Abstract-We investigated whether upregulation of Src by Ang II leads to increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and whether these processes are associated with altered activation of C-terminal Src kinase (Csk), a negative regulator of Src. Furthermore, the role of epidermal growth factor receptor (EGFR) transactivation by angiotensin II (Ang II) was determined. Ang II-mediated c-Src phosphorylation was significantly greater (Ϸ4-fold, PϽ0.01) in SHR than in Wistar-Kyoto rats (WKY). Ang II increased Csk phosphorylation 2-to 3-fold in WKY but not in SHR. Key Words: angiotensin II Ⅲ kinase Ⅲ protein kinases Ⅲ hyperplasia R emodeling of small arteries in essential and experimental hypertension is associated with increased vascular smooth muscle cell (VSMC) growth. Several vasoactive agents and growth factors have been implicated in this process, of which angiotensin II (Ang II) is one of the most important. 1-3 Ang II appears to have direct growth-promoting effects independent of blood pressure changes, 4,5 which may contribute to the vascular remodeling in hypertension. The involvement of Ang II is supported by studies demonstrating that Ang II-stimulated mitogenic actions are enhanced in cultured VSMCs from spontaneously hypertensive rats (SHR) 6,7 and that interruption of the renin-angiotensin system with ACE inhibitors or Ang II type 1 (AT 1 ) receptor blockers improves vascular structural and functional changes in experimental and human hypertension. 8 -12 Signal transduction pathways underlying Ang II-mediated growth actions involve the activation of mitogen-activated protein (MAP) kinases. 3,13 Multiple mammalian MAP kinase pathways have been identified, of which the extracellular signal-regulated kinase (ERK) cascade is the best characterized. 14 Ang II-activated ERK1/2 is responsible for the induction of early growth response genes, including c-fos, c-jun, and c-myc. 15,16 Recent studies have suggested that Ang II-dependent processes are mediated via Ca 2ϩ -sensitive transactivation of the epidermal growth factor receptor (EGFR). [17][18][19] Alterations in MAP kinase signaling may contribute to the pathological cellular processes that are associated with vascular remodeling in hypertension. We have previously shown that Ang II-induced phosphorylation of ERK1/2 is increased and that ERK1/2 activation is essential for the Ang II-stimulated growth of VSMCs in SHR. 3,20,21 Glomerular MAP kinase activity and c-fos gene expression are enhanced in Ang II-induced hypertension, 22 and in SHR, VSMC ERK1/2 phosphorylation is increased. 20,23 Received 24,25 It is also possible that alterations in regulatory tyrosine kinases (such as Src) that are upstream from ERK1/2 could be important. Src kinases are a family of nonreceptor tyrosine kinases, of which the prototype, c-Src, is the major isoform in the vasculature. 26,27 Src is activated by autophosphorylation of Tyr416 and is inactivated by carboxy...

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.

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Background: Full-length dysferlin exceeds adeno-associated virus encapsulation capacity, requiring the generation of mini-dysferlin molecules. Results: By studying the modular dispensability of the dysferlin C2 domains regarding plasmalemmal repair and localization, functional mini-dysferlin constructs were designed. Conclusion: Mini-dysferlin constructs retained a similar capacity for plasmalemmal localization and repair as full-length dysferlin. Significance: These mini-dysferlins can now be used to study their therapeutic effect in animal models of dysferlinopathy.