Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

Inhibition of NTPDase, 5′-nucleotidase, Na/K-ATPase and acetylcholinesterase activities by subchronic treatment with Casearia sylvestris

The aqueous extract of Casearia sylvestris was tested in cortical membrane preparations. C. sylvestris was obtained commercially from two different sources, designated as Sample A and Sample B. The enzymes studied in this work were NTPDase-like, 5'-Nucleotidase, Na(+)/K(+)-ATPase and acetylcholinesterase (AChE). Adult rats received aqueous extracts from C. sylvestris in a dose of 20mg/kg body wt. daily for a 75-day-period, by oral administration (gavage). Our study showed that this treatment caused an inhibition of NTPDase-like activity with both, ATP (19.41% with Sample A and 25.03% with Sample B) and ADP (41.57% with Sample A and 31.20% with Sample B) as substrates. This treatment also caused an inhibition of 5'-nucleotidase activity (28.34% with Sample A and 31.46% with Sample B) and Na(+)/K(+)-ATPase (25.08% with Sample A and 24.81% with Sample B). The rate of acetylcholine degradation was reduced, as shown by the inhibition of AChE (31.65% and 26.74%, Samples A and B, respectively). These results suggest that extracts of C. sylvestris can cause neurochemical alterations in the purinergic and cholinergic systems of the central nervous system.     

Reconstruction of the Evolutionary Dynamics of A(H3N2) Influenza Viruses Circulating in Italy from 2004 to 2012

Background

Influenza A viruses are characterised by their rapid evolution, and the appearance of point mutations in the viral hemagglutinin (HA) domain causes seasonal epidemics. The A(H3N2) virus has higher mutation rate than the A(H1N1) virus. The aim of this study was to reconstruct the evolutionary dynamics of the A(H3N2) viruses circulating in Italy between 2004 and 2012 in the light of the forces driving viral evolution.

Methods

Phylodinamic analyses were made using a Bayesian method, and codon-specific positive selection acting on the HA coding sequence was evaluated.

Results

Global and local phylogenetic analyses showed that the Italian strains collected between 2004 and 2012 grouped into five significant Italian clades that included viral sequences circulating in different epidemic seasons. The time of the most recent common ancestor (tMRCA) of the tree root was between May and December 2003. The tMRCA estimates of the major clades suggest that the origin of a new viral strain precedes the effective circulation of the strain in the Italian population by 6–31 months, thus supporting a central role of global migration in seeding the epidemics in Italy. The study of selection pressure showed that four codons were under positive selection, three of which were located in antigenic sites. Analysis of population dynamics showed the alternation of periods of exponential growth followed by a decrease in the effective number of infections corresponding to epidemic and inter-epidemic seasons.

Conclusions

Our analyses suggest that a complex interaction between the immune status of the population, migrations, and a few selective sweeps drive the influenza A(H3N2) virus evolution. Our findings suggest the possibility of the year-round survival of local strains even in temperate zones, a hypothesis that warrants further investigation.     

Multimodal Magnetic Resonance Imaging Study of Treatment-Na?ve Adults with Attention-Deficit/Hyperactivity Disorder

Background

Attention-Deficit/Hiperactivity Disorder (ADHD) is a prevalent disorder, but its neuroanatomical circuitry is still relatively understudied, especially in the adult population. The few morphometric magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) studies available to date have found heterogeneous results. This may be at least partly attributable to some well-known technical limitations of the conventional voxel-based methods usually employed to analyze such neuroimaging data. Moreover, there is a great paucity of imaging studies of adult ADHD to date that have excluded patients with history of use of stimulant medication.

Methods

A newly validated method named optimally-discriminative voxel-based analysis (ODVBA) was applied to multimodal (structural and DTI) MRI data acquired from 22 treatment-naïve ADHD adults and 19 age- and gender-matched healthy controls (HC).

Results

Regarding DTI data, we found higher fractional anisotropy in ADHD relative to HC encompassing the white matter (WM) of the bilateral superior frontal gyrus, right middle frontal left gyrus, left postcentral gyrus, bilateral cingulate gyrus, bilateral middle temporal gyrus and right superior temporal gyrus; reductions in trace (a measure of diffusivity) in ADHD relative to HC were also found in fronto-striatal-parieto-occipital circuits, including the right superior frontal gyrus and bilateral middle frontal gyrus, right precentral gyrus, left middle occipital gyrus and bilateral cingulate gyrus, as well as the left body and right splenium of the corpus callosum, right superior corona radiata, and right superior longitudinal and fronto-occipital fasciculi. Volumetric abnormalities in ADHD subjects were found only at a trend level of significance, including reduced gray matter (GM) in the right angular gyrus, and increased GM in the right supplementary motor area and superior frontal gyrus.

Conclusions

Our results suggest that adult ADHD is associated with neuroanatomical abnormalities mainly affecting the WM microstructure in fronto-parieto-temporal circuits that have been implicated in cognitive, emotional and visuomotor processes.     

HDL Size is More Accurate than HDL Cholesterol to Predict Carotid Subclinical Atherosclerosis in Individuals Classified as Low Cardiovascular Risk

Background

Misclassification of patients as low cardiovascular risk (LCR) remains a major concern and challenges the efficacy of traditional risk markers. Due to its strong association with cholesterol acceptor capacity, high-density lipoprotein (HDL) size has been appointed as a potential risk marker. Hence, we investigate whether HDL size improves the predictive value of HDL-cholesterol in the identification of carotid atherosclerotic burden in individuals stratified to be at LCR.

Methods and Findings

284 individuals (40–75 years) classified as LCR by the current US guidelines were selected in a three-step procedure from primary care centers of the cities of Campinas and Americana, SP, Brazil. Apolipoprotein B-containing lipoproteins were precipitated by polyethylene glycol and HDL size was measured by dynamic light scattering (DLS) technique. Participants were classified in tertiles of HDL size (<7.57; 7.57–8.22; >8.22 nm). Carotid intima-media thickness (cIMT) <0.90 mm (80^th percentile) was determined by high resolution ultrasonography and multivariate ordinal regression models were used to assess the association between cIMT across HDL size and levels of lipid parameters. HDL-cholesterol was not associated with cIMT. In contrast, HDL size >8.22 nm was independently associated with low cIMT in either unadjusted and adjusted models for age, gender and Homeostasis Model Assessment 2 index for insulin sensitivity, ethnicity and body mass index (Odds ratio 0.23; 95% confidence interval 0.07–0.74, p = 0.013).

Conclusion

The mean HDL size estimated with DLS constitutes a better predictor for subclinical carotid atherosclerosis than the conventional measurements of plasma HDL-cholesterol in individuals classified as LCR.     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.