Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.     

New insights on the proton pump associated with cytochrome b6f turnovers from the study of H/D substitution effects on the electrogenicity and electron transfer reactions

We have studied the effect of protium/deuterium substitution on different kinetics associated with the turnovers of cytochrome b(6)f complex in whole cells of Chlamydomonas reinhardtii. Both the oxidation of cytochrome f and the reduction of hemes b were only little affected by the isotopic substitution. Contrasting with this, the initial slope of the electrogenic phase associated with cytochrome b(6)f turnover was slowed by a factor of 4 by H(2)O/D(2)O substitution. Whereas in the presence of H(2)O the electrogenic phase developed concomitantly with cytochrome b reduction, it lagged for a few hundreds of microseconds after cytochrome b reduction in the presence of D(2)O. We propose that a proton pump is triggered by the oxidation of plastoquinol at the Q(o) site. The proton transfer is specifically delayed upon isotopic substitution, accounting for the lack of significant effect on the electron-transfer reaction as well as for the strong decrease of the initial rate of the electrogenic phase.     

Les inhibiteurs spécifiques de la recapture de la sérotonine dans la dépression de l’enfant et de l’adolescent: revue des controverses récentes

European and US drug regulatory authorities have recently warned against the use of selective serotonin reuptake inhibitors (SSRIs) in childhood and adolescent depression. Through an extensive literature review on the use of SSRIs to treat depression in children and adolescents — including psychiatric clinical trials, pharmacology, and drug safety data — we will: i) describe the main arguments justifying various regulatory decisions; ii) present published data and subsequent meta-analyses; iii) discuss how to integrate the recommendations into daily clinical practice, freeing professionals from a Manichean approach. We reach the limits of evidence-based medicine, which provide the basis of the scientific work of the regulatory authorities.     

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.     

Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM)

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.     

Are Child and Adolescent Responses to Placebo Higher in Major Depression than in Anxiety Disorders? A Systematic Review of Placebo-Controlled Trials

Background

In a previous report, we hypothesized that responses to placebo were high in child and adolescent depression because of specific psychopathological factors associated with youth major depression. The purpose of this study was to compare the placebo response rates in pharmacological trials for major depressive disorder (MDD), obsessive compulsive disorder (OCD) and other anxiety disorders (AD-non-OCD).

Methodology and Principal Findings

We reviewed the literature relevant to the use of psychotropic medication in children and adolescents with internalized disorders, restricting our review to double-blind studies including a placebo arm. Placebo response rates were pooled and compared according to diagnosis (MDD vs. OCD vs. AD-non-OCD), age (adolescent vs. child), and date of publication. From 1972 to 2007, we found 23 trials that evaluated the efficacy of psychotropic medication (mainly non-tricyclic antidepressants) involving youth with MDD, 7 pertaining to youth with OCD, and 10 pertaining to youth with other anxiety disorders (N = 2533 patients in placebo arms). As hypothesized, the placebo response rate was significantly higher in studies on MDD, than in those examining OCD and AD-non-OCD (49.6% [range: 17–90%] vs. 31% [range: 4–41%] vs. 39.6% [range: 9–53], respectively, ANOVA F = 7.1, p = 0.002). Children showed a higher stable placebo response within all three diagnoses than adolescents, though this difference was not significant. Finally, no significant effects were found with respect to the year of publication.

Conclusion

MDD in children and adolescents appears to be more responsive to placebo than other internalized conditions, which highlights differential psychopathology.     

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Summary. Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' Eco RI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).     

Tachykinin regulation of cholinergic transmission in the limbic/prefrontal territory of the rat dorsal striatum: implication of new neurokinine 1-sensitive receptor binding site and interaction with enkephalin/mu opioid receptor transmission

The tachykinin neurokinin 1 receptors (NK₁Rs) regulation of acetylcholine release and its interaction with the enkephalin/mu opioid receptors (MORs) transmission was investigated in the limbic/prefrontal (PF) territory of the dorsal striatum. Using double immunohistochemistry, we first showed that in this territory, cholinergic interneurons contain tachykinin NK₁Rs and co-express MORs in the last part of the light period (afternoon). In slices of the striatal limbic/PF territory, following suppression of the dopaminergic inhibitory control of acetylcholine release, application of the tachykinin NK₁R antagonist, SSR240600, markedly reduced the NMDA-induced acetylcholine release in the morning but not in the afternoon when the enkephalin/MOR regulation is operational. In the afternoon, the NK₁R antagonist response required the suppression of the enkephalin/MOR inhibitory control of acetylcholine release by βfunaltrexamine. The pharmacological profile of the tachykinin NK₁R regulation tested by application of the receptor agonists [[Pro⁹]substance P, neurokinin A, neuropeptide K, and substance P(6–11)] and antagonists (SSR240600, GR205171, GR82334, and RP67580) indicated that the subtype of tachykinin NK₁R implicated are the new NK₁-sensitive receptor binding site. Therefore, in the limbic/PF territory of the dorsal striatum, endogenous tachykinin facilitates acetylcholine release via a tachykinin NK₁R subtype. In the afternoon, the tachykinin/NK₁R and the enkephalin/MOR transmissions interact to control cholinergic transmission.