Correction: Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women.

This corrects the article on p. e73445 in vol. 8.]. This corrects the article "Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Non-Obese Women" , e73445. There was an error in the title of the article. The correct version of the title in the article is: Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women The correct citation is: Martin F-PJ, Montoliu I, Collino S, Scherer M, Guy P, et al. (2013) Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women. PLoS ONE 8(9): e73445. doi:10.1371/journal.pone.0073445

Cytochrome P-450 activities in human and rat brain microsomes.

The role of cytochrome P450 in the metabolism of dextromethorphan, amitriptyline, midazolam, S-mephenytoin, citalopram, fluoxetine and sertraline was investigated in rat and human brain microsomes. Depending on the parameters, the limit of quantification using gas chromatography-mass spectrometry methods was between 1.6 and 20 pmol per incubation, which generally contained 1500 microg protein. Amitriptyline was shown to be demethylated to nortriptyline by both rat and human microsomes. Inhibition studies using ketoconazole, furafylline, sulfaphenazole, omeprazole and quinidine suggested that CYP3A4 is the isoform responsible for this reaction whereas CYP1A2, CYP2C9, CYP2C19 and CYP2D6 do not seem to be involved. This result was confirmed by using a monoclonal antibody against CYP3A4. Dextromethorphan was metabolized to dextrorphan in rat brain microsomes and was inhibited by quinidine and by a polyclonal antibody against CYP2D6. Only the addition of exogenous reductase allowed the measurement of this activity in human brain microsomes. Metabolites of the other substrates could not be detected, possibly due to an insufficiently sensitive method. It is concluded that cytochrome P450 activity in the brain is very low, but that psychotropic drugs could undergo a local cerebral metabolism which could have pharmacological and/or toxicological consequences.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

Alteration of glucose metabolism in cultured astrocytes after AQP9-small interference RNA application.

Aquaglyceroporin-9 (AQP9) facilitates diffusion of water and energy substrates such as glycerol and monocarboxylates. AQP9 is present in plasma membrane and mitochondria of astrocytes and catecholaminergic neurons, suggesting that it plays a role in the energetic status of these cells. Using specific small interference RNA directed against AQP9 in astrocyte cultures, we showed that glycerol uptake is decreased which is associated with an increase in glucose uptake and oxidative metabolism. Our results not only confirm the presence of AQP9 in astrocytes but also suggest that changes in AQP9 expression alter glial energy metabolism.

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

The inflammasome, autoinflammatory diseases, and gout.

IL-1beta is a cytokine with major roles in inflammation and innate immune responses. IL-1beta is produced as an inactive proform that must be cleaved within the cell to generate biologically active IL-1beta. The enzyme caspase-1 catalyzes the reaction. Recent work showed that caspase-1 must be activated by a complex known as the inflammasome. The inflammasome comprises NALP, which is an intracellular receptor involved in innate immunity, and an ASC adapter that ensures caspase-1 recruitment to the receptor. The most extensively described inflammasome to date is formed by the NALP3 receptor within monocytes. Mutations involving the NALP3 gene cause hereditary periodic fever syndromes in humans. Increased inflammasome activity responsible for uncontrolled IL-1beta production occurs in these syndromes. Inhibition of the IL-1beta pathway by IL-1 receptor antagonist (anakinra) is a highly effective treatment for inherited periodic fever syndromes. A major role for inflammasome activity in the development of gout attacks was established recently. Urate monosodium crystals are specifically detected via the NALP3 inflammasome, which results in marked IL-1beta overproduction and initiation of an inflammatory response. This finding opens up new possibilities for the management of gouty attacks.

Engineering polyhydroxyalkanoate content and monomer composition in the oleaginous yeast Yarrowia lipolytica by modifying the ß-oxidation multifunctional protein.

Recombinant strains of the oleaginous yeast Yarrowia lipolytica expressing the PHA synthase gene (PhaC) from Pseudomonas aeruginosa in the peroxisome were found able to produce polyhydroxyalkanoates (PHA). PHA production yield, but not the monomer composition, was dependent on POX genotype (POX genes encoding acyl-CoA oxidases) (Haddouche et al. FEMS Yeast Res 10:917-927, 2010). In this study of variants of the Y. lipolytica β-oxidation multifunctional enzyme, with deletions or inactivations of the R-3-hydroxyacyl-CoA dehydrogenase domain, we were able to produce hetero-polymers (functional MFE enzyme) or homo-polymers (with no 3-hydroxyacyl-CoA dehydrogenase activity) of PHA consisting principally of 3-hydroxyacid monomers (>80%) of the same length as the external fatty acid used for growth. The redirection of fatty acid flux towards β-oxidation, by deletion of the neutral lipid synthesis pathway (mutant strain Q4 devoid of the acyltransferases encoded by the LRO1, DGA1, DGA2 and ARE1 genes), in combination with variant expressing only the enoyl-CoA hydratase 2 domain, led to a significant increase in PHA levels, to 7.3% of cell dry weight. Finally, the presence of shorter monomers (up to 20% of the monomers) in a mutant strain lacking the peroxisomal 3-hydroxyacyl-CoA dehydrogenase domain provided evidence for the occurrence of partial mitochondrial β-oxidation in Y. lipolytica.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Role and regulation of polyphosphate in leishmania parasites

Le protozoaire unicellulaire Leishmania est l'agent responsable de la leishmaniose, une maladie parasitaire humaine qui se manifeste par des lésions de la peau, se résolvant le plus souvent spontanément, jusqu'à des lésions viscérales fatales. Le parasite est transmis de l'insecte à l'hôte mammifère lors d'un repas sanguin de la mouche des sables et y réside respectivement sous formes extra- et intracellulaires. On estime que cette maladie touche environ 12 millions de personnes dans 98 pays. Etant donné que les médicaments disponibles à ce jour sont faiblement efficaces et/ou hautement toxiques, il est indispensable de consolider les connaissances sur le fonctionnement et la survie du parasite pour pouvoir développer de nouvelles stratégies de traitements et de préventions. Tous les organismes vivants, dont Leishmania, contiennent du polyphopshate (polyP). Cette molécule chargée négativement est constituée de trois jusqu'à plusieurs centaines de résidus de phosphates reliées par des liaisons à haute énergie. Le polyP sert donc de source d'énergie et de réservoir de phosphate; dans certaines espèces, il joue aussi un rôle dans l'adaptation au stress et la virulence de pathogènes. Ceci nous a amené à étudier le rôle du polyP dans le parasite Leishmania. L'enzyme responsable de la synthèse de polyP a été identifié récemment dans la levure : il s'agit de la chaperone de transport vacuolaire 4 (Vtc4). Nous avons identifié un homologue de Vtc4 chez les Trypanosomatidae, et avons donc décidé d'examiner sa fonction dans le métabolisme du polyP chez Leishmania. En éliminant l'expression de Vtc4 chez L. major et L. guyanensis, nous avons pu démontrer qu'il est indispensable pour la production de polyP chez Leishmania. De plus, nous avons constaté que ces parasites possèdent des chaînes de polyP allant de trois jusqu'à environ 300 résidus de phosphate. Le taux de polyP dans la cellule est précisément régulé et varie entre un très haut niveau durant la phase proliférative des promastigotes à un niveau bas en phase stationnaire tardive, alors que l'expression de Vtc4p reste stable. Dans les amastigotes intracellulaires, seulement des petites quantités de polyP et de Vtc4p sont détectées. En outre, l'absence de Vc4p et de polyP n'a pas d'effet significatif sur les infections in vivo de souris, ce qui indique que le polyP n'est pas nécessaire au développement de la leishmaniose. Ceci suggère que Vtc4p n 'est pas une bonne cible pour le développement de nouveaux traitements contre Leishmania. "Néanmoins, la présence du polyP favorise fortement la survie du parasite suite à un choc de température (37°C) et aide ainsi à sa persistance intracellulaire pendant les premiers jours d'infection de macrophages. En résumé, nos résultats indiquent que si le polyP a peu d'importance pendant l'infection et le développement de la leishmaniose chez la souris, il est par contre crucial pour l'adaptation à des situations de stress comme l'augmentation de la température. Le fait que le polyP a été conservé dans tous les organismes durant l'évolution suggère toutefois que cette molécule joue un rôle fondamental. Etant donné que l'absence de polyP n'a pas d'effet sur la survie des amastigotes, il pourrait être plus important dans la forme promastigote infectant la mouche des sables. - The unicellular protozoan parasite Leishmania is the causative agent of the human disease leishmaniasis, which can range from self-healing skin lesions to fatal visceral lesions. The parasite is transmitted from the insect vector to the mammalian host when the sand fly takes its blood meal and exists in an extra- and an intracellular form, respectively. The disease is estimated to affect 12 million people in 98 countries and currently available drug treatments are of relatively low potency and/or high toxicity. Thus, investigating parasite survival mechanisms and parasite adaptation to the two host environments contributes to the general understanding of Leishmania propagation and might therefore help to develop future treatments or preventions. All living cells, including Leishmania, contain a negatively charged polymer of a few up to several hundred phosphate residues. These so-called polyphosphates (polyPs) serve as an energy source and phosphate reservoir. In some organisms, polyP is also involved in adaptation to stresses and virulence of pathogens. Therefore we were interested in investigating the importance of polyP in Leishmania parasites. Recently, an eukaryotic enzyme responsible for polyP synthesis has been identified as the vacuolar transporter chaperone 4 (Vtc4) in yeast. We, and others, found a Vtc4 homologue in trypanosomatids and decided to examine its potential function in polyP metabolism. By generating VTC4 knock-out cell lines in L. major and Vtc4 knock-down cell lines in L. guyanensis, we were able to demonstrate that Vtc4p is responsible for the total amount of cellular polyP. We also observed that Leishmania polyP chain length ranges from a few up to around 300 residues and that its level is tightly regulated. PolyP abundance is highest during the logarithmic proliferating phase of promastigotes and decreases in the stationary phase, while Vtc4 protein expression remains stable during both phases. In the intracellular amastigote form, only low amounts of polyP and Vtc4p were detectable. Furthermore, absence of Vtc4p and polyP did not have a significant effect on in vivo mouse infections, indicating that polyP is not necessary for Leishmania disease progression. This suggests that Vtc4p would be a poor drug target against Leishmania infection. However, presence of the polymer strongly supported parasite survival during heat shock (37°C) and thereby promoted intracellular persistence during the first days of macrophage infections. Taken together, we found that polyP has little importance in Leishmania {in vivo) infection but that it plays a crucial role during adaptation to stress, such as heat shock. Given that polyP has been preciously conserved in all organisms during evolution it seems to play a fundamental role. Since absence of polyP does not affect amastigote survival, it might be significant for promastigote existence in the sand fly vector.

Perampanel: a significant liver enzyme inducer in some patients?

Perampanel is one of the latest released antiepileptic drugs (AEDs). Early studies suggest no significant liver enzyme induction from this compound. We report on two patients with medically resistant epilepsy, who had perampanel added to their usual regimen. Both experienced a worsening of their epilepsy and presented in convulsive status epilepticus; concurrent antiepileptic drug levels (phenytoin, phenobarbital, rufinamide) were significantly decreased (<50%) in comparison with levels prior to perampanel introduction. Intravenous load and significant increase of maintenance dosages were needed to restore therapeutic drug levels. In one patient, further increase of perampanel resulted in a new drop of phenytoin level. This suggests that perampanel could, in some subjects, induce liver enzymes and interact with concomitant AEDs; monitoring levels of concomitant compounds could be useful.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an &gt; or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.