Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.
1 Release of the tachykinin, substance P, from the peripheral terminals of polymodal afferent C-fibres is thought to be largely responsible for the vasodilatation and plasma protein extravasation described as neurogenic inflammation. The effects of CP-96,345, a non-peptide antagonist at the substance P (NK1) receptor, on these vascular reactions were investigated in the rat. 2 Intravenously (i.v.) injected CP-96,345 (0.4-3.Opmolkg-') prevented the drop in blood pressure, a measure of the peripheral vasodilatation, evoked by substance P and neurokinin A in a dose-and timedependent manner, but did not affect that elicited by the non-tachykinin peptides calcitonin gene-related peptide and vasoactive intestinal polypeptide. 3 Plasma protein extravasation evoked by i.a. infusion of substance P. antidromic stimulation of the saphenous or the vagus nerve, and stimulation of cutaneous afferent nerves with mustard oil, were each significantly inhibited by CP-96,345 (3.0-9.Opmol kg-i.v.). Furthermore, CP-96,345 was orally active in blocking mustard oil-induced plasma extravasation with an ED50 of 10,umol kg-'. 4 The inhibition of substance P-induced vasodilatation and of neurogenic plasma extravasation by CP-96,345 was stereospecific as the inactive isomer CP-96,344 (2R, 3R enantiomer of CP-96,345) had no effect. 5 Thus CP-96,345 is a specific, highly potent, long-acting and orally active inhibitor of tachykininmediated neurogenic inflammation.
Adenosine is an endogenous neuromodulator that when produced in the central and the peripheral nervous systems has anticonvulsant, anti-inflammatory, and analgesic properties. However, efforts to use adenosine receptor agonists are plagued by dose-limiting cardiovascular side effects. As an alternative, we explored the use of adenosine kinase inhibitors (AKIs) as potential antiseizure agents and demonstrated an adenosine receptor mediated therapeutic effect in the absence of overt cardiovascular side effects. These activities were associated with elevation of extracellular adenosine concentrations due to inhibition of AK in a site and event specific manner. Several tubercidin based AKIs, including the ribo- and lyxo-furanosyltubercidin analogues as well as the newly discovered erythro-furanosyltubercidin analogues, designed to prevent 5'-O-phosphorylation and associated toxicities, were tested for their analgesic activity in the rat formalin paw model. Described herein are the synthesis, enzyme inhibition structure-activity relationships (SARs) of erythro-furanosyltubercidin analogues, and SARs of analgesic activity of various classes of AKIs. Also reported is the characterization of a lead AKI, 19d (GP3966), an orally bioavailable compound (F% = 60% in dog) which exhibits broad-spectrum analgesic activities (ED50 < or = 4 mg/kg, per os) that are reversible with an adenosine receptor antagonist (theophylline).
Background: Chondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9), but molecular mechanisms underlying this activity remain unclear. Here we report that coactivatorassociated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9.
4-(Phenylamino)-5-phenyl-7-(5-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine 1 and related compounds known as "diaryltubercidin" analogues are potent inhibitors of adenosine kinase (AK) and are orally active in animal models of pain such as the rat formalin paw model (GP3269 ED50= 6.4 mg/kg). However, the utility of this compound class is limited by poor water solubility that can be attributed to the high energy of crystallization caused by stacking of the parallel C4 and C5 aryl rings in the solid state (compound 1 and GP3269 each with pH 7.4 solubility <0.05 microg/mL). To increase water solubility, the hydrophobic C4-phenylamino substituent was replaced with a more hydrophilic group, glycinamide. This modification resulted in improved water solubility while retaining AK inhibition potency. Analogues were studied where changes in the glycinamide moiety were combined with changes to the base and sugar. A lead compound, 4-N-(N-cyclopropylcarbamoylmethyl)amino-5-phenyl-7-(5-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (16c) (IC50= 3 nM and water solubility = 32 +/- 9 microg/mL at pH 7.4), was further characterized in biological assays. Compound 16c exhibited strong oral efficacy in the rat formalin paw model (ED50 of 2.5 mg/kg). In the most advanced assay, 16c was found to inhibit bradykinin-induced licking in marmoset monkeys with an ED50 estimated at 0.9 mg/kg without producing evidence of side effects such as ataxia, sedation, and emesis at this dose. However, lethal toxicity in the rat formalin paw model occurred with high doses of 16c, and further work on this series was discontinued.
Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery.
Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time‐lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62‐knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation‐mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.
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