bluuebunny/crossref_metadata_2025 huggingface dataset:
Pseudoallergic reactions in chronic urticaria are associated with altered gastroduodenal permeability
Authors: Buhner S. , Reese I. , Kuehl F. , Lochs H. , Zuberbier T.
Published: September 2004

Background:  In a subgroup of patients with chronic urticaria (CU) the disease is caused by pseudoallergic reactions to food. The aim of this study was to investigate whether disturbances of the gastrointestinal barrier function play a role in the pathomechanism of the disease.

Methods:  In 55 patients with CU gastrointestinal permeability was measured with an in vivo triple‐sugar‐test before and after 24 days of a diet low in pseudoallergens. Sucrose served as marker for gastroduodenal permeability, lactulose/mannitol ratio for intestinal permeability.

Results:  Basal gastroduodenal and intestinal permeability were significantly higher in patients with urticaria as compared to controls. In 29 of the 55 patients skin symptoms decreased or completely disappeared during the diet (responders). Compared to nonresponders (n = 26), responders had a significantly higher gastroduodenal permeability before treatment (0.36 ± 0.04 vs 0.15 ± 0.01% sucrose; P < 0.001), which decreased after the diet (0.17 ± 0.02; P < 0.001). The number of patients with Helicobacter pylori infections did not differ between the two groups.

Conclusions:  The results indicate that in a subgroup of patients with CU and pseudoallergy an impaired gastroduodenal barrier function may be of pathophysiological importance. The underlying mechanisms seem to be independent of H. pylori infection.
Sputum eosinophil counts and eosinophil cationic protein levels in cough‐variant asthma and in classic asthma, and their relationships to airway hypersensitivity or maximal airway response to methacholine
Authors: Yoo Y. , Koh Y. Y. , Kang H. , Yu J. , Nah K. M. , Kim C. K.
Published: September 2004

Background:  The aims of this study were to compare the degree of airway inflammation in cough‐variant asthma (CVA) with that in classic asthma (CA), and to examine the relationship between airway inflammation and airway hypersensitivity or maximal airway response to methacholine in both conditions.

Methods:  Sputum was induced in 41 CVA patients, in 41 methacholine PC20‐matched CA patients, and in 20 healthy children. The sputum samples were analyzed for total and differential cell counts, and for eosinophilic cationic protein (ECP). A high‐dose methacholine challenge test was performed in CVA and CA patients to determine PC20 and maximal airway response.

Results:  Sputum eosinophil percentages and ECP levels were significantly elevated in CVA and CA vs the control, but no significant differences were found between the two asthma groups. In the two asthma groups, neither sputum parameters correlated significantly with methacholine PC20. However, the absence of a maximal response plateau or its higher level, when present, was associated with increased eosinophil percentages and ECP levels in the CVA group.

Conclusions:  The degree of eosinophilic inflammation may not be causally related to differences in presented asthma manifestations. The identification of a maximal response plateau and the level of this plateau in patients with CVA may provide information pertinent to airway eosinophilic inflammation.
Haplotype analysis at the ETM2 locus in a Singaporean sample with familial essential tremor
Authors: Higgins JJ , Lombardi RQ , Tan EK , Jankovic J , Pucilowska J , Rooney JP
Published: September 2004

An ancestral haplotype on chromosome 2p24.1 described in an American sample with familial essential tremor (ET) was analyzed in a different ethnic sample from Singapore. Six polymorphic loci (etm1240, etm1231, etm1234, APOB, etm1241, and etm1242) in a 274‐kb interval within an ET gene candidate region (ETM2) were analyzed in Singaporean individuals with a family history of ET (n = 52) and compared to Singaporean controls older than age 65 (n = 49). The allele frequencies were significantly different between cases and controls for the loci etm1234 (p = 0.0001) and APOB (p = 0.0320). An extended haplotype formed by the loci etm1231, etm1234, and APOB occurred with a frequency of 31% in Singaporean cases and in 1.8% of elderly Singaporean controls (p = 0.0005). Haplotype studies in two different population samples suggest that a disease locus for ET lies near or within the 100‐kb interval between the loci etm1231 and APOB.
Genetic landmarks through philately – epilepsy and clinical genetic issues
Authors: Chodirker BN , Chudley AE
Published: September 2004

Epilepsy is a common neurologic disorder. Major advances in the understanding of the etiology and treatment have occurred. Although most cases of epilepsy do not follow a simple pattern of inheritance, recently single gene epilepsy disorders have been identified. We present some postage stamps to illustrate issues and advances in knowledge about epilepsy, as well as famous people with this disorder.
Hand‐assisted laparoscopic donor nephrectomy for pediatric kidney allograft recipients
Authors: Kim Dean Y. , Stegall Mark D. , Prieto Mikel , Chow George K. , Bohorquez Humberto E. , Covarrubias Marco A. , Heimbach Julie K. , Morgenstern Bruce Z. , Gloor James M. , Milliner Dawn S. , Weckwerth Jody A. , Weis Karla D. , Ishitani Michael B.
Published: September 2004

Abstract:  Laparoscopic donor nephrectomy (LDN) is the method of choice for procuring kidneys from living donors at many transplant centers. The aim of this study was to assess the feasibility as well as outcome of LDN in pediatric recipients. Twenty‐two pediatric patients, 18‐yr old or younger received kidneys procured by a hand‐assisted LDN technique. The mean operative time was no different (p = 0.9) and the mean length of stay was more than 1 day shorter in the LDN group (p = 0.0001) compared with the 13 pediatric patients who received kidneys by standard open nephrectomy. Body mass index (BMI), number of donor kidney vessels, or laterality of the kidney did not impact the donor operation or outcome. Actuarial 1‐yr patient survival was 100% and allograft survival was 95%, which are equivalent to registry data. There were no donor mortalities and there were five morbidities. None required hospitalization. There were no conversions from LDN to open nephrectomy. One kidney was lost because of overwhelming infection necessitating withdrawal of immunosuppression. In conclusion, hand‐assisted LDN is a safe method of procuring kidneys from potential donors with no significant negative outcomes to the pediatric recipients.
Cardioprotective action of fentanyl in a model of central sympathetic overactivity in rabbits: antiarrhythmic and anti‐ischemic effects
Authors: Lessa M. A. , Rodrigues E. , TibiriÇá E.
Published: September 2004

Background:  Sympathetic overactivity resulting from perioperative noxious stimuli elicits hyperdynamic cardiovascular responses that may lead to myocardial ischemia and/or ventricular arrhythmia, especially in patients presenting with coronary artery disease. In the present study we investigated the cardioprotective effects of clinically relevant doses of fentanyl in an experimental model of sympathetic overactivity associated with myocardial ischemia in anesthetized rabbits.

Methods:  Central sympathetic stimulation was achieved through intracerebroventricular (i.c.v.) injection of L‐glutamate (10 µmol), with simultaneous inhibition of nitric oxide (NO) synthesis through i.v. administration of N(omega)‐nitro‐l‐arginine methyl ester (L‐NAME; 40 mg kg−1).

Results:  L‐glutamate triggered ventricular arrhythmia and electrocardiographic alterations indicative of myocardial ischemia. The intravenous administration of fentanyl (5, 10 or 50 µg kg−1) reduced the incidence of ST‐segment shift (70, 20 and 10%, respectively, vs. 66.7% in controls) as well as of T‐wave inversion from 58.3% to 30, 20 and 10%, respectively. The total number of ventricular premature complexes per minute fell from 65.2 ± 16 in the control group to 6.8 ± 3, 3.5  ±  2 and 2.6 ± 1.5, respectively. The occurrence of ventricular tachycardia and bigeminy was completely abolished by fentanyl. Finally, the i.v. administration of fentanyl did not induce significant hemodynamic effects (except for dP/dtmax in the 50 µg kg−1‐dose).

Conclusion:  Fentanyl elicits significant cardioprotective effects in a model of arrhythmia resulting from the association of central sympathetic overactivity with myocardial ischemia in rabbits, independently from its systemic hemodynamic actions.
Gas embolism: an exceptional complication of radial arterial catheterization
Authors: Dube L. , Soltner C. , Daenen S. , Lemarieé J. , Asfar P. , Alquier P.
Published: September 2004

Background:  We report a rare complication of radial arterial catheterization in a 74‐year‐old man who had undergone retroperitoneal surgery for an infra‐renal aortic aneurysm. A right subclavian venous catheter and a right radial artery catheter were inserted for hemodynamic monitoring.

Results:  The patient suddenly went into a coma, with dyspnea and bradycardia, 1 day postsurgery, as a result of a cerebral gas embolism produced by the accidental entry of pressurized air into the artery via the arterial catheter. Cerebral Magnetic Resonance Imaging revealed multiple, diffuse brain lesions. Six months later the patient still had a left hemiparesis and a cognitive deficit. He walks with assistance.

Conclusion:  Misuse of an arterial catheter can lead to a severe gas embolism. The infusion system used to flush arterial catheters should be checked regularly to ensure it contains no gas.
Oleic acid lung injury: a morphometric analysis using computed tomography
Authors: Rylander C. , Högman M. , Perchiazzi G. , Magnusson A. , Hedenstierna G.
Published: September 2004

Background:  The oleic acid‐induced lung injury (OAI) model is considered to represent the early phase of acute respiratory distress syndrome (ARDS). Its inherent properties are important for the design and the interpretation of interventional studies. The aim of this study was to describe the evolution of morphometric lung changes during OAI using computed tomography (CT) analysis. Furthermore, the effect of a temporary change in positive end‐expiratory pressure (PEEP) was evaluated.

Methods:  Fifteen anaesthetized pigs were ventilated in volume‐controlled mode with a baseline PEEP of 5 cm  H2O. Helical CT scans were taken at baseline and 1 h after oleic acid injection. The PEEP was then either increased to 10 cm  H2O (n = 5), decreased to 0 cm  H2O (n = 5) or kept constant (n = 5) for 30 min. For the next 30 min, the baseline PEEP level was applied in all animals before the final CT scans 2 h after the induction of OAI. Dimensional and volumetric changes were determined from radiographical attenuation values.

Results:  There was a major decrease in gas volume and an increase in tissue volume within the first hour. A net increase in total lung volume, with a larger transverse area but no displacement of the diaphragm, was manifest after 2 h. A minor increase in volume of non‐aerated lung, located to the caudal region, was observed during the second hour. The tidal volume was redistributed to the middle and apical regions. The temporary change in PEEP did not influence the morphological progress of OAI.

Conclusion:  Decreased gas volume and increased tissue volume are the dominating morphometric characteristics of oleic acid lung injury, occurring mainly within the first hour. With these changes manifest, the course of injury is not affected by a limited period of moderately changed PEEP during the second hour. The net increase of total lung volume suggests a predominance of oedema formation over airway and alveolar collapse.
‘Protective premedication’: an option with gabapentin and related drugs?
Authors: Dahl J. B. , Mathiesen O. , Møiniche S.
Published: September 2004

Substantial progress has been made during the last decades in our understanding of acute pain mechanisms, and this knowledge has encouraged the search for novel treatments. Of particular interest has been the observation that tissue injury initiates a number of modulations of both the peripheral and the central pain pathways, which convert the system from a ‘physiological’ to a ‘pathological’ mode of processing afferent information. Gabapentin, which binds to the α2δ subunit of the voltage‐dependent calcium channel, is active in animal models of ‘pathological’ but not in models of ‘physiological’ pain. Consequently, attention has so far been focused on neuropathic pain as a target for the clinical use of gabapentin and analogues. Recently, several reports have indicated that gabapentin may have a place in the treatment of post‐operative pain. This article presents a brief summary of the potential mechanisms of post‐operative pain, and a systematic review of the available data of gabapentin and pregabalin for post‐operative analgesia. It is concluded that the results with gabapentin and pregabalin in post‐operative pain treatment published so far are promising. It is suggested that future studies should explore the effects of ‘protective premedication’ with combinations of various antihyperalgesic and analgesic drugs for post‐operative analgesia.
Electromyographic assessment of blink reflexes correlates with a clinical scale of depth of sedation/anaesthesia and BIS during propofol administration
Authors: Mourisse J. , Lerou J. , Zwarts M. , Booij L.
Published: September 2004

Background:  General anaesthesia is characterized by loss of consciousness, amnesia and obtundation of reflex responses to noxious stimuli. Quantifying the blink reflex may reflect the depression of reflex arches induced by anaesthetics and thus being informative on the anaesthetic state.

Methods:  The relation between the electrically evoked blink reflexes and the depth of sedation and anaesthesia induced with intravenous propofol was investigated. Twenty patients received propofol by target‐controlled infusion to create a stepwise deepening of sedation and anaesthesia. Depth of anaesthesia was assessed using the observer's assessment of anaesthesia and sedation (OAAS) scale, and by bispectral EEG analysis (BIS). Probit analysis was used to estimate the predicted propofol effect site concentrations producing unconsciousness, no response to noxious stimulation, and loss of blink reflex components.

Results:  Latency of the first (R1) and second (R2) blink component increased, whereas duration and area decreased with increasing depth of sedation and anaesthesia. A reasonably strong correlation between OAAS and the areas of R1 and R2 components was found (Spearman's ρ = 0.92 and 0.89). The areas of R1 and R2 and the OAAS also correlated with BIS (Spearman's ρ = 0.91, 0.88 and 0.90). EC50 and EC95 for loss of R1 were 2.8 (95% CI: 2.5–3.2) µg/ml and 4.6 (95% CI: 4.1–5.5) µg/ml, respectively.

Conclusions:  Our results suggest that the differential sensitivity of the components of the blink reflex could be useful in monitoring depth of sedation and light levels of anaesthesia during the administration of propofol. Both OAAS and BIS correlate similarly with the blink reflex components.
Presurgical intravenous parecoxib sodium and follow‐up oral valdecoxib for pain management after laparoscopic cholecystectomy surgery reduces opioid requirements and opioid‐related adverse effects
Authors: Gan T. J. , Joshi G. P. , Zhao S. Z. , Hanna D. B. , Cheung R. Y. , Chen C.
Published: September 2004

Background:  Opioids are associated with numerous adverse effects. It is unclear if reduced postoperative opioid consumption lowers the incidence and severity of opioid‐related adverse effects. This analysis – from a multicenter, randomized, double‐blind trial – tested if the reduction of opioid consumption among patients who received intravenous preoperative parecoxib 40 mg, followed by oral valdecoxib 40 mg qd postoperatively, in Days 1–4 after outpatient laparoscopic cholecystectomy surgery, reduced opioid‐related symptoms.

Methods:  Patients received intravenous fentanyl for pain before discharge, and oral acetaminophen 500 mg hydrocodone 5 mg q 4–6 h prn postdischarge for up to 7 days postsurgery. Patients also received intravenous parecoxib 40 mg administered 30–45 min preoperatively, and valdecoxib 40 mg qd up to Day 4 and prn Days 5–7 postsurgery, or placebo. Patients completed an opioid‐related Symptoms Distress Scale (SDS) questionnaire every 24 h for 7 days. Opioid use was converted to morphine‐equivalent doses (MEDs). Clinically meaningful events (CMEs) for 12 opioid‐related symptoms were assessed by three ordinal measures: frequency, severity, and bothersomeness. Reduction of CMEs on Day 1 and number of patient‐days with CMEs on Days 1–4 were examined.

Results:  Cumulative MEDs on Day 0, Day 1, and Days 1–4 were significantly lower in the parecoxib/valdecoxib group compared with the placebo group (P < 0.001). At the end of Day 1, parecoxib/valdecoxib‐treated patients had significantly lower SDS scores (P < 0.02), a significantly reduced incidence of CMEs (P < 0.05), and significantly fewer patient‐days with CMEs in Days 1–4 than placebo patients (P < 0.05). Patients in the parecoxib/valdecoxib group were less likely to have CMEs for multiple symptoms than those in the placebo group (P < 0.001).

Conclusions:  Treatment with parecoxib and valdecoxib significantly reduced the cumulative MED requirements, the incidence of opioid‐related adverse effects, and patient‐days with CMEs.
Efficacy of oral rofecoxib versus intravenous ketoprofen as an adjuvant to PCA morphine after urologic surgery
Authors: Cabrera M. C. , Schmied S. , Derderian T. , White P. F. , Vega R. , Santelices E. , Verdugo F.
Published: September 2004

Background:  Adjunctive use of nonsteroidal anti‐inflammatory drugs has become increasingly popular in the perioperative period because of their opioid‐sparing effects. This randomized, controlled, double‐dummy study was designed to evaluate the cost‐effectiveness of using oral rofecoxib as an alternative to intravenous ketoprofen for pain management in patients undergoing urologic surgery.

Methods:  Seventy patients were randomly assigned to receive either a placebo (Control) or rofecoxib 50 mg po (Rofecoxib) 1 h prior to surgery. After a standardized spinal anesthetic, patients in the Control group received ketoprofen 100 mg IV q 8 h for 24 h, while the Rofecoxib group received an equivolume of saline at 8‐h intervals for 24 h. Both groups were allowed to self‐administer morphine (1 mg IV boluses) using a PCA delivery system. The need for ‘rescue’ analgesic medication, as well as pain scores [using an 11‐point verbal rating scale (VRS) (0 = none to 10‐severe)], were recorded at 1, 2, 6, 12, and 24‐h intervals after surgery. In addition, the incidences of side‐effects were recorded at the end of the study period.

Results:  Total amount of morphine required in the initial 24‐h postoperative period was nonsignificantly reduced in the Rofecoxib group (29 ± 2 vs. 37 ± 4 mg). More importantly, the percentage of patients reporting moderate‐to‐severe pain (VRS score ≥4) during the study period was lower in the Rofecoxib group (12 vs. 22%, P < 0.05). The daily cost of rofecoxib (USD 1.14 for 50‐mg dose) was also significantly less than ketoprofen (USD 3.06 for three 100‐mg doses).

Conclusion:  Premedication with oral rofecoxib (50 mg) is a cost‐effective alternative to the parenteral nonselective NSAID, ketoprofen (100 mg q 8 h), when used as an adjuvant to PCA morphine for pain management after urologic surgery.
Peripheral venous pressure is an alternative to central venous pressure in paediatric surgery patients
Authors: Anter A. M. , Bondok R. S.
Published: September 2004

Background:  Peripheral venous pressure (PVP) is easily and safely measured. In adults, PVP correlates closely with central venous pressure (CVP) during major non‐cardiac surgery. The objective of this study was to evaluate the agreement between CVP and PVP in children during major surgery and during recovery.

Methods:  Fifty patients aged 3–9 years, scheduled for major elective surgery, each underwent simultaneous measurements of CVP and PVP at random points during controlled ventilation intraoperatively (six readings) and during spontaneous ventilation in the postanaesthesia care unit (three readings). In a subset of four patients, measurements were taken during periods of hypotension and subsequent fluid resuscitation (15 readings from each patient).

Results:  Peripheral venous pressure was closely correlated to CVP intraoperatively, during controlled ventilation (r=0.93), with a bias of 1.92 (0.47) mmHg (95% confidence interval = 2.16–1.68). In the postanaesthesia care unit, during spontaneous ventilation, PVP correlated strongly with CVP (r = 0.89), with a bias of 2.45 (0.57) mmHg (95% confidence interval = 2.73–2.17). During periods of intraoperative hypotension and fluid resuscitation, within‐patient changes in PVP mirrored changes in CVP (r = 0.92).

Conclusion:  In children undergoing major surgery, PVP showed good agreement with CVP in the perioperative period. As changes in PVP parallel, in direction, changes in CVP, PVP monitoring may offer an alternative to direct CVP measurement for perioperative estimation of volume status and guiding fluid therapy.
Impact of a critical care outreach team on critical care readmissions and mortality
Authors: Garcea G. , Thomasset S. , Mcclelland L. , Leslie A. , Berry D. P.
Published: September 2004

Aims:  The aim of a critical care outreach team is to facilitate discharges from critical care beds, educate ward staff in the management of deteriorating patients, facilitate transfer to critical care and reduce readmission rates to critical care. Although intuitively a good idea, there are few data to support outreach in terms of reducing the readmission rate to critical care and subsequent patient mortality.

This retrospective observational study attempted to determine the change in the critical care readmission rate, an indicator of the quality of critical care, critical care mortality and in‐hospital mortality following the introduction of a critical care outreach team in a major teaching hospital.

Methods:  A retrospective review of 1380 discharges from critical care was undertaken and the readmissions identified (n = 176). Readmission rate, mortality and other demographic data were compared between the pre and postoutreach periods.

Results:  Critical care mortality, in‐hospital mortality and 30‐day mortality were all reduced in the postoutreach period amongst readmissions to critical care. There was also a decease in the overall mortality of all patients admitted to critical care. There were no apparent causative factors for this reduction in mortality before and following outreach.

Conclusions:  There are many confounding factors in assessing the impact of outreach teams in hospitals. This study tentatively concludes that outreach teams may have a favourable impact on mortality rate amongst readmissions to critical care, but more data is needed from multicentre trials.
Fetal bradycardia and disseminated coagulopathy: atypical presentation of amniotic fluid emboli
Authors: Levy R. , Furman B. , Hagay Z. J.
Published: September 2004

We present a parturient who developed an atypical case of amniotic fluid emboli presented by sudden fetal bradycardia, followed by maternal disseminated coagulopathy. The typical feature of cardiopulmonary collapse was absent in this patient implying that in some cases of amniotic fluid emboli (AFE), fetal hypoxia or acidemia is unrelated to maternal cardiopulmonary status.
Impaired Photosystem II in a Mutant of Chlamydomonas Reinhardtii Defective in Sulfoquinovosyl Diacylglycerol
Authors: Sato Norihiro , Sonoike Kintake , Tsuzuk Mikio , Kawaguchi Akihiko
Published: September 2004

The photosynthetic apparatus was characterized in a mutant of Chlamydomonas reinhardtii, hf‐2, defective in the synthesis of a chloroplast‐specific lipid, sulfoquinovosyl diacylglycerol (SQui‐acyl2Gro). hf ‐2 showed reduced photosystem II (PSII) activity with little effect on photosystem I (PSI) activity, as compared with the parent. PAGE in the presence of dodecyl β‐D‐maltoside (DodGlc2) of C. reinhardtii thylakoid membranes was used to isolate chlorophyll‐protein complexes without chlorophyll (Chi) release in order to examine lipid species bound to these complexes. The four complexes obtained were shown to be the PSI complex, the PSII core complex and the two groups of the light‐harvesting complex of PSII by analyses of 77‐K emission spectra of Chi fluorescence and of subunit compositions. Lipid analysis of Chl‐protein complexes in the parent revealed the localization of SQui‐acyl2Gro in the PSII core complex and the two groups of the light‐harvesting complex of PSII, but not in the PSI complex. These results suggest that SQui‐acyl2Gro is responsible for PSII activity by associating with the core and light‐harvesting complexes of PSII.
Cartography of Ribosomal Proteins of the 30S Subunit from the Halophilic Haloarcula Marismortui and Complete Sequence Analysis of Protein HS26
Authors: Engemann Sabine , Noelle Ruth , Herfurth Eike , Briesemeister Ulrike , Grelle Gerlinde , Wittmann‐Liebold Brigitte
Published: September 2004

By two‐dimensional polyacrylamide gel electrophoresis of 30S ribosomal subunit proteins (S proteins) from Haloarcula marismortui we identified 27 distinct spots and analyzed all of them by protein sequence analysis. We demonstrated that protein HmaS2 (HS2) is encoded by the open reading frame orfMSG and has sequence similarities to the S2 ribosomal protein family. The proteins HmaS5 and HmaS14 were identified as spots HS7 and HS21/HS22, respectively. Protein HS4 was characterized by amino‐terminal sequence analysis. The spot HS25 was recognized as an individual protein and also characterized by sequence analysis. Furthermore, the complete primary sequence of HS26 is reported, showing similarity only to eukaryotic ribosomal proteins. The sequence data of a further basic protein shows a high degree of similarity to ribosomal protein S12, therefore it was designated HmaS12. Slightly different results compared to published sequence data were obtained for the proteins HS12 and HmaS19. The putative ‘ribosomal’ protein HSH could not be localized in the two‐dimensional pattern of the total 30S ribosomal subunit proteins of H. marismortui. Therefore, it seems to be unlikely that this protein is a real constituent of the H. marismortui ribosome.
Site‐Directed Mutagenesis and Sugar‐Binding Properties of the Wheat Germ Agglutinin Mutants Tyr73Phe and Phe116Tyr
Authors: Nagahora Hitoshi , Harata Kazuaki , Muraki Michiro , Jigami Yoshifumi
Published: September 2004

Wheat germ agglutinin is a dimeric lectin composed of two identical subunits. Each subunit consists of four homologous hevein‐like domains of 42 or 43 amino acids each. Amino acid residues at the same position in each domain involved in sugar binding are thought to play a similar role in sugar binding. In order to clarify the role of the amino acid residue at domain position 30 of wheat germ agglutinin isolectin 2 (WGA2) in sugar binding, two WGA2 variants each containing a mutation, either Tyr73→Phe (domain B) or Phell6→Tyr (domain C), were produced. The binding activity for (GlcNAc)3 and the three‐dimensional structure of these mutants were characterized by comparing with the properties of wild‐type WGA2. Equilibrium dialysis experiments using (GlcNAc)3 indicated that the mutation Tyr73→Phe reduced the overall sugar‐binding activity at both pH5.9 and pH 4.7. In addition, positive cooperativity toward (GlcNAc)3 binding was observed at pH 4.7. In contrast, the mutation of Phe116→Tyr increased the overall sugar‐binding activity at pH 5.9, but reduced this activity at pH 4.7 without changing the number of sugar‐binding sites. Positive cooperativity was not observed at pH 5.9 or pH 4.7. X‐ray crystallographic analysis of mutant WGA2 revealed that the mutation of Tyr73→Phe caused a side chain movement of the Glu115 residue of the opposite subunit that formed a hydrogen bond with Tyr73 in wild‐type WGA2. No changes were observed in the backbone structure and the disposition of the benzene ring of Phe73. The mutation Phell6→Tyr caused the formation of a new hydrogen bond between Tyr116 and Glu72 of the opposite subunit. The changes in the sugar‐binding properties in WGA2 mutants are discussed in relation to the structural change at the binding site.
Purification and Properties of Human Placental ATP Diphosphohydrolase
Authors: Christoforidis Savvas , Papamarcaki Thomais , Galaris Dimitrios , Kellner Roland , Tsolas Orestes
Published: September 2004

ATP diphosphohydrolase activity (ATP‐DPH) has been previously identified in the paniculate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1–8]. In the present study we have purified to homogeneity and characterized this activity. A 260‐fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL‐6B and 5′‐AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity.

The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8–azido‐[α‐32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg21. The Km values for ATP and ADP were determined to be 10μM and 20 μM, respectively. Maximum activity was found at pH 7.0–7.5 with ATP as substrate, and pH 7.5–8.0 with ADP. The enzymic activity is inhibited by NaN3 NaF3 adenosine 5′‐[β,γ‐imido]triphosphate and adenosine 5′‐[α,β‐methylene]triphosphate.

Protein sequence analysis showed ATP‐DPH to be N‐terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins.

ATP‐DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.
DNA Binding through Distinct Domains of Zinc‐Finger‐Homeodomain Protein AREB6 has Different Effects on Gene Transcription
Authors: Ikeda Keiko , Kawakami Kiyoshi
Published: September 2004

Transcription factor AREB6 has a unique structure composed of two zinc‐finger clusters in N‐ and C‐terminal regions, and one homeodomain in the middle. AREB6 has been known to regulate the expression of the Na, K‐ATPase α1 subunit, interleukin 2 and δ‐crystallin genes. We determined the optimal binding sites for the N‐terminal zinc‐finger cluster as GTCACCTGT or TGCACCTGT and for the C‐terminal zinc‐finger cluster as C/TACCTG/TT by the CASTing method (cyclic amplification and selection of targets). The additional consensus sequence GTTTC/G, in conjunction with the CACCTGT sequence, was selected by the second CASTing for the entire coding region. The N‐terminal zinc‐finger cluster binds to DNA strongly when the DNA has GTTTC/G in conjunction with the CACCTGT sequence. The homeodomain had no specific DNA binding activity but was found to interact with the N‐terminal zinc‐finger cluster. Analyses of zinc‐finger mutation proteins revealed that the contribution to DNA binding of each N‐terminal zinc‐finger motif is altered depending on the presence of the additional consensus. Transient transfection assays showed that AREB6 repressed the human 70‐kDa heat‐shock gene promoter harboring the CACCTGT sequence together with the additional consensus, and that AREB6 activated the promoter harboring the CACCTGT sequence without the additional consensus. These results suggest that AREB6 has multiple conformational states, leading to positive and negative regulations of gene transcription.
Phosphotyrosine Residues in the Nerve‐Growth‐Factor Receptor (Trk‐A)
Authors: Baxter Ruth M. , Cohen Philip , Obermeier Axel , Ullrich Axel , Downes C. Peter , Doza Yair N.
Published: September 2004

PC12 cells, which lack platelet derived‐growth‐factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the β‐PDGF receptor and the intracellular and transmembrane domains of the nerve‐growth‐factor receptor Trk‐A (termed PT‐R). Mutation of the Trk‐A residue Tyr490 to phenylalanine prevents the association with Shc, while similar mutations at Tyr751 or Tyr785 are reported to prevent interaction of Trk‐A with the p85 subunit of inositol phospholipid 3‐kinase and phospholipase C‐γ1, respectively. The strong and sustained activation of p42 and p44 mitogen‐activated‐protein kinases induced by PDGF‐B/B in PC12/PT‐R cells was unaffected by mutation of Tyr785 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr785. Mutation of Tyr490 reduced by 70% the PDGF‐induced increase in inositol phospholipid 3‐kinase activity immunoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF‐induced increase in the intracellular products of inositol phospholipid 3‐kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutation of Tyr490 or Tyr751) abolished PDGF‐stimulated hydrolysis of phosphatidylinositol 4,5‐bisphosphate. Mutation of Tyr490, alone or in combination with mutation of Tyr751 and Tyr785, had no effect on the PDGF‐induced activation of p70 S6 kinase (p70S6K). However, the activation of p70S6K by PDGF (or nerve growth factor), but not the activation of mitogen‐activated‐protein kinase, was prevented by two structurally unrelated inhibitors of inositol phospholipid 3‐kinase, wortmannin or LY294002. Our results demonstrate the following: (1) the phosphorylation of Tyr490 plays a major role in the activation of inositol phospholipid 3‐kinase and formation of 3‐phosphorylated inositol lipids and confirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C‐γ1 in vivo. (2) Tyr490 phosphorylation (but not inositol phospholipid 3‐kinase activation) is also required for strong and sustained activation of mitogen‐activated‐protein kinase and neuronal differentiation, while the smaller and more transient activation of mitogen‐activated‐protein kinase, produced by the activation of phospholipase C‐γ1 is insufficient to trigger the neuronal differentiation of PT‐R cells. (3) Inositol phospholipid 3‐kinase is required for the activation of p70S6K, but only a small increase in inositol phospholipid 3‐kinase activity and the level of 3‐phosphorylated inositol lipids is required for maximal p70s6K activation.
Diphenylene Iodonium Inhibits the Induction of Erythropoietin and Other Mammalian Genes by Hypoxia
Authors: Gleadle Jonathan M. , Ebert Benjamin L. , Ratgliffe Peter J.
Published: September 2004

Recent studies on the induction of erythropoietin gene expression by hypoxia have indicated that erythropoietin forms part of a widely operative system of gene regulation by oxygen. Similar responses to hypoxia, cobaltous ions and desferrioxamine have indicated that the action of these agents is closely connected with the mechanism of oxygen sensing. To consider further the mechanisms underlying these responses, the effect of iodonium compounds was tested on five genes which show oxygen‐regulated expression; erythropoietin, vascular endothelial growth factor (VEGF), lactate dehydrogenase‐A (LDH‐A), glucose transporter‐1 (GLUT‐1) and placental growth factor (PLGF). In each case, the response to hypoxia was specifically inhibited by low doses of diphenylene iodonium (Ph2I+). This occurred irrespective of whether the hypoxic response was induction of gene expression (erythropoietin, vascular endothelial growth factor, lactate dehydrogenase‐A, glucose transporter‐1) or inhibition of gene expression (PLGF). In contrast, the induction of gene expression by cobaltous ions or desferrioxamine was not inhibited by PhJ+. The differential action of Ph2I+ on the response to hypoxia versus the response to cobaltous ions or desferrioxamine must reflect a difference in the mechanism of action of these stimuli, which will require accommodation in any model of the oxygen‐sensing mechanism. Based on the known properties of Ph2I+, the implication of these findings is that the mechanism of oxygen sensing most probably involves the operation of a flavoprotein oxidoreductase
Purification and Biochemical Characterization of Myomesin, a Myosin‐Binding and Titin‐Binding Protein, from Bovine Skeletal Muscle
Authors: Obermann Wolfgang M. J. , Plessmann Uwe , Weber Klaus , Fürst Dieter O.
Published: September 2004

We report a method for isolating homogeneous myomesin from mammalian skeletal muscle. The identity of the purified bovine protein was confirmed by its reactivity with myomesin‐specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the amino‐terminal and carboxy‐terminal ends of the sequence predicted by the human myomesin cDNA. All partial sequences obtained from bovine myomesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods with a molecular length of about 50 nm. Circular dichroism spectra showed a high degree of β structure as expected for a member of the immunoglobulin superfamily of proteins. Alignment of the sequences of the class I and II domains of myomesin with the sequences of domains of known three‐dimensional structure provides a more detailed model of myomesin. In agreement with this view, the cleavage sites observed by limited proteolysis locate primarily between individual domains.

In a solid‐phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy‐terminal 30‐kDa fragment of LMM. The myosin‐binding site seemed to be confined to the amino‐terminal 240 residues of the molecule. The cross‐reactivity of myomesin with the phosphorylation‐dependent monoclonal neurofilament antibody NE14 [Shaw, G. E., Debus, E. & Weber, K. (1984) Eur. J. Cell Biol. 34, 130–136] was analyzed. NE14 reactivity of myomesin was abolished by alkaline phosphatase. Reactivity of the antibody on stable proteolytic fragments of myomesin showed that the phosphorylation site must reside within the carboxy‐terminal 60 residues.
A Two‐Binding‐Site Kinetic Model for the Ribonuclease‐T1‐Catalysed Transesterification of Dinucleoside Phosphate Substrates
Authors: Steyaert Jan , Engelborghs Yves
Published: September 2004

Ribaonucleases have been found to have subsites that confer large rate enhancements but do not contribute to substrate binding. In this study, we present a kinetic model that formally explains how subsite binding energy is converted into chemical activation energy. The proposed mechanism takes into account a primary specificity site and a subsite, both of which must be occupied for chemical turnover. An unstable reaction intermediate is formed upon binding of the polymeric substrate monomers at the corresponding subsites. The structure of this reaction intermediate resembles the transition state of the catalysed transphosphorylation reaction. Similar mechanisms may be used by other depolymerizing enzymes including nucleases, glycosidases, and proteases.
Functional Consequences of the Ser334→Pro Mutation in a Human Factor X Variant (Factor XMarseille)
Authors: Bezeaud Annie , Miyata Toshiyuki , Helley Dominique , Zeng Yan‐Zhen , Kato Hisao , Aillaud Marie‐Françoise , Juhan‐Vague Irène , Guillin Marie‐Claude
Published: September 2004

A factor X molecular variant was identified in a 55‐year‐old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV‐X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV‐X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S‐2765 (D‐Arg‐Gly‐Arg‐NH‐Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV‐X was the consequence of a threefold decrease in the Kcat for the activation of factor XMarseille while the Km of RVV‐X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single‐strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T→C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three‐dimensional models of various serine proteases, it appears that Ser334 is located within a surface‐exposed variable region of factor X. This observation suggests that the Ser334→Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser→Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.
The Affinity of Human Erythrocyte Porphobilinogen Synthase for Zn2+ and Pb2+
Authors: Simons Timothy J. B.
Published: September 2004

Porphobilinogen synthase activity has been measured in human erythrocyte lysates supplemented with metal‐ion buffers to control free Zn2+ and Pb2+ concentrations. The enzyme is activated by Zn2+ with a Km, of 1.6 pM and inhibited by Pb2+ with a K1 of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal‐binding site. The half‐time for loss of Zn2+ from the active site, or replacement of Pb2+ by Zn2+, were in the 10–20‐min range at 37°C. Zn2+ did not affect the affinity for the substrate 5‐aminolevulinate, but Pb2+ reduced it non‐competitively.

All the experiments were conducted with a blood sample of the common 1–1 phenotype [Astrin, K. H., Bishop, D. P., Wetmur, J. G., Kaul, B., Davidow, B. & Desnick, R. J. (1987) Ann. NY Acad. Sci. 514, 23–29]
Purification and Characterization of Protein PB of Betaine Reductase and its Relationship to the Corresponding Proteins Glycine Reductase and Sarcosine Reductase from Eubacterium Acidaminophilum
Authors: Meyer Manfred , Granderath Katrin , Andreesen Jan R.
Published: September 2004

Simple complementation assay systems were developed for the substrate‐specific proteins PB of glycine reductase, sarcosine reductase, and betaine reductase, in which acetyl phosphate was detected as the product in all three cases. The betaine‐specific subunits of protein B (PB betaine) responsible for betaine reductase activity were purified to homogeneity from cells of Eubacterium acidaminophilum. The molecular masses of the two different subunits were 45 kDa and 48 kDa according to SDS/PAGE. The molecular mass of the native protein was about 200 kDa, indicating an α2β2 structure. The glycine‐specific protein B (PB glycime) was partially purified and subunits of 47 kDa and 27 kDa were N‐terminally sequenced. The latter subunits cross‐reacted with antibodies raised against PB betaine and showed high sequence similarity to the 45‐kDa and 48‐kDa subunits of PB betaine, respectively. (2‐14C]Glycine could be covalently coupled to the 47‐kDa subunit by treatment with borohydride. By the same procedure, [2‐14C]sarcosine labeled a protein of the same size. Like the sarcosine reductase activity, this protein was not present in glycine‐grown cells, indicating its specific involvement in sarcosine metabolism. The labile viologen‐dependent formate dehydrogenase purified with the respective PB proteins and could be tentatively assigned to a 95‐kDa protein.
Characterization of Neutrophil NADPH Oxidase Activity Reconstituted in a Cell‐Free Assay Using Specific Monoclonal Antibodies Raised Against Cytochrome b558
Authors: Batot Géraldine , Martel Cécile , Capdeville Nicolas , Wientjes Frans , Morel Françoise
Published: September 2004

The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell‐free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane‐bound and octyl‐glucoside‐extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91‐phox cytochrome b558, two were selected, both of which were found to bind to the β subunit of cytochrome b558, and to inhibit superoxide formation in the oxidase reconstituted cell‐free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X‐linked chronic granulomatous disease patients did not produce O2− in the reconstituted system and did not bind to the antibodies.
Tissue Distribution and Intracellular Localisation of the 75‐kDa Inositol Polyphosphate 5‐Phosphatase
Authors: Speed Caroline J. , Matzaris Maria , Bird Phillip I. , Mitchell Christina A.
Published: September 2004

The 75‐kDa inositol polyphosphate 5‐phosphatase (75‐kDa 5‐phosphatase) hydrolyses several important mediators of intracellular calcium homeostasis, including inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2].

Northern analysis of various human tissues revealed the 75‐kDa 5‐phosphatase has a ubiquitous expression, where differential splicing may occur in specific tissues. Prominent expression of a 4.4‐kb transcript was noted in human lung, thymus, testes and placenta, and a 4.6‐kb transcript was observed in heart, brain, kidney, ovary and colon. Determination of the intracellular location of the enzyme by indirect immunofluorescence, demonstrated that the 75‐kDa 5‐phosphatase was associated with mitochondrial and cytosolic cellular compartments. Immunoprecipitation of the total cell homogenate of human lung carcinoma cells (A549) with anti‐(recombinant 75‐kDa 5‐phosphatase) antibodies revealed that the 75‐kDa 5‐phosphatase is the major PtdIns(4,5)P2 5‐phosphatase in this cell line. Analysis of PtdIns(4,5)P2 5‐phosphatase activity in subcellular fractions of A549 cells revealed peak 75‐kDa 5‐phosphatase enzyme activity in the cytosolic and mitochondrial enriched fractions. Immunoblot analysis further confirmed the mitochondrial location of the enzyme.

This study demonstrates the tissue distribution and intracellular location of the 75‐kDa 5‐phosphatase and reveals a novel location for an enzyme involved in phosphatidylinositol turnover.