The gene order in the nuo-operon is not essential for the assembly of E. coli complex I.

Energy-converting NADH: ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular energy metabolism. Bacterial complex I is generally composed of 14 different subunits, seven of which are membranous and the other seven are globular proteins. They are encoded by the nuo-operon, whose gene order is strictly conserved in bacteria. The operon starts with nuoA encoding a membranous subunit followed by genes encoding globular subunits. To test the idea that NuoA acts as a seed to initiate the assembly of the complex in the membrane, we generated mutants that either lacked nuoA or contain nuoA at a different position within the operon. To enable the detection of putative assembly intermediates, the globular subunit NuoF and the membranous subunit NuoM were individually decorated with the fluorescent protein mCherry. Deletion of nuoA led to the assembly of an inactive complex in the membrane containing NuoF and NuoM. Re-arrangement of nuoA within the nuo-operon led to a slightly diminished amount of complex I in the membrane that was fully active. Thus, nuoA but not its distinct position in the operon is required for the assembly of E. coli complex I. Furthermore, we detected a previously unknown assembly intermediate in the membrane containing NuoM that is present in greater amounts than complex I.

c-ANCA-induced neutrophil-mediated lung injury: a model of acute Wegener's granulomatosis.

Anti-neutrophil cytoplasmic antibodies (c-ANCA) targeting proteinase 3 (PR3) are implicated in the pathogenesis of Wegener's granulomatosis (WG). Fulminant disease can present as acute lung injury (ALI). In this study, a model of ALI in WG was developed using isolated rat lungs. Isolated human polymorphonuclear leukocytes (PMNs) were primed with tumour necrosis factor (TNF) to induce surface expression of PR3. Co-perfusion of TNF-primed neutrophils and monoclonal anti-PR3 antibodies induced a massive weight gain in isolated lungs. This effect was not observed when control immunoglobulin G was co-perfused with TNF-primed PMNs. The c-ANCA-induced oedema formation was paralleled by an increase in the capillary filtration coefficient as a marker of increased pulmonary endothelial permeability. In contrast, pulmonary artery pressure was not affected. In the presence of the oxygen radical scavenger superoxide dismutase and a NADPH oxidase inhibitor, c-ANCA-induced lung oedema could be prevented. Inhibition of neutrophil elastase was equally effective in preventing c-ANCA-induced lung injury. In conclusion, anti-PR3 antibodies induced neutrophil mediated, elastase- and oxygen radical-dependent ALI in the isolated lung. This experimental model supports the hypothesis of a pathogenic role for c-ANCA in WG and offers the possibility of the development of therapeutic strategies for the treatment of lung injury in fulminant WG.

[Improved survival by guideline compliant cardiopulmonary resuscitation: analysis of primary survival rates in the Hamburg emergency medical service]. / Verbessertes Überleben durch leitliniengerechte kardiopulmonale Reanimation : Analyse der primären Überlebensrate im Hamburger Rettungsdienst.

BACKGROUND: In 2005 revised guidelines for cardiopulmonary resuscitation (CPR) were published by the European Resuscitation Council replacing the guidelines implemented in the year 2000. The aim of this study was to test the compliance with valid guidelines and to establish the quality of pre-hospital CPR provided by paramedics over a period of 38 months. PATIENTS AND METHODS: A total of 299 CPRs performed by paramedics of the emergency medical services of Hamburg, Germany between 1(st) November 2004 and 31(st) December 2007 were analyzed. Digital recordings of automated external defibrillators and emergency protocol data were analyzed in detail. CPR was judged as incorrect if the defibrillation energy level did not correspond to the valid guidelines or if the interval between defibrillations exceeded a tolerance range of more than 30% compared to the valid guidelines. RESULTS: All CPRs (299) were included in the analysis of which 197 (65.9%) were intended to follow the 2000 guidelines and 102 (34.1%) the 2005 guidelines. Return of spontaneous circulation (ROSC) was achieved in 164 cases (54.8%) and survival to hospital admission in 125 cases (41.8%). CPR was performed accurately according to guidelines in only 26 cases (8.7%). In 273 cases (91.3%) the guidelines were not followed completely. Concerning the translation of guidelines into practice most faults occurred due to wrong intervals (89.3%), wrong defibrillation energy (33.4%) and medical errors, such as defibrillating an asystolic patient (7.0%). Primary survival rates were not significantly different when CPR accurately followed the 2000 or 2005 guidelines (40.1% versus 45.1%). Comparing primary survival rates of cases in which the guidelines were followed completely, there was no significant difference between the 2000 guidelines (15 out of 21 cases 71.4%) and 2005 guidelines (4 out of 5 cases 80.0%). However, compliance with valid guidelines significantly increased primary survival rates compared to non-compliance with valid guidelines (19 out of 26 cases 73.1% versus 106 out of 273 cases 38.8%; p=0.007). This effect was independent of the duration of CPR. Comparing CPR with monophasic defibrillation (189 cases) or biphasic defibrillation (58 cases), there was a significantly higher rate of ROSC (56.1% versus 72.4%) and a significantly higher rate of primary survival (41.3% versus 56.9%) in favour of biphasic defibrillation. CONCLUSION: The results of our study show that compliance with valid guidelines is low and furthermore suggest that compliance with guidelines significantly reduces mortality. Future research may be warranted into the question of how to increase compliance with current CPR guidelines in pre-hospital emergency care.

Effect of sirolimus on the metabolism of apoB100- containing lipoproteins in renal transplant patients.

BACKGROUND: Sirolimus (Rapamune, rapamycin, RAPA) is a potent immunosuppressive drug that has reduced the rate of acute rejection episodes by more than 40% in phase III trials when added to an immunosuppression regimen of cyclosporine (CsA) and prednisone. However, RAPA treatment tends to increase lipid levels, particularly among patients with pre-existing hyperlipidemia. METHODS: To identify the metabolic pathway(s) leading to RAPA-mediated hyperlipidemia, five patients with renal transplants maintained on CsA+/-prednisone+/- azathioprine (AZA) were studied before and after 6 weeks of treatment with RAPA (off RAPA and on RAPA, respectively). Each study patient was infused with a single bolus of [2H4]-lysine to derive metabolic parameters for apoB100-containing lipoproteins by using kinetic analysis based upon quantitation of isotopic enrichment by gas chromatography-mass spectrometry. RESULTS: Serial lipid measurements revealed that four patients displayed increased plasma triglyceride levels after RAPA treatment, which coincided with significantly higher plasma VLDL-apoB100 concentrations (21.7+/-12.1 mg/dl off RAPA vs. 38.7+/-14.8 mg/dl on RAPA, mean+/-SD, P<0.05). Kinetic analysis showed that the RAPA-induced increase in VLDL-apoB100 concentrations was due to a significant reduction in the fractional catabolic rate (FCR) of very low-density lipoprotein (VLDL) apoB100 (0.83+/-0.65 off RAPA vs. 0.24+/-0.10 on RAPA, mean+/-SD, P<0.05), rather than an enhanced VLDL-apoB100 synthesis. In one patient, RAPA treatment induced hypercholesterolemia but not hypertriglyceridemia. This hypercholesterolemia was due to elevated low-density lipoprotein (LDL) cholesterol levels, which coincided with a decreased FCR of LDL-apoB100. Heparin-induced lipoprotein lipase activity was significantly lower in the immunosuppressed hyperlipidemic patients than in normolipidemic controls. However, RAPA treatment did not significantly alter basal lipoprotein lipase activity in renal transplant patients in this study. CONCLUSIONS: This study indicates that for renal transplant patients in whom RAPA treatment induces hyperlipidemia, this effect is the result of reduced catabolism of apoB100-containing lipoproteins.

Phylogeny of the third component of complement, C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species.

The third component of complement, C3, binds to several other complement proteins. To study the diverse reactivities of C3, we analyzed the conservation of structural and functional features in the C3 from different species. First, we developed a method to purify swine (Po), rabbit (Rb), mouse (Mo), cobra (Co), Xenopus (Xe), axolotl (Ax), and trout (Tr) C3 from plasma. This involved protein precipitation by polyethylene glycol, followed by anion-exchange, gel filtration, and cation exchange chromatography. All C3's tested were comprised of two chains (alpha/beta-chain) and contain a thiolester bond within the alpha-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved (as detected by ConA binding) in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the beta-chain and Po and Mo C3 only in the alpha-chain. Co C3, in contrast to cobra venom factor (CVF), lacks ConA binding carbohydrates in both chains. N-terminal amino acid sequence analysis of the alpha-, alpha'-, and beta-chains showed varying degrees of similarity within the different C3's. The N-termini of the Xe and Ax C3 beta-chains were found to be blocked. The conservation of binding sites in the different C3's for human complement receptors type one (CR1) and two (CR2) and for factors H and B was investigated due to the structural and functional similarities of these molecules and to the ability of some of them to bind to the same domain(s) in human C3.(ABSTRACT TRUNCATED AT 250 WORDS)

Ritanserin attenuates the in vitro effects of the 5-HT2 receptor agonist DOI on skeletal muscles from malignant hyperthermia-susceptible patients.

STUDY OBJECTIVES: To study the in vitro effects of the serotonin2 (5-HT2) receptor agonist 1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in skeletal muscle specimens from malignant hyperthermia-susceptible (MHS) and normal (MHN) patients following pretreatment with the 5-HT2 receptor antagonist ritanserin. DESIGN: Prospective study. SETTING: Malignant hyperthermia (MH) laboratory at a university hospital. PATIENTS: 41 patients undergoing in vitro contracture test for diagnosis of MH susceptibility. INTERVENTIONS: Skeletal muscle biopsies in adult patients were performed with a 3-in-1 nerve block with 40 ml prilocaine 1%. In children, general anesthesia was induced with 50 micrograms/kg alfentanil intravenously (i.v.) and 2 to 2.5 micrograms/kg propofol i.v. and maintained with a continuous infusion of propofol (< or = 150 micrograms/kg/min) and nitrous oxide (66%) in oxygen. MEASUREMENTS AND MAIN RESULTS: Patients were first classified as MHS or MHN by the in vitro contracture test according to the European MH protocol. Surplus muscle specimens of 21 MHS and 20 MHN patients were used in this study. At first, DOI was added to the organ bath at a concentration of 0.02 mM. In the second part of the study, muscle specimens were preincubated with ritanserin 0.01 mM for 10 minutes before DOI 0.02 mM was added to the bath. Muscle specimens from all patients developed contractures after administration of DOI. The onset of contractures was significantly faster in MHS muscles, and the magnitude of contracture was significantly greater than in MHN. The muscle twitch decreased significantly in both groups after DOI. After pretreatment with ritanserin, start of contracture was significantly delayed in MHS muscles. MHN muscles failed to develop contractures. The maximum level of contracture was significantly reduced in MHS. Muscle twitch decreased also in both MHS and MHN groups. CONCLUSIONS: The findings may indicate that stimulation of 5-HT2 receptors is involved in MH induction. Furthermore, 5-HT2 receptor antagonists could possibly be effective in preventing MH. Additional studies are required to determine if administration of 5-HT2 receptor antagonists could be of additional value in the treatment or prevention of anesthetic-induced MH.

Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR2 interaction may suggest a possible immunoregulatory role of HSV glycoprotein C.

An in-house coordinator program to increase organ donation in public trauma hospitals.

A 4-year retrospective study was conducted regarding the donor potential, consent rates, and organ recovery at a large 500-bed public trauma hospital. An independent organ procurement organization hired two in-house coordinators, one white and one black, to work exclusively in the hospital. The duties of the in-house coordinators included the following: working with nurses, physicians, and residents to identify donors; closely managing and coordinating the consent process; and assisting organ procurement coordinators in donor management. Following the program's implementation and the use of race-specific requesters, a 64% increase in consent rate resulted along with an overall increase of 94% in the number of organ donors. The consent rate of blacks increase 115%, whereas the number of black organ donors increased 154%. The Hispanic consent rate increased 48% with a corresponding increase of 83% in the number of Hispanic organ donors. In addition, the white consent rate increased from 55% (the 3-year average from 1993 to 1995) to 75% in 1996, resulting in a 36% increase following the implementation of the program. The investment of dedicated race-sensitive personnel in large urban county trauma facilities can result in a significant increase in donor conversion rates.