Protein N -glycosylation in the endoplasmic reticulum (ER) and in the Golgi apparatus is an essential process in eukaryotic cells. Although the N -glycosylation pathway in the ER has been shown to regulate protein quality control, salt tolerance, and cellulose biosynthesis in plants, no biological roles have been linked functionally to N -glycan modifications that occur in the Golgi apparatus. Herein, we provide evidence that mutants defective in N -glycan maturation, such as complex glycan 1 ( cgl1 ), are more salt-sensitive than wild type. Salt stress caused growth inhibition, aberrant root-tip morphology, and callose accumulation in cgl1 , which were also observed in an ER oligosaccharyltransferase mutant, staurosporin and temperature sensitive 3a ( stt3a ). Unlike stt3a , cgl1 did not cause constitutive activation of the unfolded protein response. Instead, aberrant modification of the plasma membrane glycoprotein KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/RSW2) that is necessary for cellulose biosynthesis occurred in cgl1 and stt3a . Genetic analyses identified specific interactions among rsw2 , stt3a , and cgl1 mutations, indicating that the function of KOR1/RSW2 protein depends on complex N -glycans. Furthermore, cellulose deficient rsw1-1 and rsw2-1 plants were also salt-sensitive. These results establish that plant protein N -glycosylation functions beyond protein folding in the ER and is necessary for sufficient cell-wall formation under salt stress.

More than 20 genes in the Arabidopsis genome encode proteins similar to phosphatases that act on the carboxyl-terminal domain (CTD) of RNA polymerase II. One of these CTD-phosphatase-like (CPL) proteins, CPL2, dephosphorylates CTD-Ser5-PO4 in an intact RNA polymerase II complex and contains a double-stranded (ds)-RNA-binding motif (DRM). Although the dsRNA-binding activity of CPL2 DRM has not been shown to date, T-DNA insertion mutants that express CPL2 variants lacking either a part of DRM (cpl2-1) or the entire DRM (cpl2-2) exhibited leaf expansion defects, early flowering, low fertility, and increased salt sensitivity. cpl2 mutant plants produced shorter hypocotyls than wild-type plants in the light, but were indistinguishable from wild type in the dark. CPL2 was expressed in shoot and root meristems and vasculatures, expanding rosette leaves, and floral organs suggesting a focal role for growth. Microarray and RT-PCR analyses revealed that basal levels of several auxin-responsive transcripts were reduced in cpl2. On the other hand, the levels of endogenous auxin and its conjugates were similar in wild type and cpl2. Overexpression of ARF5 but not all activator ARF transcription factors restored the auxin-responsive DR5-GUS reporter gene expression and the leaf expansion of cpl2 mutant plants but not early flowering phenotype. These results establish CPL2 as a multifunctional regulator that modulates plant growth, stress, and auxin responses.

Age-associated loss of muscle mass and function is a major cause of morbidity and mortality in the elderly adults. Muscular atrophy can also be induced by disuse associated with long-term bed rest or disease. Although miRNAs regulate muscle growth, regeneration, and aging, their potential role in acute muscle atrophy is poorly understood. Furthermore, alterations in circulating miRNA levels have been shown to occur during aging but their potential as noninvasive biomarkers for muscle atrophy remains largely unexplored. Here, we report comprehensive miRNA expression profiles by miRNA-seq analysis in tibialis anterior muscle and serum of a disuse-induced atrophy mouse model, mimicking the acute atrophy following long-term bed rest, as compared to those of young and old mice. Comparative analysis and validation studies have revealed that miR-455-3p was significantly decreased in muscle of both induced-atrophy model and old mice, whereas miR-434-3p was decreased in both serum and muscle of old mice, as compared to young mice. Furthermore, upregulation of miR-455-3p in fully differentiated C2C12 myoblasts induced a hypertrophic phenotype. These results suggest that deregulation of miR-455-3p may play a functional role in muscle atrophy and miR-434-3p could be a candidate serum biomarker of muscle aging.

Exercise has positive effects on health and improves a variety of disease conditions. An in vitro model of exercise has been developed to better understand its molecular mechanisms. While various conditions have been used to mimic in vivo exercise, no specific conditions have matched a specific type of in vivo exercise. Here, we screened various electrical pulse stimulation (EPS) conditions and compared the molecular events under each condition in myotube culture with that obtained under voluntary wheel running (VWR), a mild endurance exercise, in mice. Both EPS and VWR upregulated the mRNA levels of genes involved in the slow-type twitch ( Myh7 and Myh2) and myogenesis ( Myod and Myog) and increased the protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α, which is involved in mitochondrial biogenesis. These changes were accompanied by activation of p38 and AMPK. However, neither condition induced the expression of muscle-specific E3 ligases such as MAFbx and MuRF1. Both EPS and VWR consistently induced antioxidant genes such as Sod3 and Gpx4 but did not cause similar changes in the expression levels of the calcium channel/pump-related genes Ryr and Serca. Furthermore, both EPS and VWR reduced glycogen levels but not lactate levels as assessed in post-EPS culture medium and post-VWR serum, respectively. Thus we identified an in vitro EPS condition that effectively mimics VWR in mice, which can facilitate further studies of the detailed molecular mechanisms of endurance exercise in the absence of interference from multiple tissues and organs. NEW & NOTEWORTHY This study establishes an optimal condition for electrical pulse stimulation (EPS) in myotubes that shows a similar molecular signature as voluntary wheel running. The specific EPS condition 1) upregulates the mRNA of slow-twitch muscle components and myogenic transcription factors, 2) induces antioxidant genes without any muscle damage, and 3) promotes peroxisome proliferator-activated receptor-γ coactivator-1α and its upstream regulators involved in mitochondrial biogenesis.

The blood exhibits a dynamic flux of proteins that are secreted by the tissues and cells of the body. To identify novel aging-related circulating proteins, we compared the plasma proteomic profiles of young and old mice using tandem mass spectrometry. The expression of 134 proteins differed between young and old mice. We selected seven proteins that were expressed at higher levels in young mice, and confirmed their plasma expression in immunoassays. The plasma levels of anthrax toxin receptor 2 (ANTXR2), cadherin-13 (CDH-13), scavenger receptor cysteine-rich type 1 protein M130 (CD163), cartilage oligomeric matrix protein (COMP), Dickkopf-related protein 3 (DKK3), periostin, and secretogranin-1 were all confirmed to decrease with age. We then investigated whether any of the secreted proteins influenced bone metabolism and found that CDH-13 inhibited osteoclast differentiation. CDH 13 treatment suppressed the receptor activator of NF-κB ligand (RANKL) signaling pathway in bone marrow-derived macrophages, and intraperitoneal administration of CDH-13 delayed age-related bone loss in the femurs of aged mice. These findings suggest that low plasma CDH-13 expression in aged mice promotes aging-associated osteopenia by facilitating excessive osteoclast formation. Thus, CDH-13 could have therapeutic potential as a protein drug for the prevention of osteopenia.

Background The microRNAs (miRNAs) down-regulated in aged mouse skeletal muscle were mainly clustered within the delta-like homologue 1 and the type III iodothyronine deiodinase (Dlk1-Dio3) genomic region. Although clustered miRNAs are coexpressed and regulate multiple targets in a specific signalling pathway, the function of miRNAs in the Dlk1-Dio3 cluster in muscle aging is largely unknown. We aimed to ascertain whether these miRNAs play a common role to regulate age-related muscle atrophy. Methods To examine anti-atrophic effect of miRNAs, we individually transfected 42 miRNA mimics in fully differentiated myotubes and analysed their diameters. The luciferase reporter assay using target 3′ untranslated region (UTR) and RNA pull-down assay were employed to ascertain the target predicted by the TargetScan algorithm. To investigate the therapeutic potential of the miRNAs in vivo, we generated adeno-associated virus (AAV) serotype 9 expressing green fluorescent protein (GFP) (AAV9-GFP) bearing miR-376c-3p and infected it into the tibialis anterior muscle of old mice. We performed morphometric analysis and measured ex vivo isometric force using a force transducer. Human gluteus maximus muscle tissues (ages ranging from 25 to 80 years) were used to investigate expression levels of the conserved miRNAs in the Dlk1-Dio3 cluster. Results We found that the majority of miRNAs (33 out of 42 tested) in the cluster induced anti-atrophic phenotypes in fully differentiated myotubes with increasing their diameters. Eighteen of these miRNAs, eight of which are conserved in humans, harboured predicted binding sites in the 3′ UTR of muscle atrophy gene-1 (Atrogin-1) encoding a muscle-specific E3 ligase. Direct interactions were identified between these miRNAs and the 3′ UTR of Atrogin-1, leading to repression of Atrogin-1 and thereby induction of eIF3f protein content, in both human and mouse skeletal muscle cells. Intramuscular delivery of AAV9 expressing miR-376c-3p, one of the most effective miRNAs in myotube thickening, dramatically ameliorated skeletal muscle atrophy and improved muscle function, including isometric force, twitch force, and fatigue resistance in old mice. Consistent with our findings in mice, the expression of miRNAs in the cluster was significantly down-regulated in human muscle from individuals > 50 years old.

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Cinnamic acid (CA) and its derivatives have a broad therapeutic spectrum that includes antimicrobial, antifungal, and antitumoral activities. However, the vasodilative effect of CA has not been demonstrated. The present study characterizes the vasodilative activity and the mechanism of CA in rat thoracic aorta. The vasomotion of aortic strips following CA treatment was measured in an organ bath system. In addition, vascular strips and human umbilical vein endothelial cells (HUVECs) were used in organ bath, Western blot, nitrite, and cyclic guanosine monophosphate (cGMP) measurements. CA relaxed phenylephrine-precontracted aortic strips in an endothelium-dependent manner. Pretreatment of the endothelium-intact aortic strips with N(G) -nitro-l-arginine methyl ester (10(-4)  M), 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10-one, (10(-6)  M) and methylene blue (10(-5)  M) inhibited CA-induced vasorelaxation. CA also increased the phosphorylation of endothelial nitric oxide synthase and nitric oxide generation in a concentration-dependent manner in HUVECs. In addition, cGMP generation and cGMP-dependent protein kinase G (PKG) expression in aortic strips were increased by CA treatment. Furthermore, CA-induced vasorelaxation was inhibited by the PKG inhibitor KT5823 (0.3 μM) and the Ca(2+) -activated K(+) channel inhibitor tetraethylammonium (10(-3)  M). These findings suggest that CA exerts an endothelium-dependent vasodilation effect via the nitric oxide-cGMP-PKG-mediated pathway in rat thoracic aorta.