Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major.

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

Chemical profiling of different hashish seizures by gas chromatography-mass spectrometry and statistical methodology: a case report

Limited information is available regarding the methodology required to characterize hashish seizures for assessing the presence or the absence of a chemical link between two seizures. This casework report presents the methodology applied for assessing that two different police seizures were coming from the same block before this latter one was split. The chemical signature was extracted using GC-MS analysis and the implemented methodology consists in a study of intra- and inter-variability distributions based on the measurement of the chemical profiles similarity using a number of hashish seizures and the calculation of the Pearson correlation coefficient. Different statistical scenarios (i.e., a combination of data pretreatment techniques and selection of target compounds) were tested to find the most discriminating one. Seven compounds showing high discrimination capabilities were selected on which a specific statistical data pretreatment was applied. Based on the results, the statistical model built for comparing the hashish seizures leads to low error rates. Therefore, the implemented methodology is suitable for the chemical profiling of hashish seizures.

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor.

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.

CHARACTERIZATION OF EPSTEIN-BARR VIRUS-ENCODED LATENT MEMBRANE PROTEIN 1

Le virus d'Epstein-Barr (EBV), un virus de la famille des gammaherpesvirus, infecte plus de 95% de la population adulte mondiale. EBV est associé à plusieurs types de cancers dont le lymphome de Hodgkin, le lymphome de Burkitt et le carcinome nasopharyngé. La protéine membranaire de latence 1 (LMP1), l'oncogène principal d'EBV, est une protéine membranaire intégrale composée d'une petite extrémité N-terminale cytoplasmique, six segments transmembranaires (TMs) lié par de petites boucles et un long domaine C-terminale cytoplasmique. Le gène de LMP1, BNLF-1, est très polymorphe et plusieurs variants de la protéine LMP1 ont été décrits. Parmi les variants de LMP1 la majeure différence décrite est leur capacité à activer le facteur de transcription NF-κB. Nous avons défini des polymorphismes permettant aux variants d'avoir une activation accrue de NF-κB comparé au prototype B95-8 LMP1. Tous les polymorphismes cruciaux identifiés dans notre étude se trouvent dans les TMs 4 et 5 de LMP1. Nous avons étudié l'implication de chaque paire de TMs dans l'association à la membrane, l'auto-agrégation, la liaison aux partenaires cellulaires de LMP1 TRAF3 et β-TrCP, ainsi que pour NF-κB. De plus, nous avons décrit un nouveau rôle pour LMP1 consistant à inhiber l'activation contrôlée par MAVS de ISRE et du promoteur d'IFNβ. En résumé, nous avons observé que les différentes paires de TMs, ainsi que les deux boucles intracellulaires, ne sont pas équivalents. Dans l'ensemble, notre étude a montré que les TMs jouent un rôle clé dans les interactions protéine-protéine et la signalisation et qu'ils peuvent être considérés comme des régulateurs essentiels des activités de LMP1.

Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Role of vitreoretinal surgery in maximizing treatment outcome following complications after proton therapy for uveal melanoma.

PURPOSE: To assess the role of vitreoretinal surgery in maximizing treatment outcome following complications after proton therapy for uveal melanoma and to evaluate its safety. METHODS: Retrospective chart study on 21 patients (2% of a total of 1,005 treated by proton therapy between January 2003 and August 2007) who had developed a complication requiring vitreoretinal surgery. Mean/median total follow-up after irradiation was 43/43 months (range, 12-70 months). RESULTS: Indications for surgery included vitreous hemorrhage (n = 13), epimacular membrane (n = 5), rhegmatogenous retinal detachment (n = 1), combined vitreous hemorrhage with total serous retinal detachment (n = 1), and vitritis (n = 1). Mean/median interval for vitreoretinal surgery after irradiation was 21/20 months (range, 4-45 months), and mean/median follow-up after pars plana vitrectomy was 22/23 months (range, 2-56 months). Pars plana vitrectomy was combined with retinal photocoagulation (n = 5), air/gas (n = 5), or silicone oil tamponade (n = 1). Mean Snellen visual acuity was 20/200 (0-20/40) before and 20/100 (0-20/25) after pars plana vitrectomy. A transient postoperative rise in intraocular pressure was measured in seven patients. Four patients developed phthisis bulbi. CONCLUSION: Vitreoretinal surgery was efficient in maximizing treatment outcome after proton therapy, as it allowed a better oncologic follow-up. Pars plana vitrectomy permitted panretinal photocoagulation to avoid neovascular glaucoma or retinal detachment repair. Macular surgery improved visual acuity, especially in anterior melanoma, whereas repeated surgery may increase the risk of enucleation.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Initial clinical experience with the admiral oxygenator combined with separated suction.

The Admiral, a new microporous membrane oxygenator with a low surface area, decreased priming volume and two separate reservoirs, was tested in 30 adult patients. This study was undertaken to evaluate blood path resistance, gas exchange capabilities and blood trauma in clinical use, with and without shed blood separation. Patients were divided into 3 groups. Group 1 had valve surgery without separation of suction, Group 2 had coronary artery bypass grafting (CABG) with direct blood aspiration and Group 3 had coronary artery bypass grafting with shed blood separation. The suctioned, separated, cardiotomy blood in Group 3 was treated with an autotransfusion device at the end of bypass before being returned to the patient. Theoretical blood flow could be achieved in all cases without problem. The pressure drop through the oxygenator averaged 88 +/- 13 mmHg at 4 l/min and 109 +/- 12 mmHg at 5 l/min. O(2) transfer was 163 +/- 27 ml/min. Free plasma haemoglobin rose in all groups, but significantly less in group 3. Lactate dehydrogenase (LDH) rose significantly in Groups 1 and 2. Platelets decreased in all groups without significant differences. Clinical experience with this new oxygenator was safe, the reduced membrane surface did not impair gas exchange and blood trauma could be minimized easily by separating shed blood, using the second cardiotomy reservoir.

Molecular determinants for membrane association of the hepatitis C virus NS2 protease domain

Background: Hepatitis C virus (HCV) nonstructural protein 2 (NS2) plays essential roles in particle assembly and polyprotein processing. It harbors an N-terminal membrane domain comprising three putative transmembrane s egments ( amino acids [aa] 1-93) a nd a C-terminal cysteine protease domain (aa 94-217). Given that the latter has been predicted to be membrane-associated, we aimed to identify molecular determinants for membrane association of the NS2 protease domain. Methods: A comprehensive panel of NS2 deletion constructs was analyzed by fluorescence microscopy, selective membrane extraction, and m embrane flotation assays. Candidate aa r esidues involved in membrane association were substituted by site-directed mutagenesis. Results: The NS2 protease domain alone was found to associate with membranes. Two N-terminal α-helices comprising aa 102-114 and aa 123-136 were found to m ediate this a ssociation, w ith c onserved hydrophobic and positively charged aa residues representing the key determinants. I nterestingly, m utagenesis analyses r evealed that electrostatic interactions involving a positively charged aa residue in α-helix aa 123-136 are required for membrane association. Mono- and bicistronic (i.e. NS2 c leavage-independent) HCV constructs were prepared to i nvestigate the effect o f these substitutions on RNA replication and infectious viral particle formation. Conclusions: T he NS2 protease d omain itself harbors m olecular determinants for membrane association within α-helices aa 102-114 and aa 1 23-136 which may contribute to p roper p ositioning of t he active site. These results provide new insights i nto the membrane topology and t he p oorly understood f unction of t his essential viral protease.