Migration education and literacy in Brazil with special reference to the North East and Sao Paulo

<p>Cells were cultured, incubated with NECA and assayed for mRNA expression by quantitative realtime PCR as described in Materials and Methods. Data given in A and B are means of five replica culture dishes ±SEM. Two repetitions of the experiment gave similar results. Data given in C are means of 3 different experiments done in four replica culture dishes. *p<0.05, ** p<0.01, *** p<0.001, student’s t-test, two-tailed.</p

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

<div><p>The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.</p></div

Concentration-response relationship (A) and time course (B) of the effect of NECA on expression of RGS2 protein in cultured rat astrocytes.

<p>Cells were cultured, incubated with NECA (concentrations as indicated in A; 1 μM in B) and assayed for protein expression by western blotting as described in Materials and Methods. Shown are the results of one typical experiment, one repetition gave similar results.</p

Effects of adenosine agonists and the adenosine A2A and A2B receptor antagonists, MRS1706 (500nM) and ZM241385 (1μM), on the expression of mRNA of RGS2 (A,B,C), RGS3 (D,E,F) and RGS4 (G) in rat astrocytes.

<p>Cells were cultured, incubated with agonists/antagonists and assayed for mRNA expression by quantitative realtime PCR as described in Materials and Methods. Data given are means of 4 (NECA, CGS21680), 3 (CPA, IB-MECA, CGS21680/ZM241385) or 5 (NECA/MRS1706) experiments each done with 4 replica culture dishes. * p<0.05, ** p<0.01, *** p<0.001, student’s t-test, two-tailed.</p

Effects of adenosine agonists and the adenosine A2B receptor antagonist MRS1706 (500 nM) on the expression of mRNA of RGS2 (A,B), RGS3 (C,D) and RGS4 (E,F) in human astrocytoma cells U373 MG.

<p>Cells were cultured, incubated with agonists/antagonists and assayed for mRNA expression by quantitative realtime PCR as described in Materials and Methods. Data given are means of 5 experiments (NECA, NECA/MRS1706) or 2 experiments (CPA, IB-MECA, CGS21680) each done in five replica culture dishes.* p<0.05, ** p<0.01, *** p<0.001, student’s t-test, two-tailed.</p

Diazoxide increases Nrf2 nuclear translocation in NSC-34 motoneurons and prevents endogenous oxidative damage.

<p>Western blot showed an increase of Nrf2 signaling in the nuclear extracts from NSC-34 neurons treated with different doses of diazoxide for 24 h. The higher increase of Nrf2 was found at lower doses (10 and 1 µM) (A). Cell viability of NSC-34 cells was measured after 24 h AAPH oxidative stress activation and results demonstrated that diazoxide treatment effectively ameliorates cell viability at low doses (B). Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05, ** p<0.01. Scale bar  =  30 µm</p

Diazoxide effects in NMDA-induced neurodegeneration in hippocampal slice culture.

<p>At 7 DIV, treatment with 10 µM NMDA for 4 h induced a region-specific increase neuronal cell death of the CA1 layer compared to undamaged slices, as determined as PI uptake (PI, red) in colocalization with the neuronal nuclear marker NeuN (green) (A, B). Treatment 30 min before NMDA lesion with diazoxide prevented cell death, significantly at 100 µM but also at lowest dose 1 µM (C, D). Upon quantification, results showed a significant decrease at 100 µM (65±5% in PI uptake compared to control) and 1 µM (67±7% in PI uptake compared to control (E). When microglia was removed from the slices, cell protection remained at the lowest dose 1 µM (62±10% in PI uptake compared to control) (F-J). Data are a summary of four to seven individual experiments with 6 slices per conditions analyzed in each experiment. Results are shown as mean ± SEM. * p<0.05, ** p<0.01. Scale bar 300 µm</p

Diazoxide prevents neuronal damage after different neurotoxic insults.

<p>Representative images of differentiated NSC-34 motoneuron cells line after glutamate damage and diazoxide treatment (A). Diazoxide inhibited glutamate mediated cell death by 24 h in differentiated NSC-34 motoneuron cell line (B). Diazoxide inhibited H₂O₂ mediated cell death by 24 h (C) and also when used as pretreatment during 2 h (survival analysis performed 24 h after toxic insult) (D). Diazoxide inhibited inflammatory BV2 microglial mediated cell death by 24 h in NSC-34 motoneuron. Conditioned Medium (CM): Neurons damaged with activated BV2 medium. CM (Dxz to NSC-34): Neurons treated with diazoxide and damaged with activated BV2 medium afterwards. CM (Dzx to BV2): Neurons damaged with diazoxide treated BV2 activated medium (E). Diazoxide treatment 100 µM. Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05. Scale bar =  30 µm</p

Additional file 1: Table S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Primer sequences which were used for RT-PCR. (DOC 40 kb

Additional file 3: Figure S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb