Real-time PCR analysis: sequence of the oligonucleotide pairs, annealing temperature (Ta) and product size.

<p>Real-time PCR analysis: sequence of the oligonucleotide pairs, annealing temperature (Ta) and product size.</p

Immunostaining of DDAH-2 in the diencephalon of <i>T. b. b.-</i>infected rats.

<p>(A) Immunohistochemistry for DDAH-2 observed in different thalamic structures: submedius thalamic nucleus (Sub); mediodorsal thalamic nucleus (MD); ventral posterolateral and posteromedial thalamic nucleus (VPL, VPM); laterodorsal thalamic nucleus (LD); paracentral thalamic nucleus (PC); posterior thalamic nuclear group (Po); ventromedial thalamic nucleus (VM); ventrolateral thalamic nucleus (VL); centrolateral thalamic nucleus (CL). (B) Comparison of the DDAH-2 protein expression shown by immunohistochemistry in the ventral posterior nucleus of the thalamus between infected animals and control healthy rats. (C) Immunostaining of DDAH-2 (B-b, d) in the kidney of healthy rats (control of specificity of anti-DDAH-2). The specificity of the secondary antibody was verified by incubating kidney sections without anti-DDAH-2 (B-a, c). Number of animals, n = 3. The microscope used was a Nikon Eclipse E400 equipped with Sony DXC-390P camera (objective: X20, X40). Abbreviations: DDAH-2, N^G, N^G-dimethylarginine dimethylaminohydrolase isoform 2; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016891#pone-0016891-g002" target="_blank">figure 2</a>.</p

Specific parameters used for identification of amino acid in MS/MS positive ion mode.

<p>AA: Amino acid.</p><p>CV, CE correspond to the cone voltage (in V), and collision energy (in eV), respectively.</p><p>AA* corresponds to the stable labelled amino acid (AA) chosen as internal standard for the quantification.</p><p>RT corresponds to the time of retention (in min) of each analyte under selected chromatographic conditions.</p><p>ADMA: asymmetric dimethylarginine. GABA: γ-aminobutyric acid.</p

Relative changes in activity, mRNA and protein expression of DDAH and arginase in the brain during the course of infection in <i>Trypanosoma brucei brucei</i> (<i>T. b. b.</i>) infected rats.

<p>DDAH (A) and arginase (D) activities were measured in diencephalic (hypothalamus/thalamus) biopsies. Results (mean value ± SEM, n = 6 per experimental day) were normalized with corresponding values measured in control rats (n = 6 per experimental day). Control values (100%), delineated by the dotted line, correspond to 269.66±7.09 pmol/min/mg for DDAH and to 26.34±0.40 pmol/min/mg for arginase. Relative intensity of DDAH (B) and arginase (E) bands in western blot was calculated for each band and the results expressed as a mean value for control and different experimental days (β-actin was used as an internal standard). The significant up-regulation taking place for <i>DDAH-2</i> transcription (C) is not observed for <i>DDAH-1</i>(C), <i>ARG-1</i> or <i>ARG-2 (F)</i>. The transcript levels for <i>DDAH-1</i>, <i>DDAH-2</i>, <i>ARG-1 and ARG-2</i> were estimated by real-time quantitative RT-PCR and normalized to 18S rRNA as endogenous control. The graphs represent the mean ± SEM of 4 independent experiments (n = 4 per experimental day). Statistics: ANOVA followed by post-hoc Fisher's test (<i>*p<0.05</i>, **p<0.001 compared to healthy rats). Abbreviations: DDAH, N^G, N^G-dimethylarginine dimethylaminohydrolase; <i>DDAH-1</i>, DDAH isoform 1; <i>DDAH-2</i>, DDAH isoform 2; Ctrl: Control; <i>ARG-1</i>, arginase isoform 1; <i>ARG-2</i>, arginase isoform 2.</p

Schematic representation of the enzymatic pathways involved in the control of trypanosome entry into the brain during the later stage of the disease.

<p>In the periphery (blood), the arginase is strongly expressed in the activated macrophages and competes with iNOS for a common substrate, L-arginine. This phenomenon, under control of cytokines, leads to a decrease in iNOS activity and consequently in the production of NO, a gaseous messenger possessing trypanocidal properties. Such a mechanism, favoring the growth and multiplication of the parasites, is strengthened by the accumulation of the endogenous inhibitor of iNOS, ADMA, as observed in the blood. An opposite mechanism is observed in the brain since the activity of iNOS increases in the three cell types: neurons, microglia and astrocytes. DDAH-2 is over expressed in neurons, likely by decreasing the ADMA pool, and may enhance iNOS activity and, consequently, NO production. Concomitantly, the activity and expression of the arginase remain unchanged. Abbreviations: NO, nitric oxide; iNOS, inducible nitric oxide synthase; ADMA, asymmetric dimethylarginine; DDAH, N^G, N^G-dimethylarginine dimethylaminohydrolase. Small arrows indicate an increase in the given product when pointing upwards and a decrease when pointing downwards. Hyphen is used to indicate that the amount in the given molecule is stable.</p

Immunostaining of DDAH-1 in the diencephalon of <i>T. b. b.-</i>infected rats.

<p>(A) Immunoreactivity of DDAH-1 observed in different hypothalamic and thalamic structures: perifornical nucleus (PeF), magnocellular nucleus of the lateral hypothalamus (MCLH); lateral hypothalamic area (LH); subincertal nucleus (SubI); zona incerta (ZI); reuniens thalamic nucleus (Re); central medial thalamic nucleus (CM); posterior thalamic nuclear group (Po); ventral posterolateral (VPL) and posteromedial (VPM) thalamic nucleus; paracentral thalamic nucleus (PC); laterodorsal thalamic nucleus (LD); and habenular nucleus (Hb). (B) Immunostaining of DDAH-1 (B-b, d) in kidney of healthy rats (control of specificity for anti-DDAH-1). For control for the specificity of the secondary antibody, kidney sections were incubated without anti-DDAH-1 (B-a, c). Number of animals, n = 3. The microscope used was a Nikon Eclipse E400 equipped with Sony DXC-390P camera (objective: X20, X40). Abbreviations: DDAH-1, N^G, N^G-dimethylarginine dimethylaminohydrolase isoform 1; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016891#pone-0016891-g002" target="_blank">figure 2</a>.</p

Changes occurring in the amino acid content in the brain during the course of infection in <i>T. b. b.</i>-infected rats.

<p>The variations of the concentration of amino acids (mean value ± SEM) in the diencephalon (hypothalamus/thalamus) of infected rats were normalized with the corresponding values obtained in healthy control animals. 100% of the control value (delineated by the dotted line) corresponds to: 34±2 µM for L-arginine; 10±1 µM for L-citrulline; 892±36 µM for L-glutamate; 796±80 µM for L-GABA; 16±1 µM for L-proline and 5±0.4 µM for L-ornithine. Concentration of ADMA was undetectable. Statistics: ANOVA followed by post-hoc Fisher's test (<i>*p<0.05</i>, <i>**p<0.001</i> compared to healthy rats); n = 6 animals in each group at the different times post-infection: D5, D10, D16 and D22. Abbreviations: GABA, γ-aminobutyric acid; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016891#pone-0016891-g002" target="_blank">figure 2</a>.</p

Relative changes in the content of amino acid in blood during the course of infection in <i>T. b. b.</i>-infected rats.

<p>The changes in the concentration of amino acids (mean value ± SEM) in the blood of infected rats were normalized with the corresponding values obtained in healthy control animals. 100% of the control values (delineated by the dotted line) correspond to: 110±3 µM for L-arginine; 100±4 µM for L-citrulline; 95±4 µM for L-glutamate and 0.96±0.1 µM for ADMA. Statistics: ANOVA followed by post-hoc Fisher's test (<i>*p<0.05</i>, <i>**p<0.001</i>, <i>***p<0.0001</i> compared to healthy rats); n = 6 animals in each group at the different times post-infection: D5, D10, D16 and D22. Abbreviations: ADMA, asymmetric dimethylarginine; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016891#pone-0016891-g002" target="_blank">figure 2</a>.</p

Simplified scheme of L-arginine pathways in mammalian host cells.

<p>L-arginine is a crossroad for multiple pathways since it is the common substrate of arginase and NOS that catalyzes the production of L-ornithine, L-citrulline and NO, respectively. ADMA, a product of the proteolysis of methylated proteins by PRMT-1, is a potent inhibitor of NOS. Its concentration is regulated by DDAH that catalyzes the production of L-citrulline and dimethylamine. L-ornithine and L-glutamate serve as precursors of polyamines and glutathione. These two metabolites will be further utilized by the trypanosomes for the synthesis of trypanothione, an essential element protecting the parasites from free radicals. Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; ADMA, asymmetric dimethylarginine; DDAH, N^G,N^G-dimethylarginine dimethylaminohydrolase; PRMT-1, Protein arginine N-methyltransferase 1; GABA, γ-aminobutyric acid.</p

Morphometric parameters for differentiation of DDAH-1 and DDAH-2 cell groups in <i>Trypanosoma brucei brucei</i>-infected Wistar rats.

<p>*The values given represent the mean ± standard deviation; a Fisher's post-hoc analysis was achieved.</p><p>FF: The form factor.</p><p>**N.S: Non significant.</p