Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class

Agam (Anopheles gambiae) relies on its olfactory system to target human prey, leading eventually to the injection of Plasmodium falciparum, the malaria vector. OBPs (odorant-binding proteins) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. In the present study, we undertook a large-scale analysis of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the three-dimensional structures of several Agam OBPs, either alone or in complex with model compounds. In the present paper, we report the first three-dimensional structure of a member of the C-plus class of OBPs, AgamOBP47, which has a longer sequence than classical OBPs and contains six disulfide bridges. AgamOBP47 possesses a core of six α-helices and three disulfide bridges, similar to the classical OBP fold. Two extra loops and the N- and C-terminal extra segments contain two additional α-helices and are held in conformation by three disulfide bridges. They are located either side of the classical OBP core domain. The binding site of OBP47 is located between the core and the additional domains. Two crevices are observed on opposite sides of OBP47, which are joined together by a shallow channel of sufficient size to accommodate a model of the best-tested ligand. The binding sites of C-plus class OBPs therefore exhibit different characteristics, as compared with classical OBPs, which should lead to markedly diverse functional implications.     

The Increased Risk of Road Crashes in Attention Deficit Hyperactivity Disorder (ADHD) Adult Drivers: Driven by Distraction? Results from a Responsibility Case-Control Study

Background and Objective

Both distractions (external and internal) and attention-deficit/hyperactivity disorder (ADHD) are serious risk factors for traffic crashes and injuries. However, it is still unknown if ADHD (a chronic condition) modifies the effect of distractions (irregular hazards) on traffic crashes. The objective of this study was to assess the effects of distractions and ADHD on traffic crash responsibility.

Methods

A responsibility case-control study was conducted in the adult emergency department of Bordeaux University Hospital, France. Subjects were recruited among drivers injured in a motor vehicle crash between April 2010 and August 2011. Responsibility levels were estimated using a standardized method. Frequencies of exposures were compared between drivers responsible and drivers not responsible for the crash. Independent risk factors were identified using a multivariate logistic regression including test interactions between distractions and ADHD.

Results

A total of 777 subjects were included in the analysis. Factors associated with responsibility were distraction induced by an external event (adjusted OR (aOR)  = 1.47; 95% confidence interval (CI) [1.06–2.05]), distraction induced by an internal thought (aOR = 2.38; CI: [1.50–3.77]) and ADHD (aOR = 2.18 CI: [1.22–3.88]). The combined effect of ADHD and external distractions was strongly associated with responsibility for the crash (aOR = 5.79 CI: [2.06–16.32]). Interaction assessment showed that the attributable proportion due to the interaction among participants with both exposures was 68%.

Discussion

Adults with ADHD are a population at higher risk of being responsible for a road traffic crash when exposed to external distractions. This result reinforces the need to diagnose adult ADHD and to include road safety awareness messages delivered by the physician. Developing advanced driver assistance systems devoted to the management of attention lapses is also increasingly relevant for these drivers.     

Preferential Transfer of 2-Docosahexaenoyl-1-Lysophosphatidylcholine Through an In Vitro Blood-Brain Barrier Over Unesterified Docosahexaenoic Acid

Abstract : The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an in vitro model of the blood-brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC-DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC-DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC-DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC-DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood-brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC-DHA.     

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.     

Rapid evolution of goat and sheep globin genes following gene duplication

Statistical analyses of DNA sequences of globin genes (beta A, beta C, and gamma) from goat and sheep (including new sequence information for the second intron of sheep beta A and gamma, kindly provided by A. Davis and A. W. Nienhuis) indicate that the rates of nonsynonymous substitution in these genes have been greatly accelerated following the gene duplication separating gamma and the ancestor of beta A and beta C and the gene duplication separating beta A and beta C. In both cases the acceleration was apparently due to relaxation of purifying selection (functional constraints) rather than advantageous mutations because acceleration occurred only in less important parts of the beta globin chain. The rates of nonsynonymous substitution in these genes are estimated to be about 2.3 x 10(-9) per site per year, which is three times higher than that for the divergence between human beta and mouse beta major globin genes. Our analyses further suggest that the rate of synonymous substitution in functional genes and the rate of substitution in pseudogenes are approximately equal and are between 2.8 x 10(-9) and 5.0 x 10(-9) and that the rate of substitution in introns is about 3.0 x 10(-9). The divergence time between beta A and beta C and that between gamma and the beta A-beta C pair are about 12 and 30 million years, respectively. The proportion of transition mutations is estimated to be 64%, two times higher than expected under random mutation but considerably lower than the 96% estimated for animal mitochondrial DNA.      

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Holes photogeneration in phthalocyanine-doped polysilane

Polysilane polymers are attractive photoconductive materials, due to the high mobility of the charge carriers (holes). The photoconductivity in the visible region is greatly enhanced by the addition of a phthalocyanine, because the dopant increases the absorption of light and the quantum yield of the holes photogeneration. The quantum yield has been measured by the technique of xerographic discharge at several values of the polarisation field. From these data the intrinsic quantum yield ₀ and the thermalisation distance r ₀ were calculated. r ₀ is similar to the value measured in the trinitrofluorenone doped polyvinylcarbazole system (27.5 ) while ₀ is much lower (2.8·10^–2 compared to 0.11). In fact, r ₀ is assumed to be mainly dependent on the polymeric environment, while ₀ depends on the nature of the coupling between the dopant and the polymer.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.