| 1. |
- Utsugisawa, K, et al.
(författare)
-
Candesartan prevents angiotensin II-induced facilitation of hypoxic neuronal damage through PKC delta inhibition
- 2005
-
Ingår i: Brain Research. Molecular Brain Research. - : Elsevier BV. - 0169-328X. ; 135:1-2, s. 134-140
-
Tidskriftsartikel (refereegranskat)abstract
- To investigate the role of protein kinase C delta (PKC delta) in angiotensin II-induced facilitation mechanisms of hypoxic neuronal damage and whether candesartan, an AT1 receptor antagonist, can suppress these mechanisms, we performed in vitro experiments which were free from vascular components using PC12 cells under hypoxic (12 h)/reoxygenation (0-48 h) conditions. Angiotensin II apparently increased the basal expression level of PKC delta phosphorylated at Ser(643) before hypoxia, promoted the cleavage of PKC delta to its catalytic fragment, and fostered the progression of DNA fragmentation after hypoxia. Candesartan inhibited both phosphorylation and cleavage of PKC delta and suppressed the angiotensin II-induced facilitation of DNA fragmentation under hypoxic/reoxygenation conditions. However, PD123319, an AT2 receptor antagonist, influenced neither PKC delta nor the angiotensin II-induced facilitation of DNA fragmentation. Furthermore, in PC12 cells expressing the ATP-binding mutant of PKC delta (PKC delta(K376R)) acting as a dominant-negative protein, both phosphorylation and cleavage of PKC delta were attenuated and DNA fragmentation was markedly suppressed regardless of the presence of angiotensin II. These findings suggest that angiotensin II-induced facilitation of DNA fragmentation under hypoxic conditions is mediated by PKC delta, and the mechanisms can be suppressed by the candesartan mediated blockade of the AT1 receptor.
|
|
| 2. |
- Blank Savukinas, Ulrika, et al.
(författare)
-
Smad7 promotes self-renewal of hematopoietic stem cells in vivo.
- 2006
-
Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 108:13, s. 4246-4254
-
Tidskriftsartikel (refereegranskat)abstract
- The Smad-signaling pathway downstream of the transforming growth factor–beta superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.
|
|
| 3. |
- Flygare, Johan, et al.
(författare)
-
Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia
- 2005
-
Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 105:12, s. 4627-4634
-
Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34+ umbilical cord blood (CB) and bone marrow (BM) cells with 3 lentiviral vectors expressing small interfering RNA (siRNA) against RPS19 and 1 scrambled control vector. All vectors also express green fluorescent protein (GFP). Transduction with the siRNA vectors reduced RPS19 mRNA levels to various degrees, which resulted in erythroid defects, correlating to the degree of RPS19 down-regulation, and was rescued by expression of an siRNA-resistant RPS19 transcript. Erythroid colony formation capacity conjointly decreased with RPS19 levels in CD34+ CB and BM cells. In liquid culture supporting erythroid differentiation, RPS19-silenced as well as DBA patient CD34+ cells exhibited reduced proliferative capacity and impaired erythroid differentiation resulting in fewer erythroid colony-forming units (CFU-Es). When assaying myeloid development, a less pronounced influence on proliferation was seen. This study shows for the first time that RPS19 silencing decreases the proliferative capacity of hematopoietic progenitors and leads to a defect in erythroid development.
|
|
| 4. |
- Matsson, H, et al.
(författare)
-
Erythropoiesis in the Rps19 disrupted mouse: Analysis of erythropoietin response and biochemical markers for Diamond-Blackfan anemia.
- 2006
-
Ingår i: Blood Cells Mol Dis. - : Elsevier BV. ; 36:2, s. 259-264
-
Tidskriftsartikel (refereegranskat)abstract
- The human ribosomal protein S19 gene (RPS19) is mutated in approximately 20% of patients with Diamond-Blackfan anemia (DBA), a congenital disease with a specific defect in erythropoiesis. The clinical expression of DBA is highly variable, and subclinical phenotypes may be revealed by elevated erythrocyte deaminase (eADA) activity only. In mice, complete loss of Rps19 results in early embryonic lethality whereas Rps19+/− mice are viable and without major abnormalities including the hematopoietic system. We have performed a detailed analysis of the Rps19+/− mice. We estimated the Rps19 levels in hematopoietic tissues and we analyzed erythrocyte deaminase activity and globin isoforms which are used as markers for DBA. The effect of a disrupted Rps19 allele on a different genetic background was investigated as well as the response to erythropoietin (EPO). From our results, we argue that the loss of one Rps19 allele in mice is fully compensated for at the transcriptional level with preservation of erythropoiesis.
|
|
| 5. |
|
|
| 6. |
- Miyake, Koichi, et al.
(författare)
-
RPS19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia
- 2008
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 26:2, s. 323-329
-
Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed the effects of RPS19 deficiency on EPO induced signal transduction, cell cycle, and apoptosis in RPS19-deficient TF-1 cells. We did not find any abnormality in EPO induced signal transduction. However, RPS19 deficient-TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin-V analysis also increased compared to control cells (13% vs 3.1%, p<0.05). Western blot analysis of apoptotic related proteins showed that the level of bcl-2 and Bad was decreased and Bax was increased in RPS19-deficient TF1 cells. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generated a higher number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). Finally, we show that while RPS19 silencing reduces EPO induced development of erythroid progenitors expressing Glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.
|
|
| 7. |
|
|
