Journal: PLoS ONE

Article Title: Cerebral Changes Occurring in Arginase and Dimethylarginine Dimethylaminohydrolase (DDAH) in a Rat Model of Sleeping Sickness

doi: 10.1371/journal.pone.0016891

Figure Lengend Snippet: 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 DDAH-2 transcription (C) is not observed for DDAH-1 (C), ARG-1 or ARG-2 (F) . The transcript levels for DDAH-1 , DDAH-2 , ARG-1 and ARG-2 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 ( *p<0.05 , **p<0.001 compared to healthy rats). Abbreviations: DDAH, N G , N G -dimethylarginine dimethylaminohydrolase; DDAH-1 , DDAH isoform 1; DDAH-2 , DDAH isoform 2; Ctrl: Control; ARG-1 , arginase isoform 1; ARG-2 , arginase isoform 2.

Article Snippet: Rabbit polyclonal anti-DDAH-1 antibody (1/500, Orbigen, San Diego, USA) and goat polyclonal anti-DDAH-2 antibody (1/500, Abcam, Paris, France) were used as primary antibody (2×24 h at 4°C).

Techniques: Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Cerebral Changes Occurring in Arginase and Dimethylarginine Dimethylaminohydrolase (DDAH) in a Rat Model of Sleeping Sickness

doi: 10.1371/journal.pone.0016891

Figure Lengend Snippet: (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 .

Article Snippet: Rabbit polyclonal anti-DDAH-1 antibody (1/500, Orbigen, San Diego, USA) and goat polyclonal anti-DDAH-2 antibody (1/500, Abcam, Paris, France) were used as primary antibody (2×24 h at 4°C).

Techniques: Immunohistochemistry, Expressing, Infection, Immunostaining, Microscopy

Journal: PLoS ONE

Article Title: Cerebral Changes Occurring in Arginase and Dimethylarginine Dimethylaminohydrolase (DDAH) in a Rat Model of Sleeping Sickness

doi: 10.1371/journal.pone.0016891

Figure Lengend Snippet: 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.

Article Snippet: Rabbit polyclonal anti-DDAH-1 antibody (1/500, Orbigen, San Diego, USA) and goat polyclonal anti-DDAH-2 antibody (1/500, Abcam, Paris, France) were used as primary antibody (2×24 h at 4°C).

Techniques: Activity Assay, Expressing