidence points to dyslexia as a multi-system deficit possibly based on a fundamental incapacity of the brain in performing tasks requiring processing of brief stimuli in rapid temporal succession. It is proposed that this so-called 'temporal processing impairment' theory of dyslexia could also account for at least some of the perceptual, motor and cognitive symptoms very often associated with the learning disorder, a coincidence that has remained unexplained so far.
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11099443
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Identification of a pathway for intelligible speech in the left temporal lobe.
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It has been proposed that the identification of sounds, including species-specific vocalizations, by primates depends on anterior projections from the primary auditory cortex, an auditory pathway analogous to the ventral route proposed for the visual identification of objects. We have identified a similar route in the human for understanding intelligible speech. Using PET imaging to identify separable neural subsystems within the human auditory cortex, we used a variety of speech and speech-like stimuli with equivalent plexity but varying intelligibility. We have demonstrated that the left superior temporal sulcus responds to the presence of phonetic information, but its anterior part only responds if the stimulus is also intelligible. This novel observation demonstrates a left anterior temporal pathway for prehension.
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11099444
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Acute disseminated encephalomyelitis, multiphasic disseminated encephalomyelitis and multiple sclerosis in children.
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Forty-eight children with disseminated demyelination of the CNS, 28 with acute disseminated encephalomyelitis (ADEM), seven with multiphasic disseminated encephalomyelitis (MDEM) and 13 with multiple sclerosis were studied for a mean follow-up period of 5.64 years. The presentation findings of the ADEM/MDEM group pared with those of the multiple sclerosis group. The following findings were monly seen in ADEM/MDEM pared with the multiple sclerosis presentations: predemyelinating infectious disease (74 versus 38%, P: < 0.05); polysymptomatic presentation (91 versus 38%, P: < 0.002); pyramidal signs (71 versus 23%, P: < 0.01); encephalopathy (69 versus 15%, P: < 0.002); and bilateral optic neuritis (23 versus 8%, not significant). Seizures occurred only in the ADEM/MDEM group (17 versus 0%, not significant). Unilateral optic neuritis occurred only in the multiple sclerosis patients (23 versus 0%, P: < 0.01). There were no differences in the frequencies of transverse myelitis, brainstem involvement, cerebellar signs and sensory disturbance between the two groups. ADEM/MDEM patients were more likely to have blood leucocytosis (64 versus 22%, P: < 0.05), CSF lymphocytosis (64 versus 42%, not significant) and CSF protein elevation (60 versus 33%, not significant). Patients presenting with multiple sclerosis were more likely to have intrathecal synthesis of oligoclonal bands on presentation (64 versus 29%, not significant). MRI showed that subcortical white matter lesions were almost universal in both groups, though periventricular lesions were mon in multiple sclerosis (92 versus 44%, P: < 0.01). By contrast, in ADEM/MDEM there was absolute and relative periventricular sparing in 56 and 78% of patients, respectively. Follow-up MRI plete or partial lesion resolution in 90% and no new lesions in the ADEM/MDEM group. All of the multiple sclerosis patients had new lesions on repeat MRI (five during relapse and six during asymptomatic convalescent phases). The e in the ADEM patients was mixed; 57% of patients made plete recovery. The mean follow-up for the 35 ADEM/MDEM patients was 5.78 years (range 1.0-15.4 years). Eight of the 13 multiple sclerosis patients relapsed within the first year; 11 had a relapsing-remitting course, one a primary progressive course and one a secondary progressive course. These differences in the presentation of pared with multiple sclerosis may help in the prognosis given to families regarding the possibility of later development of multiple sclerosis.
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11099445
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Brain iron deposition in Parkinson's disease imaged using the PRIME magnetic resonance sequence.
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Iron content of the basal ganglia was investigated in 25 patients with idiopathic Parkinson's disease and 14 matched healthy control subjects using a partially refocused interleaved multiple echo sequence on a 1.5 Tesla MRI system. R(2)* (1/T(2)*) and R(2)' (1/T(2)') relaxation rates were higher in the substantia nigra of patients with Parkinson's disease, which indicates that iron content is elevated in this region. R(2)' was lower in the putamen, indicating reduced iron levels; reduction in this region was positively correlated with disease duration. Iron-related oxidative stress may contribute to the neurodegeneration of Parkinson's disease, which may lead to alterations in the iron levels of the striatum. We describe a simple, non-invasive technique for measuring iron content.
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11099446
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The reorganization of sensorimotor function in children after hemispherectomy. A functional MRI and somatosensory evoked potential study.
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Children who have suffered extensive unilateral brain injury early in life may show a remarkable degree of residual sensorimotor function. It is generally believed that this reflects the high capacity of the immature brain for cerebral reorganization. In this study, we investigated 17 patients who had undergone hemispherectomy for relief from seizures; eight of the patients had congenital brain damage and nine had sustained their initial insult at the age of 1 year or older. Sensorimotor functions of the hand were investigated using functional MRI (fMRI) during a passive movement task, somatosensory evoked potentials (SEPs) arising from electrical and vibration stimulation, and behavioural tests including grip strength, double simultaneous stimulation and joint position sense. On fMRI, two of the eight patients studied with this technique (one with congenital damage and one with damage acquired at the age of 3 years) showed activation in the sensorimotor cortex of the remaining hemisphere with passive movement of the hemiplegic hand. The location of the ipsilateral brain activation was similar to that found on movement of the normal contralateral hand, although the latter was greater in spatial extent. In one of these patients, a greater role was demonstrated for the ipsilateral secondary sensorimotor area (compared with the ipsilateral primary sensorimotor area) for movement of the hemiplegic hand than for movement of the normal hand. Median nerve stimulation of the hemiplegic hand showed reproducible early-latency ipsilateral ponents in the remaining sensorimotor cortex in 10 of the 17 patients (five with congenital and five with acquired disease). Five of the patients who demonstrated ipsilateral electrical SEPs also showed ipsilateral vibration SEPs (two with congenital and three with acquired disease). The behavioural tests revealed residual sensorimotor function in 14 of the patients; however, not all of the patients who exhibited ipsilateral SEP or fMRI responses had residual sensorimotor function in the hemiplegic hand. Ipsilateral sensorimotor responses were demonstrated both in patients with congenital disease and those with acquired disease, suggesting that factors additional to aetiology and age at injury may influence the degree of residual sensorimotor function and cerebral reorganization.
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11099447
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Failed surgery for epilepsy. A study of persistence and recurrence of seizures following temporal resection.
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From a series of 282 consecutive temporal resections for medically intractable epilepsy associated with mesial temporal sclerosis (MTS), dysembryoplastic neuroepithelial tumour (DNT) or non-specific pathology (NSP), 51 patients had persistent or recurrent seizures occurring at least monthly. Of these patients, 44 underwent detailed assessment of their postoperative seizures, which included clinical evaluation, interictal and ictal EEG and high-resolution MRI. Of the 20 patients with MTS in the original pathology, 14 (70%) had postoperative seizures arising in the hemisphere of the resection, the majority (12 patients) in the temporal region. Although MRI demonstrated residual hippocampus in five of these 12 patients, only one patient was considered to have seizures arising there, whilst the remainder had electroclinical evidence of seizure onset in the neocortex. In contrast, five of the MTS relapses (25%) had seizure onset exclusively in the contralateral temporal region. Among the 14 patients with non-specific pathology, relapse was also predominantly from the ipsilateral hemisphere (64%), but more relapsed from extratemporal pared with the MTS cases, including two with NSP who had occipital structural abnormalities. Although 70% of the 10 patients with DNT had postoperative partial seizures arising in the ipsilateral hemisphere, many (60%) had evidence of a more diffuse disorder with additional generalized seizures, cognitive and behavioural disturbance and multifocal and generalized EEG abnormalities. Nine patients (20%) had immediate postoperative seizure-free periods of at least 1 year, and seven of these had MTS in the operative specimen. Of these seven patients, four had ipsilateral temporal seizures and three had contralateral temporal seizures. Overall, few missed lesions were discovered on postoperative MRI and reoperations were performed or considered possible in a minority of cases. Despite well-defined preoperative electroclinical syndromes of temporal lobe epilepsy, many patients relapsed unexpectedly, either immediately or remotely from the time of surgery. Maturing epileptogenicity in a surgical scar was not, however, considered to be a significant primary mechanism in patients who relapsed after a seizure-free interval.
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11099448
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Pathology of early-onset Alzheimer's disease cases bearing the Thr113-114ins presenilin-1 mutation.
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Most cases of familial presenile Alzheimer's disease are caused by mutations in the presenilin-1 (PSEN-1) gene, most of these mutations being missense mutations. A mutation in the splice donor site of intron 4 of PSEN-1 has been described recently which results in aberrant splicing of PSEN-1 mRNA, causing insertion of an additional amino acid, Thr113-114ins, into the protein. We studied the neuropathology of four cases bearing this mutation in an attempt to clarify the pathology of this hereditary form of Alzheimer's disease and to determine whether it differs from other familial forms of the disease. The disease presented as a progressive cognitive decline, myoclonus and seizures developing later in the disease, a mon to PSEN-1-linked Alzheimer's disease. The course of the disease was relatively rapid, death occurring approximately 6 years after onset. Pathology in the intron 4 cases demonstrated a severe Alzheimer's disease pathology with abundant deposition of ss-amyloid (Ass) 1-42 senile plaques and the formation of neurofibrillary tangles. Amyloid angiopathy was present in these cases and was readily demonstrated by Ass 1-40 staining, particularly in the cerebellum. Cases with the intron 4 mutation appear clinically and pathologically similar to other cases of early-onset Alzheimer's disease bearing PSEN-1 mutations.
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11099449
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Multimodal EEG analysis in man suggests impairment-specific changes in movement-related electric brain activity after stroke.
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Movement-related slow cortical potentials and event-related desynchronization of alpha (alpha-ERD) and beta (beta-ERD) activity after self-paced voluntary triangular finger movements were studied in 13 ischaemic supratentorial stroke patients and 10 age-matched control subjects during movement preparation and actual performance. The stroke patients suffered from central arm paresis (n = 8), somatosensory deficits (n = 3) or ideomotor apraxia (n = 2). The multimodal EEG analysis suggested impairment-specific changes in the movement-related electrical activity of the brain. The readiness potential of paretic subjects was centred more anteriorly and laterally; during movement, they showed increased beta-ERD at left lateral frontal recording sites. Patients with somatosensory deficits showed reduced alpha-ERD and beta-ERD during both movement preparation and actual performance. Patients with ideomotor apraxia showed more lateralized frontal movement-related slow cortical potentials during both movement preparation and performance, and reduced left parietal beta-ERD during movement preparation. We conclude that (i) disturbed motor efference is associated with an increased need for excitatory drive of pyramidal cells in motor and premotor areas or an attempt to drive movements through projections from these areas to brainstem motor systems during movement preparation; (ii) an undisturbed somatosensory afference might contribute to the release of relevant cortical areas from their 'idling' state when movements are prepared and performed; and (iii) apraxic patients have a relative lack of activity of the mesial frontal motor system and the left parietal cortex, which is believed to be part of a network subserving ideomotor praxis.
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11099450
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Evidence of functional somatotopy in GPi from results of pallidotomy.
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The objective of this study was to explore the functional anatomy of the globus pallidus internus (GPi) by studying the effects of unilateral pallidotomy on parkinsonian 'off' signs and levodopa-induced dyskinesias (LID). We found significant positive correlations between the preoperative levodopa responsiveness of motor signs and the levodopa responsiveness of scores in timed tests (Core Assessment Program for Intracerebral Transplantations) in the contralateral limbs and the improvement in these scores after surgery, whereas there was no correlation with the improvement in LID. We also found a highly significant correlation (P: < 0.0001, r = 0.8) between the volume of the ventral lesion in the GPi and the improvement in LID in the contralateral limbs, whereas there was no correlation between the ventral volume and the improvement in parkinsonian 'off' signs. The volumes of the total lesion cylinder and the dorsal lesion did not correlate with the e of either dyskinesias or parkinsonian 'off' signs. The differential predictive value of levodopa responsiveness for the e of parkinsonian 'off' signs and LID and the different correlations of ventral lesion volume with dyskinesias and parkinsonian 'off' signs indicate that different anatomical or pathophysiological substrates may be responsible for the generation of parkinsonian 'off' signs and dyskinesias. Whereas cells in a wider area of the GPi may be implicated in parkinsonism, the ventral GPi seems to be crucial for the manifestation of LID. We suggest that our observations are additional proof of the functional somatotopy of the systems within the GPi that mediate parkinsonism and dyskinesias, especially along the dorsoventral trajectory used in pallidotomy. The e of pallidotomy in which the lesion involves the ventral and dorsal GPi could be the net effect of alteration in the activity of pathways which mediate different symptoms, and hence could be variable.
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11099451
|
Anterior and posterior callosal contributions to simultaneous bimanual movements of the hands and fingers.
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In order to study the role of the corpus callosum in two-handed coordination we tested callosotomy subjects while they attempted to initiate simultaneous discrete movements with both hands. We observed four split-brain patients, including one pre- and post-operatively, as well as normal and epileptic control subjects. Split-brain patients made button presses that were less synchronous than either normal or epileptic controls. Although split-brain patients' average performance did not always differ from control subjects, callosotomy resulted in a 3-fold increase in the variability with which 'simultaneous' movements were initiated. The one subject tested pre- and post-callosotomy showed distinct changes in movement initiation synchrony after both the anterior and the posterior stages of the surgery. These changes suggest that anterior and posterior callosal fibres may make unique contributions to bimanual synchronization, depending on whether responses are self-initiated or in reaction to a visual stimulus. This study demonstrates that munication across anterior and posterior fibres of the corpus callosum strongly influences the temporal precision of bimanual coordination. Specifically, callosal transmission affects the degree of bilateral synchrony with which simple simultaneous hand and finger movements are initiated.
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11099452
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Handedness and hemispheric language dominance in healthy humans.
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In most people the left hemisphere of the brain is dominant for language. Because of the increased incidence of atypical right-hemispheric language in left-handed neurological patients, a systematic association between handedness and dominance has long been suspected. To clarify the relationship between handedness and language dominance in healthy subjects, we measured lateralization directly by functional transcranial Doppler sonography in 326 healthy individuals using a word-generation task. The incidence of right-hemisphere language dominance was found to increase linearly with the degree of left-handedness, from 4% in strong right-handers (handedness = 100) to 15% in ambidextrous individuals and 27% in strong left-handers (handedness = -100). The relationship could be approximated by the formula: f1.gif" BORDER="0">. These results clearly demonstrate that the relationship between handedness and language dominance is not an artefact of cerebral pathology but a natural phenomenon.
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11099454
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Neural consequences of acting in near versus far space: a physiological basis for clinical dissociations.
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We used PET to determine which brain regions are implicated when normal volunteers bisect horizontal lines and point to dots in near (peripersonal) or far (extrapersonal) space. Studies of line bisection in patients with right hemisphere lesions have shown that bisection performance can be severely impaired in either near or far space while remaining within normal limits in the other spatial domain. Likewise, clinical dissociations between pointing to objects in near and far space have been reported. The normal functional anatomy of these dissociations has not been demonstrated convincingly. Regional cerebral blood flow measurements using PET were carried out in 12 healthy right-handed male volunteers who bisected lines or pointed to dots in near or far space, using a laser pen. Subjects performing either task in near space showed neural activity in the left dorsal occipital cortex, left intraparietal cortex, left ventral premotor cortex and left thalamus. In far space, subjects performing either task showed activation of the ventral occipital cortex bilaterally and the right medial temporal cortex. These data provide physiological support for the clinically observed dissociations demonstrating that attending to and acting in near space differentially employs dorsal visuomotor processing areas, whereas attending to and acting in far space differentially draws on ventral visuoperceptual processing areas, even when the ponents of the tasks are identical when performed in the two spaces.
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11099453
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Distribution of ataxin-7 in normal human brain and retina.
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Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in the protein ataxin-7. We developed antibodies directed against two different parts of the ataxin-7 protein and studied its distribution in brain and peripheral tissue from healthy subjects. Normal ataxin-7 was widely expressed in brain, retina and peripheral tissues, including striated muscle, testis and thyroid gland. In the brain, expression of ataxin-7 was not limited to areas in which neurones degenerate, and the level of expression was not related to the severity of neuronal loss. Immunoreactivity was low in some vulnerable populations of neurones, such as Purkinje cells. In neurones, ataxin-7 was found in the cell bodies and in processes. Nuclear labelling was also observed in some neurones, but was not related to the distribution of intranuclear inclusions observed in an SCA7 patient. In this patient, the proportion of neurones with nuclear labelling was higher, on average, in regions with neuronal loss. Double immunolabelling coupled with confocal microscopy showed that ataxin-7 colocalized with BiP, a marker of the endoplasmic reticulum, but not with markers of mitochondria or the trans-Golgi network.
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11099455
|
Effects of membrane polarization and ischaemia on the excitability properties of human motor axons.
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Multiple nerve excitability measurements have been proposed for clinical testing of nerve function, since excitability measures can provide evidence of altered axonal membrane properties and plementary to conventional nerve conduction studies. An important determinant of excitability is membrane potential, and this study was undertaken to determine the changes in a range of excitability properties associated with alterations in membrane potential. Membrane potential was varied directly using DC polarizing currents and indirectly by ischaemia. The median nerve was stimulated at the wrist and the pound muscle action potentials recorded from abductor pollicis brevis. Stimulus-response behaviour, strength-duration time constant (tau(SD)), threshold electrotonus to 100-ms polarizing currents, a current-threshold relationship and the recovery of excitability following supramaximal activation were each followed in four normal subjects during the two manoeuvres, using a recently described protocol. Membrane depolarization and ischaemia produced an increase in axonal excitability, an increase in the slope of the current-threshold relationship, a 'fanning in' of responses during threshold electrotonus, a decrease in super-excitability, and increases in both tau(SD) and the refractory period. Changes in the opposite direction occurred with membrane hyperpolarization and during the post-ischaemic period. One excitability parameter differentiated between the direct and indirect changes in membrane potential: late subexcitability was sensitive to polarizing currents but relatively insensitive to ischaemia, probably because pensatory changes in extracellular potassium ions. These results should enable multiple excitability measurements to be used as a tool to identify changes in axonal membrane potential in neuropathy.
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11099456
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Brain responses to nouns, verbs and class-ambiguous words in context.
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Recent neuropsychological and imaging data have implicated different brain networks in the processing of different word classes, nouns being linked primarily to posterior, visual object-processing regions and verbs to frontal, motor-processing areas. However, as most of these studies have examined words in isolation, the consequences of such anatomically based representational differences, if any, for the processing of these items in sentences remains unclear. Additionally, in some languages many words (e.g. 'drink') are class-ambiguous, i.e. they can play either role depending on context, and it is not yet known how the brain stores and uses information associated with such lexical items in context. We examined these issues by recording event-related potentials (ERPs) in response to unambiguous nouns (e.g. 'beer'), unambiguous verbs (e. g. 'eat'), class-ambiguous words and pseudowords used as nouns or verbs within two types of minimally contrastive sentence contexts: noun-predicting (e.g. 'John wanted THE [target] but.') and verb-predicting ('John wanted TO [target] but.'). Our results indicate that the nature of neural processing for nouns and verbs is a function of both the type of stimulus and the role it is playing. Even when the pletely specifies their role, word class-ambiguous items differ from unambiguous ones over frontal regions by approximately 150 ms. Moreover, whereas pseudowords elicit larger N400s when used as verbs than when used as nouns, unambiguous nouns and ambiguous words used as nouns elicit more frontocentral negativity than unambiguous verbs and ambiguous words used as verbs, respectively. Additionally, unambiguous verbs elicit a left-lateralized, anterior positivity (approximately 200 ms) not observed for any other stimulus type, though only when these items are used appropriately as verbs (i.e. in verb-predicting contexts). In summary, the pattern of neural activity observed in response to lexical items depends on their general probability of being a verb or a noun and on the particular role they are playing in any given sentence. This implicates more than a simple two-way distinction of the brain networks involved in their storage and processing. Experience, as well as context during on-line language processing, clearly shapes the neural representations of nouns and verbs, such that there is no single neural marker of word class. Our results further suggest that the presence and nature of the word class-based dissociations observed after brain damage are similarly likely to be a function of both the type of stimulus and the context in which it occurs, and thus must be assessed accordingly.
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11099458
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In vivo imaging of the vesicular acetylcholine transporter and the vesicular monoamine transporter.
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Validation of the vesicular acetylcholine transporter (VAChT) and the neuronal vesicular monoamine transporter (VMAT2) as important molecular targets in the cholinergic and dopamine neurons, respectively, has sparked interest in the development of radiotracers for studying these markers in vitro and in vivo. Currently, a number of selective high-affinity radiotracers are available for studying these targets in vivo with positron emission tomography (PET) or single photon puted tomography (SPECT). PET studies of VMAT2 in neuropathology reveal changes in the density of this marker that can be verified independently. Similarly, in vivo studies with VAChT ligands suggest that the latter are potentially useful in detecting cholinergic lesions in vivo; however, additional development is required to fully realize the potential of these radioligands.
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11099459
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Neurogenetics of vesicular transporters in C. elegans.
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The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our understanding of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of mutations causing a suggestive phenotype (1, 2). The phenotypes of eat-4 mutants and the cell biology of the EAT-4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure associated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be assayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter-specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mutants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport.
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11099460
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Transport mechanisms in acetylcholine and monoamine storage.
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Sequence-related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure-function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single-site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence-defined superfamily, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage.
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11099461
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Chemical neuroanatomy of the vesicular amine transporters.
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Acetylcholine, catecholamines, serotonin, and histamine are classical neurotransmitters. These small molecules also play important roles in the endocrine and immune/inflammatory systems. Serotonin secreted from enterochromaffin cells of the gut epithelium regulates gut motility; histamine secreted from basophils and mast cells is a major regulator of vascular permeability and skin inflammatory responses; epinephrine is a classical hormone released from the adrenal medulla. Each of these molecules is released from neural, endocrine, or immune/inflammatory cells only in response to specific physiological stimuli. Regulated secretion is possible because amines are stored in secretory vesicles and released via a stimulus-dependent exocytotic event. Amine storage-at concentrations orders of magnitude higher than in the cytoplasm-is plished in turn by specific secretory vesicle transporters that recognize the amines and move them from the cytosol into the vesicle. Immunohistochemical visualization of specific vesicular amine transporters (VATs) in neuronal, endocrine, and inflammatory cells provides important new information about how amine-handling cell phenotypes arise during development and how vesicular transport is regulated during homeostatic response events. Comparison of the chemical neuroanatomy of VATs and amine biosynthetic enzymes has also revealed cell groups that express vesicular transporters but not enzymes for monoamine synthesis, and vice versa: their function and regulation is a new topic of investigation in mammalian neurobiology. The chemical neuroanatomy of the vesicular amine transporters is reviewed here. These and similar data emerging from the study of the localization of the recently characterized vesicular inhibitory and excitatory amino acid transporters will contribute to understanding chemically coded synaptic circuitry in the brain, and amine-handling neuroendocrine and immune/inflammatory cell regulation.
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11099462
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Molecular analysis of vesicular amine transporter function and targeting to secretory organelles.
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Vesicular transporters are responsible for the loading of neurotransmitters into specialized secretory organelles in neurons and neuroendocrine cells to make them available for regulated neurosecretion. The exocytotic release of neurotransmitter therefore depends on the functional activity of the vesicular transporters and their efficient sorting to these secretory organelles. Molecular analysis of vesicular transport proteins has revealed important information regarding structural domains responsible for their functional properties, including substrate specificity and trafficking to various classes of secretory vesicles. These studies have established the existence of an important functional relationship between transporter activity and presynaptic quantal neurosecretion.
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11099463
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The VMAT2 gene in mice and humans: amphetamine responses, locomotion, cardiac arrhythmias, aging, and vulnerability to dopaminergic toxins.
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partmentalization in monoaminergic neurons uses serial action of the plasma membrane and vesicular monoamine (VAMT2) transporters. We can now define the sequences of the genes encoding these transporters in mice and humans, examine influences of deletions of this gene and alteration in its expression levels in transgenic mice, and identify sequence polymorphisms in the human VMAT2 gene. Examination of VMAT2 variants can provide potential insights into roles for allelic variants at these loci in variant drug responses and in diseases linked to monoaminergic systems, including substance abuse and Parkinson's disease.
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11099464
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The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release.
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We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca(2+), membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca(2+) change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X(7), as confirmed by ability of dendritic cells to e permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X(7) receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca(2+) increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X(7) antagonist KN-62 inhibited both permeabilization and Ca(2+) changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y(1), P2Y(2), P2Y(5), P2Y(11) and P2X(1), P2X(4), P2X(7). Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1 beta and TNF-alpha. Purinoceptors may provide a new avenue to modulation of dendritic cells function.
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11099465
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Halofuginone: a potent inhibitor of critical steps in angiogenesis progression.
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We have previously demonstrated that halofuginone, a low molecular weight quinazolinone alkaloid, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also effectively suppresses tumor progression and metastasis in mice. These results together with the well-documented role of extracellular matrix ponents and matrix degrading enzymes in formation of new blood vessels led us to investigate the effect of halofuginone on the angiogenic process. In a variety of experimental system, representing sequential events in the angiogenic cascade, halofuginone treatment resulted in profound inhibitory effect. Among these are the abrogation of endothelial cell MMP-2 expression and basement membrane invasion, capillary tube formation, and vascular sprouting, as well as deposition of subendothelial ECM. The most conclusive anti-angiogenic activity of halofuginone was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor (bFGF) -induced neovascularization in response to systemic administration of halofuginone, either i.p. or in the diet. The ability of halofuginone to interfere with key events in neovascularization, together with its oral bioavailability and safe use as an anti-parasitic agent, make it a promising drug for further evaluation in the treatment of a wide range of diseases associated with pathological angiogenesis.
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11099466
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High-efficiency transient transfection of endothelial cells for functional analysis.
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The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5-fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl-terminal domain of the beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.
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11099467
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Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I--effects of proliferative senescence.
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Oxidized and cross-linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the panying paper, ref 1 ) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no itant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was panied by dramatic increases in the accumulation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross-linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross-linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever-increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross-linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross-linked proteins in aging, is further explored during postmitotic senescence in the panying paper (1).
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11099468
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Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II--aging of nondividing cells.
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Oxidized/cross-linked intracellular protein materials, known as ceroid pigment, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is unclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxidized soluble cytoplasmic and nuclear proteins and, we propose, for the prevention of such damage accumulations. The present investigation was undertaken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cells. Since panion paper shows that both proteasome activity and the overall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to use the same human BJ fibroblasts, this time at confluency, at different PD levels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that the activity of the cytosolic proteasome declines dramatically during senescence of nondividing BJ fibroblasts. The peptidyl-glutamyl-hydrolyzing activity was particularly affected. This decline in proteasome activity was panied by a decrease in the overall turnover of short-lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was found. The decline in the activity of the proteasome was also panied by an increased accumulation of oxidized proteins, especially of oxidized and cross-linked material. Unlike the loss of lysosomal function seen in our panying studies of proliferative senescence (1), however, the present study of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very 'oldest' postmitotic cells. parative studies of proliferating (1) and nonproliferating (this paper) human BJ fibroblasts reveal a good correlation between the accumulation of oxidized/cross-linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence, which may predict a causal relationship during actual cellular aging.
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11099469
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S-adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver.
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Methionine metabolism starts with the formation of S-adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver-specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose-dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3-deazaadenosine and L-ethionine, but not D-ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte.
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11099470
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Mechanism of the antimicrobial drug trimethoprim revisited.
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We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [(3)H]-p-aminobenzoic acid before treatment with trimethoprim. Folates in untreated E. coli were present as tetrahydrofolate coenzymes. In trimethoprim-treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed plete conversion of all forms of folate to cleaved catabolites (pteridines and para-aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para-aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one-carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.
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11099471
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Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.
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It has been postulated that an inflammatory response after cutaneous wounding is a prerequisite for healing, and inflammatory cytokines, such as interleukin-6 (IL-6), might be intimately involved in this process. IL-6-deficient transgenic mice (IL-6 KO) displayed significantly delayed cutaneous wound pared with wild-type control animals, requiring up to threefold longer to heal. This was characterized by minimal epithelial bridge formation, decreased inflammation, and granulation tissue formation. Using electrophoretic mobility shift assays of wound tissue from IL-6 KO mice, decreased AP-1 transcription factor activation was pared with wild-type mice 16 h after wounding. In situ hybridization of wound tissue from wild-type mice revealed IL-6 mRNA expression primarily in the epidermis at the leading edge of the wound. Delayed wound healing in IL-6 KO mice was reversed with a single dose of binant murine IL-6 or intradermal injection of an expression plasmid containing the full-length murine IL-6 cDNA. Treatment with rmIL-6 also reconstituted wound healing in dexamethasone-treated immunosuppressed mice. The results of this study may indicate a potential use for IL-6 therapeutically where cutaneous wound healing is impaired.
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