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Background: Although chronic treatment with glucocorticoids, such as dexamethasone, is frequently associated with muscle atrophy, effective and safe therapeutics for treating muscle atrophy remain elusive. Jakyak-gamcho-tang (JGT), a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, has long been used to relieve muscle tension and control muscle cramp-related pain. However, the effects of JGT on glucocorticoid-induced muscle atrophy are yet to be comprehensively clarified., Purpose: The objective of the current study was to validate the protective effect of JGT in dexamethasone-induced muscle atrophy models and elucidate its underlying mechanism through integrated in silico - in vitro - in vivo studies., Study Design and Methods: Differential gene expression was preliminarily analyzed using the RNA-seq data to determine the effects of JGT on C2C12 myotubes. The protective effects of JGT were further validated in dexamethasone-treated C2C12 myotubes by assessing cell viability, myotube integrity, and mitochondrial function or in C57BL/6 N male mice with dexamethasone-induced muscle atrophy by evaluating muscle mass and physical performance. Transcriptomic pathway analysis was also performed to elucidate the underlying mechanism., Results: Based on preliminary gene set enrichment analysis using the RNA-seq data, JGT regulated various pathways related to muscle differentiation and regeneration. Dexamethasone-treated C2C12 myotubes and muscle tissues of atrophic mice displayed substantial muscle protein degradation and muscle loss, respectively, which was efficiently alleviated by JGT treatment. Importantly, JGT-mediated protective effects were associated with observations such as preservation of mitochondrial function, upregulation of myogenic signaling pathways, including protein kinase B/mammalian target of rapamycin/forkhead box O3, inhibition of ubiquitin-mediated muscle protein breakdown, and downregulation of inflammatory and apoptotic pathways induced by dexamethasone., Conclusion: To the best of our knowledge, this is the first report to demonstrate that JGT could be a potential pharmaceutical candidate to prevent muscle atrophy induced by chronic glucocorticoid treatment, highlighting its known effects for relieving muscle spasms and pain. Moreover, transcriptomic pathway analysis can be employed as an efficient in silico tool to predict novel pharmacological candidates and elucidate molecular mechanisms underlying the effects of herbal medications comprising diverse biologically active ingredients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7-8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Cysteine-rich protein 2 (CSRP2) is discovered as oncogene. The study aims to investigate the clinical significance and potential mechanism of CSRP2 in head and neck squamous cell carcinoma (HNSCC)., Methods: CSRP2 expression was explored by immunohistochemistry tissue microarrays and Western blotting in HNSCC. The effect of CSRP2 on the cancer stemness and epithelial-to-mesenchymal transition (EMT) of HNSCC cells was investigated by sphere formation, wound healing, and transwell assays. The vitro and vivo experiments revealed that CSRP2 modulated cancer stemness and EMT phenotypes in HNSCC., Results: CSRP2 was overexpressed in HNSCC patients and presented poor prognosis. CSRP2 knockdown inhibited the migration and invasion ability of the HNSCC cells. And CSRP2 expression was closely associated with CSCs markers, EMT-transcription factor, new oncoprotein, and immune checkpoint., Conclusion: The overexpression of CSRP2 indicates poor prognosis and plays a key role in maintaining the cancer cell stemness and EMT., (© 2023 Wiley Periodicals LLC.)

Alcoholic liver cirrhosis (ALC) is accompanied by sarcopenia. The aim of this study was to investigate the acute effects of balanced parenteral nutrition (PN) on skeletal muscle protein turnover in ALC. Eight male patients with ALC and seven age- and sex-matched healthy controls were studied for 3 h of fasting followed by 3 h of intravenous PN (SmofKabiven 1,206 mL: amino acid = 38 g, carbohydrates = 85 g, and fat = 34 g) 4 mL/kg/h. We measured leg blood flow and sampled paired femoral arteriovenous concentrations and quadriceps muscle biopsies while providing a primed continuous infusion of [ring- 2 d 5 ]-phenylalanine to quantify muscle protein synthesis and breakdown. Patients with ALC exhibited shorter 6-min walking distance (ALC: 487 ± 38 vs. controls: 722 ± 14 m, P < 0.05), lower hand-grip strength (ALC: 34 ± 2 vs. controls: 52 ± 2 kg, P < 0.05), and computed tomography (CT)-verified leg muscle loss (ALC: 5,922 ± 246 vs. controls: 8,110 ± 345 mm 2 , P < 0.05). Net leg muscle phenylalanine uptake changed from negative (muscle loss) during fasting to positive (muscle gain) in response to PN (ALC: -0.18 ± +0.01 vs. 0.24 ± 0.03 µmol/kg muscle·min -1 ; P < 0.001 and controls: -0.15 ± 0.01 vs. 0.09 ± 0.01 µmol/kg muscle·min -1 ; P < 0.001) but with higher net muscle phenylalanine uptake in ALC than controls ( P < 0.001). Insulin concentrations were substantially higher in patients with ALC during PN. Our results suggest a higher net muscle phenylalanine uptake during a single infusion of PN in stable patients with ALC with sarcopenia compared with healthy controls. NEW & NOTEWORTHY Muscle protein turnover responses to parenteral nutritional (PN) supplementation have not previously been studied in stable alcoholic liver cirrhosis (ALC). We applied stable isotope tracers of amino acids to directly quantify net muscle protein turnover responses to PN in sarcopenic males with ALC and healthy controls. We found a higher net muscle protein gain in ALC during PN, thereby providing the physiological rationale for future clinical trials of PN as a potential countermeasure to sarcopenia.

Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function. Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels. Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group. Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study. Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups. Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-β1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro. Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.

Age-related loss of muscle mass, strength, and performance, commonly referred to as sarcopenia, has wide-ranging detrimental effects on human health, the ramifications of which can have serious implications for both morbidity and mortality. Various interventional strategies have been proposed to counteract sarcopenia, with a particular emphasis on those employing a combination of exercise and nutrition. However, the efficacy of these interventions can be confounded by an age-related blunting of the muscle protein synthesis response to a given dose of protein/amino acids, which has been termed "anabolic resistance." While the pathophysiology of sarcopenia is undoubtedly complex, anabolic resistance is implicated in the progression of age-related muscle loss and its underlying complications. Several mechanisms have been proposed as underlying age-related impairments in the anabolic response to protein consumption. These include decreased anabolic molecular signaling activity, reduced insulin-mediated capillary recruitment (thus, reduced amino acid delivery), and increased splanchnic retention of amino acids (thus, reduced availability for muscular uptake). Obesity and sedentarism can exacerbate, or at least facilitate, anabolic resistance, mediated in part by insulin resistance and systemic inflammation. This narrative review addresses the key factors and contextual elements involved in reduction of the acute muscle protein synthesis response associated with aging and its varied consequences. Practical interventions focused on dietary protein manipulation are proposed to prevent the onset of anabolic resistance and mitigate its progression., (© The Author(s) 2022. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The poor gel strength and microbial infection of conventional chicken myofibrillar protein (CMP) gels have severely limited the application. Here, plasma activated water (PAW) instead of normal water was used to prepare CMP gels. PAW prepared by treating deionized water with plasma jet was incubated with CMPs and followed by heating to prepare CMP gels. Effects of PAW on CMP gels were assessed in terms of basic physicochemical properties, network structure, and antibacterial activity. The results showed that PAW treatment accelerated the aggregation of CMPs and increased the strength and water holding capacity of CMP gels. Due to the presence of NO and NO 2 free radicals in PAW, the prepared CMP gels were endowed with antibacterial activity against Salmonella Enteritidis and Staphylococcus aureus. The new method of PAW-induced CMP gels will have the prospect of improving the quality of gels and extending the shelf life of chicken gel products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Proteasomal activity is compromised in diabetic hearts that contributes to proteotoxic stresses and cardiac dysfunction. Osteocrin (OSTN) acts as a novel exercise-responsive myokine and is implicated in various cardiac diseases. Herein, we aim to investigate the role and underlying molecular basis of OSTN in diabetic cardiomyopathy (DCM). Mice received a single intravenous injection of the cardiotrophic adeno-associated virus serotype 9 to overexpress OSTN in the heart and then were exposed to intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for consecutive 5 days to generate diabetic models. Neonatal rat cardiomyocytes were isolated and stimulated with high glucose to verify the role of OSTN in vitro. OSTN expression was reduced by protein kinase B/forkhead box O1 dephosphorylation in diabetic hearts, while its overexpression significantly attenuated cardiac injury and dysfunction in mice with STZ treatment. Besides, OSTN incubation prevented, whereas OSTN silence aggravated cardiomyocyte apoptosis and injury upon hyperglycemic stimulation in vitro. Mechanistically, OSTN treatment restored protein kinase G (PKG)-dependent proteasomal function, and PKG or proteasome inhibition abrogated the protective effects of OSTN in vivo and in vitro. Furthermore, OSTN replenishment was sufficient to prevent the progression of pre-established DCM and had synergistic cardioprotection with sildenafil. OSTN protects against DCM via restoring PKG-dependent proteasomal activity and it is a promising therapeutic target to treat DCM.

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs., Methods: Peptides were prepared from the muscles of monkfish ( Lophius litulon ), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities., Results: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H 2 O 2 -related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels., Conclusion: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC., (© 2019 John Wiley & Sons Ltd.)

Fibrosis is a key pathological event during muscle aging that accelerates the development of sarcopenia. We show that sarcolipin (SLN) is highly expressed during aging, promotes intracellular calcium overload and participates in impaired myogenic differentiation. d-Galactose (D-gal) was used to induce senescence in C2C12 myoblasts. Conventional AAV-mediated SLN knockdown cells were used to study the role of SLN in muscle physiology and pathophysiology. C2C12 cells were treated with D-gal, which promoted fibrosis and SLN upregulation. The expression of TGF-β1 and α-SMA, which participate in myogenic transdifferentiation, were also elevated. C2C12 cells with reduced sarcolipin expression produced decreased amounts of collagen. Our study identified an unrecognized role of SLN in regulating myogenic transdifferentiation during aging-associated skeletal muscle cell fibrosis. Targeting SLN may be a novel therapeutic strategy to relieve sarcopenia-associated muscle fibrosis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin's role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin's involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca 2+ -homeostasis and excitation contraction (EC) coupling. While its role in Ca 2+ -homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.

Background: Myopalladin (MYPN) is a striated muscle-specific, immunoglobulin-containing protein located in the Z-line and I-band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss-of-function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe., Methods: Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro., Results: MKO mice were 13% smaller compared with wild-type controls and exhibited a 48% reduction in myofibre cross-sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild-type mice, MKO mice showed progressively decreased exercise capability, Z-line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z-line widening starting from 8 months of age. RNA-sequencing analysis revealed down-regulation of serum response factor (SRF)-target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF-cofactor myocardin-related transcription factor A (MRTF-A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF-A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF-A-mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway., Conclusions: Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z-line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise., (© 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

MDR and tumor migration and invasion are still the main obstacles to effective breast cancer chemotherapies. Transgelin 2 has recently been shown to induce drug resistance, tumor migration, and invasion. The aim of this study was to determine the biological functions of Transgelin 2 and the mechanism underlying how Transgelin 2 induces paclitaxel (PTX) resistance and the migration and invasion of breast cancer. We detected that the protein level of Transgelin 2 was significantly upregulated in breast cancer tissues compared with adjacent nontumor tissues. A bioinformatics analysis indicated that Transgelin 2 was significantly related to clinicopathologic parameters and patient prognosis. Overexpression of Transgelin 2 enhanced the migration and invasion of human breast cancer cells and decreased the sensitivity of breast cancer cells to paclitaxel. Meanwhile, the tumorigenesis and metastasis of breast cancer cells were also enhanced by Transgelin 2 overexpression in vivo Moreover, Transgelin 2 overexpression activated the PI3K/Akt/GSK-3β pathway by increasing the phosphorylation levels of Akt and GSK-3β and decreasing the expression of PTEN. We also found that Transgelin 2 could directly interact with PTEN and was located upstream of PTEN. Furthermore, the PI3K/Akt pathway inhibitor MK-2206 reversed the resistance to paclitaxel and inhibited the migration and invasion of breast cancer cells. These findings indicate that Transgelin 2 promotes paclitaxel resistance and the migration and invasion of breast cancer by directly interacting with PTEN and activating the PI3K/Akt/GSK-3β pathway. Transgelin 2 may therefore be useful as a novel biomarker and therapeutic target for breast cancer., (©2019 American Association for Cancer Research.)

Key Points: The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied. Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform. Recovery from desensitization is determined by the AChR isoform-specific cytoplasmic M3-M4 domain. The co-expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering. These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions., Abstract: The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle-type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole-cell currents in muscle-type (TE671/CN21) and non-muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3-M4 cytoplasmic loops of the γ- and ε-subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ-R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Rationale: Physical exercise provides benefits for various organ systems, and some of systemic effects of exercise are mediated through modulation of muscle-derived secreted factors, also known as myokines. Myonectin/C1q (complement component 1q)/TNF (tumor necrosis factor)-related protein 15/erythroferrone is a myokine that is upregulated in skeletal muscle and blood by exercise., Objective: We investigated the role of myonectin in myocardial ischemic injury., Methods and Results: Ischemia-reperfusion in myonectin-knockout mice led to enhancement of myocardial infarct size, cardiac dysfunction, apoptosis, and proinflammatory gene expression compared with wild-type mice. Conversely, transgenic overexpression of myonectin in skeletal muscle reduced myocardial damage after ischemia-reperfusion. Treadmill exercise increased circulating myonectin levels in wild-type mice, and it reduced infarct size after ischemia-reperfusion in wild-type mice, but not in myonectin-knockout mice. Treatment of cultured cardiomyocytes with myonectin protein attenuated hypoxia/reoxygenation-induced apoptosis via S1P (sphingosine-1-phosphate)-dependent activation of cAMP/Akt cascades. Similarly, myonectin suppressed inflammatory response to lipopolysaccharide in cultured macrophages through the S1P/cAMP/Akt-dependent signaling pathway. Moreover, blockade of S1P-dependent pathway reversed myonectin-mediated reduction of myocardial infarct size in mice after ischemia-reperfusion., Conclusions: These data indicate that myonectin functions as an endurance exercise-induced myokine which ameliorates acute myocardial ischemic injury by suppressing apoptosis and inflammation in the heart, suggesting that myonectin mediates some of the beneficial actions of exercise on cardiovascular health.

Dietary proteins harbour bioactive peptides that exert various physiological activities. Chicken meat prepared from spent layers from the egg industry is an inexpensive source of protein for the production of bioactive peptides. This study explored the effect of hen muscle-derived peptides prepared by enzymatic hydrolysis on immune functions. The hydrolysate was incorporated into the diet of weanling Sprague-Dawley rats (n = 8 per diet) for 3 weeks at 2% or 5% addition (w/w diet). At a dose of 5% (w/w) the hydrolysate exhibited immunomodulatory effects on splenocytes, including a lower proportion of OX6+ (professional antigen presenting cells) and a higher proportion of CD11b/c+ cells (macrophages/monocytes) (p < 0.05) compared to the isonitrogenous control diet. Meanwhile, the production of anti-inflammatory cytokine interleukin (IL)-10 by splenocytes stimulated ex vivo with mitogens was significantly higher from hydrolysate treatment; there was no significant difference in the other cytokines (IL-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6 and IL-2) investigated. Supplementing with the hydrolysate did not alter the growth, food intake and organ weights in young rodents. These results indicated that the spent hen muscle protein hydrolysate has the potential to be developed for value-added products with anti-inflammatory properties.

Excessive concentrations of angiotensin-converting enzyme (ACE) can give rise to high blood pressure, and is harmful to the body. ACE inhibitory peptides from food proteins are considered good sources of function food. However, the preparation of ACE inhibitory peptides by classical method faces many challenges. Three novel ACE inhibitory peptides were identified by in silico methods, and showed potent activity against ACE in vitro. The simulation hydrolysis of nebulin was performed with ExPASy PeptideCutter program. Potential activity, solubility, and absorption, distribution, metabolism, excretion, and toxicity properties of generated peptides were predicted using program online. Molecular docking displayed that EGF, HGR, and VDF were docked into the S 1 and S 2 pockets of ACE. Meanwhile, Phe and Arg at the C-terminal enhance ACE affinity. The IC 50 values of EGF, HGR, and VDF were 474.65 ± 0.08, 106.21 ± 0.52, and 439.27 ± 0.09 μM, respectively. Three peptides EGF, HGR, and VDF from Oncorhynchus mykiss nebulin were identified, and the molecular mechanism between ACE and peptides was clarified using in silico methods. The results suggested that Oncorhynchus mykiss nebulin would be an attractive raw material of antihypertensive nutraceutical ingredients., Practical Application: This study has shown the potential of Oncorhynchus mykiss nebulin as good sources for producing ACE inhibitory peptides. According to this finding, in silico approach is the feasible way for prediction and identification of food-derived ACE inhibitory peptides in emerging nutraceutical field., (© 2018 Institute of Food Technologists®.)

This study aims to examine the effects of non-hydrolyzed octopus (Octopus vulgaris) muscle proteins (NHOPs) and their hydrolysates (OPHs) on alloxan induced diabetes in Wistar rats (AIDR). Animals were allocated into seven groups of six rats each: control group (C), diabetic group (D) and diabetic rats treated with acarbose (D + Acar), non-hydrolyzed octopus proteins (D + NHOPs) and octopus proteins hydrolysates (D + OPHs) groups. The diabetic rats presented a significant increase in glycemic status such as α-amylase activity (in plasma, pancreas and intestine), hepatic glycogen, blood glucose and glycated hemoglobin (HbA1c) levels, as well as a significant decrease in the levels of plasma insulin and total hemoglobin compared to control group. In addition, plasma and liver contents in total cholesterol, triglycerides and LDL-cholesterol significantly increased in AIDR compared to control group. However, the daily administration of OPHs for 30 days improved the glucose tolerance test, the glycemic status of diabetic rats and corrected the lipid profiles. Further, a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase as well as in the level of plasma bilirubin on diabetic status was observed, indicating considerable hepatocellular injury. OPHs treatment was found to attenuate the increased activities of the plasma enzymes produced by diabetes and caused a subsequent recovery towards normalization compared to the control group. By contrast, the NHOPs treatment was found to increase the glucose metabolic disorders in AIDR. These beneficial effects of OPHs were confirmed by histological findings in the hepatic and pancreatic tissues of diabetic treated rats. Indeed, they avoid lipid accumulation in the hepatocytes and protect the pancreatic β-cells from degeneration. Our results thus suggest that OPHs may be helpful in the preventing from diabetic complications by reversing hepatotoxicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dystrophin (Dp) is a multidomain protein that links the actin cytoskeleton to the extracellular matrix through the dystrophin associated proteins complex (DAPC). Dp of 71 kDa (Dp71), corresponding to the COOH-terminal domain of dystrophin, and α1-syntrophin (α1Syn) as the principal component of the DAPC, are strongly expressed in the brain. To clarify their involvement in the central control of osmotic homeostasis, we investigated the effect of 14 days of salt loading (with drinking water containing 2% NaCl) and then reversibly to 30 days of normal hydration (with drinking water without salt), first on the expression by western-blotting and the distribution by immunochemistry of Dp71 and α1Syn in the SON of the rat and, second, on the level of some physiological parameters, as the plasma osmolality, natremia and hematocrit. Dp71 is the most abundant form of dystrophin revealed in the supraoptic nucleu (SON) of control rat. Dp71 was localized in magnocellular neurons (MCNs) and astrocytes, when α1Syn was observed essentially in astrocytes end feet. After 14 days of salt-loading, Dp71 and α1Syn signals decreased and a dual signal for these two proteins was revealed in the astrocytes processes SON surrounding blood capillaries. In addition, salt loading leads to an increase in plasma osmolality, natremia and hematocrit. Reversibly, after 30 days of normal hydration, the intensity of the signal for the two proteins, Dp71 and α1Syn, increased and approached that of control. Furtheremore, the levels of the physiological parameters decreased and approximated those of control. This suggests that Dp71 and α1Syn may be involved in the functional activity of the SON. Their localization in astrocyte end feet emphasizes their importance in neuronal-vascular-astrocyte interactions for the central detection of osmolality. In the SON, Dp71 and α1Syn may be involved in osmosensitivity., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Background and Objective: Human periodontal ligament cells (HPDLCs) express transforming growth factor-β1 (TGF-β1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells., Material and Methods: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription-polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-β1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-β1 were assessed by WST-1 assay., Results: In microarray and quantitative reverse transcription-polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-β1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-β1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-β1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA., Conclusion: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-β1 stimulation., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hepatic ischaemia/reperfusion (HIR) induces severe damage on hepatocyte cell membrane, which leads to hepatocyte death and the subsequent HIR injury. In this study, we investigated the role and the mechanism of mitsugumin-53 (MG53), a novel cell membrane repair protein, in protecting the liver against HIR injury. Rats were subjected to sham operation or 70% warm HIR with or without recombined MG53 (rhMG53), caudal vein-injected 2 hrs before inducing HIR. In vitro, cultured hepatocyte AML12 cells were subjected to hypoxia/reoxygenation (H/R) in the presence of rhMG53 and/or dysferlin gene shRNAs or adenovirus transfection. HIR resulted in severe liver injury manifested as severe liver histological changes and increased AST and ALT release. Post-ischaemic hepatic oxidative stress was significantly enhanced demonstrated by elevated dihydroethidium level, increased 4-hydroxynonenal, enhanced 15-F2t-isoprostane and decreased SOD activity. rhMG53 administration attenuated post-HIR liver injury, decreased liver oxidative stress and further enhanced dysferlin protein expression and its colocalization with MG53. Similarly, H/R induced AML12 cell injury and oxidative stress, which were abolished by either rhMG53 or dysferlin overexpression but were exacerbated by dysferlin gene knockdown. Dysferlin overexpression further increased H/R-induced increased colocalization of MG53 and dysferlin. In conclusion, MG53 was anchored by dysferlin to reduce oxidative stress and cell death and attenuate HIR injury., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The natriuretic peptide (NP) system is a critical endocrine, autocrine, and paracrine system and has been investigated for potential use against cardiovascular and metabolic diseases. The clearance of NPs is regulated by the proteolysis of neutral endopeptidase (NEP) and by endocytosis via natriuretic peptide receptor-3 (NPR3). A linear NPR3-selective peptide, [Cha 8 ]-ANP(7-16)-NH 2 (1), showed potent binding affinity for NPR3 but poor predicted chemical stability due to its free thiol group. A 12-mer peptide (9) without a thiol group was designed by the hybridization of two NPR3-binding peptides: a linear ANP fragment peptide analog and musclin, a murine member of the bHLH family of transcription factors, possessed high binding affinity and strict selectivity for NPR3. To increase the proteolytic resistance of 9, amino acid substitutions at the cleavage sites led to hydroxyacetyl-[d-Phe 5 ,d-Hyp 7 ,Cha 8 ,d-Ser 9 ,Hyp 11 ,Arg(Me) 14 ]-ANP(5-15)-NHCH 3 (23), showing high and selective binding affinity for NPR3 over NPR1 and excellent stability in mouse serum. Compound 23 increased intracellular cGMP concentrations in primary cultured adipocytes, and continuous administration induced substantial plasma cGMP elevation in mice, suggesting its potential to clarify the physiological role of NPR3 and its therapeutic application., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently characterized. The peptide could selectively interfere with the calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase, similar to 11R-VIVIT. RCAN-11R did not affect cell viability when it was used at a higher concentration than the toxic concentration of 11R-VIVIT. RCAN-11R could therefore be useful as a therapeutic agent that is less toxic than current drugs or 11R-VIVIT.

Tuna muscle consists of light and dark muscle in approximately equal proportions. However, besides for the light muscle of tuna, cod, sardine, and salmon, few researches have assessed the health-promoting functions of fish protein. Therefore, we evaluated the mechanisms underlying the alteration of lipid storage and cholesterol metabolism following the intake of tuna dark muscle protein (TDMP) by obese type-2 diabetic/obese mice. Four-week-old male KK-A y mice were separated into 2 dietary groups, with one group receiving a casein-based diet and the other receiving a diet with the substitution of part of the protein (50%, w/w) by TDMP (TDMP diet) for 4 wk. The TDMP diet significantly increased the content of serum high-density lipoprotein cholesterol, partly due to the reduction of the expression of scavenger receptor class B member 1 in epididymal white adipose tissue. In addition, dietary TDMP decreased the content of hepatic triacylglycerol, which could be due to the enhancement of carnitine palmitoyltransferase-2 activity through the activation of the expression of the peroxisome proliferative activated receptor-α in the liver. These results suggest that TDMP could have the potential to prevent the development of obesity-related diseases by suppressing the storage of hepatic triacylglycerol and cholesterol., (© 2017 Institute of Food Technologists®.)

The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24h. After 8h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC 50 value of 0.54mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3-4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC 50 value of 0.29μM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fish protein hydrolysate was prepared from muscle of small red scorpionfish (Scorpaena notata) by treatment with a protease from the fungus Penicillium digitatum. Protein hydrolysate was found to strongly inhibit the angiotensin I converting enzyme and exhibited high antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. After ultrafiltration, peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high-performance liquid chromatography with a high purification yield of 2.5 mg of peptide per gram of initial protein. Two major peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS), corresponding to the following sequences: Leu-Val-Thr-Gly-Asp-Asp-Lys-Thr-Asn-Leu-Lys (1,204.665 Da) and Asp-Thr-Gly-Ser-Asp-Lys-Lys-Gln-Leu (992.511 Da). These peptides, mainly composed of hydrophilic amino acids, showed high antioxidative and angiotensin I converting enzyme inhibitory activities. These data suggest that the two novel peptides isolated from the muscle hydrolysate of small red scorpionfish can be a beneficial ingredient for functional foods or pharmaceuticals against hypertension and oxidative stress., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Micropeptides function as master regulators of calcium-dependent signaling in muscle. Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), the membrane pump that promotes muscle relaxation by taking up Ca 2+ into the sarcoplasmic reticulum, is directly inhibited by three muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN), and sarcolipin (SLN). The widespread and essential function of SERCA across diverse cell types has raised questions as to how SERCA is regulated in cells that lack MLN, PLN, and SLN. We identified two transmembrane micropeptides, endoregulin (ELN) and another-regulin (ALN), that share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirrored that of SERCA isoform-encoding transcripts in nonmuscle cell types. Our findings identify additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca 2+ dynamics in both muscle and nonmuscle cell types., (Copyright © 2016, American Association for the Advancement of Science.)

This study investigated the in vitro anti-inflammatory and antioxidant properties, protein quality, and other related characteristics obtained by the single-cycle and two-cycle high hydrostatic pressure (HHP at 200, 400 and 600 MPa) treatment of squids (Todarodes pacificus). The soluble protein nitrogen content and in vitro protein digestibility increased significantly (p<0.05) after all HHP treatments, and the two-cycle 600 MPa HHP treatments yielded the highest values, 7.59% and 84.42%, respectively. The estimated protein efficiency ratios, and antioxidant and anti-inflammatory properties of squids significantly increased by all HHP treatments. (1)H nuclear magnetic resonance (NMR) showed that the main spectral changes associated to the anti-inflammatory properties of proteins following HHP treatment were in the range of 3.00-3.19 and 3.60-3.79 ppm. This indicates that the HHP treatments modified the protein and functional properties of squids and gave the relevant chemical shifts in NMR signals, either migrated or disappeared., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein 3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide (H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein (Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species (ROS), DNA fragmentation and reduced mitochondrial membrane potential (ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3 and -8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

In the digestive tract of humans, bioactive peptides, i.e. protein fragments impacting the physiological activity of the body, may be released during the digestion of food proteins, including those of fish. The aim of the study was to establish the method of human ex vivo digestion of carp muscle tissue and evaluate the angiotensin I-converting enzyme inhibitory and antioxidant activities of hydrolysates obtained after digestion. It was found that the hydrolysates of carp muscle tissue obtained with the three-stage method of simulated ex vivo digestion showed ACE inhibitory as well as antioxidative activities. It was demonstrated that the degree of hydrolysis depended on the duration of individual stages and the degree of comminution of the examined material. Although the applied gastric juices initiated the process of hydrolysis of carp muscle tissue, the duodenal juices caused a rapid increase in the amount of hydrolysed polypeptide bonds. The antihypertensive and antioxidative activities of the hydrolysates of carp muscle tissue increased together with progressive protein degradation. However, the high degree of protein hydrolysis does not favour an increase in the activity of free radical scavenging. The presented results are an example of the first preliminary screening of the potential health-promoting biological activity of carp muscle tissue in an ex vivo study.

Lipotoxicity resulting from a high concentration of saturated fatty acids is closely linked to development of insulin resistance, as well as apoptosis in skeletal muscle. CTRP5, an adiponectin paralog, is known to activate AMPK and fatty acid oxidation; however, the effects of CTRP5 on palmitate-induced lipotoxicity in myocytes have not been investigated. We found that globular domain of CTRP5 (gCTRP5) prevented palmitate-induced apoptosis and insulin resistance in myocytes by inhibiting the activation of caspase-3, reactive oxygen species accumulation, and IRS-1 reduction. These beneficial effects of gCTRP5 are mainly attributed to an increase in fatty acid oxidation through phosphorylation of AMPK. These results provide a novel function of CTRP5, which may have preventive and therapeutic potential in management of obesity, insulin resistance, and type 2 diabetes mellitus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cells turn on autophagy, an intracellular recycling pathway, when deprived of nutrients. How autophagy is regulated by hormonal signals in response to major changes in metabolic state is not well understood. Here, we provide evidence that myonectin (CTRP15), a skeletal muscle-derived myokine, is a novel regulator of cellular autophagy. Starvation activated liver autophagy, whereas nutrient supplementation following food deprivation suppressed it; the former and latter correlated with reduced and increased expression and circulating levels of myonectin, respectively, suggestive of a causal link. Indeed, recombinant myonectin administration suppressed starvation-induced autophagy in mouse liver and cultured hepatocytes, as indicated by the inhibition of LC3-dependent autophagosome formation, p62 degradation, and expression of critical autophagy-related genes. Reduction in protein degradation is mediated by the PI3K/Akt/mTOR signaling pathway; inhibition of this pathway abrogated the ability of myonectin to suppress autophagy in cultured hepatocytes. Together, our results reveal a novel skeletal muscle-liver axis controlling cellular autophagy, underscoring the importance of hormone-mediated tissue cross-talk in maintaining energy homeostasis.

Musclin is a novel skeletal muscle-derived secretory factor found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region homologous to the natriuretic peptide family. Thus, musclin is found to bind with the natriuretic peptide clearance receptors. However, the role of musclin in vascular regulation remains unclear. In this study, we aim to investigate the direct effect of musclin on vascular tone and to analyze its role in hypertension using the spontaneously hypertensive rats (SHR). In aortic strips isolated from SHR, musclin induced contractions in a dose-dependent manner. We found that the musclin-induced vasoconstriction was more marked in SHR than in normal rats (WKY). Moreover, this contraction was reduced by blockade of natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation of the natriuretic peptide receptor in musclin-induced vasoconstriction can be considered. In addition, similar to the natriuretic peptide receptor, expression of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of musclin markedly increased the blood pressure in rats that can be inhibited by anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in SHR than in WKY as in its expression. Taken together, these results suggest that musclin is involved in blood pressure regulation. The higher expression of musclin in hypertension indicates that musclin could be used as a new target for the treatment of hypertension in the future.

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+). In the absence of Ca(2+) and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca(2+)), and if millimolar Mg(2+) is present, one Mg(2+) is expected to occupy one of the Ca(2+)-binding sites with a millimolar dissociation constant. This Mg(2+) accelerates the reaction cycle, not permitting phosphorylation without Ca(2+) binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg(2+) state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin, a small regulatory membrane protein of Ca(2+)-ATPase, is bound, stabilizing the E1·Mg(2+) state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca(2+) regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

Alterations in intracellular Ca(2+) transients of cardiomyocytes in orchidectomized (ORX) rats could be a cause of cardiac dysfunction in the hypogonadal condition. To investigate the role of male sex hormones in intracellular Ca(2+) homeostasis during relaxation, Ca(2+)-handling activities by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and the Na(+)/Ca(2+) exchanger (NCX) were evaluated in the ventricular muscle of 10-wk-old ORX rats with and without testosterone supplementation (2.5 mg/kg testosterone propionate, 2 times/wk). ORX induced a 50% decrease in contraction force accompanied by a prolonged time to achieve 50% relaxation (T(50)) in isolated intact ventricular trabeculae, which was partially corrected by testosterone administration. Maximum active tension was also suppressed in ORX rats without changes in myofilament Ca(2+) sensitivity and passive stiffness of the heart. Using a sarcoplasmic reticulum-enriched membrane preparation, the maximum thapsigargin-sensitive SERCA activity of the ORX rat was 27% lower with an increased Ca(2+) sensitivity, which was prevented by testosterone treatment. However, neither changes in SERCA content nor its modulating components, sarcolipin and heat shock protein 20, were detected in the ORX rat, but there was a significant decrease in the phosphorylated Thr(17) form of phospholamban. Despite a lower level of NCX protein in the heart of ORX rats, prolonged T(50) disappeared after an incubation with thapsigargin (10 μM), implying a lack of effect of male sex hormone deficiency on NCX function. These findings indicate that male sex hormones can regulate cardiac relaxation by acting mainly through SERCA. However, a detailed mechanism of SERCA modulation under male sex hormone deficiency status remains to be explored.

Background & Aims: Acetaminophen (AAP) overdose is the most frequent cause of drug-induced liver failure. c-Jun N-terminal kinase (JNK) is thought to play a central role in AAP-induced hepatocellular necrosis. The apoptosis repressor with caspase recruitment domain (ARC) is a death repressor that inhibits death receptor and mitochondrial apoptotic signaling. Here, we investigated ARC's therapeutic effect and molecular mechanisms on AAP-induced hepatocellular necrosis., Methods: We tested the in vivo and in vitro effects of ARC fused with the transduction domain of HIV-1 (TAT-ARC) on murine AAP hepatotoxicity., Results: Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by AAP overdose in C57BL/6 mice. AAP triggered caspase-independent necrosis, as evidenced by liver histology, elevated serum transaminases, and secreted HMGB1 that was inhibited by ARC. ARC-mediated hepatoprotection was not caused by an alteration of AAP metabolism, but resulted in reduced oxidative stress. AAP overdose led to induction of RIP-dependent signaling with subsequent JNK activation. Ectopic ARC inhibited JNK activation by specific interactions between ARC and JNK1 and JNK2. Importantly, survival of mice was even preserved when ARC therapy was initiated in a delayed manner after AAP administration., Conclusions: This work identifies for the first time ARC-JNK-binding with subsequent inhibition of JNK signaling as a specific mechanism of ARC to interfere with AAP-dependent necrosis. Our data suggests that AAP-mediated induction of RIP signaling serves as a critical switch for hepatocellular necrosis. The efficacy of TAT-ARC protein transduction in murine AAP hepatotoxicity suggests its therapeutic potential for reversing AAP intoxication also in humans., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Trefoil factor 2 (TFF2) is a small peptide with an important role in mucosal repair. TFF2 is up-regulated in asthma, suggesting a role in asthma pathogenesis. Given its known biological role in promoting epithelial repair, TFF2 might be expected to exert a protective function in limiting the progression of airway remodeling in asthma. The contribution of TFF2 to airway remodeling in asthma was investigated by examining the expression of TFF2 in the airway and lung, and evaluating the effects of recombinant TFF2 treatment on established airway remodeling in a murine model of chronic allergic airways disease (AAD). BALB/c mice were sensitized and challenged with ovalbumin (OVA) or saline for 9 weeks, whereas mice with established OVA-induced AAD were treated with TFF2 or vehicle control (intranasally for 14 d). Effects on airway remodeling, airway inflammation, and airway hyperresponsiveness were then assessed, whereas TFF2 expression was determined by immunohistochemistry. TFF2 expression was significantly increased in the airways of mice with AAD, compared with expression levels in control mice. TFF2 treatment resulted in reduced epithelial thickening, subepithelial collagen deposition, goblet-cell metaplasia, bronchial epithelium apoptosis, and airway hyperresponsiveness (all P < 0.05, versus vehicle control), but TFF2 treatment did not influence airway inflammation. The increased expression of endogenous TFF2 in response to chronic allergic inflammation is insufficient to prevent the progression of airway inflammation and remodeling in a murine model of chronic AAD. However, exogenous TFF2 treatment is effective in reversing aspects of established airway remodeling. TFF2 has potential as a novel treatment for airway remodeling in asthma.

Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased-without change in current amplitude-when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with MβCD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn's AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR's mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.

This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. Exploring the molecular mechanisms of pathophysiology in anti-Thy1 nephritis may elucidate the pathogeneses of mesangial proliferation. We examined the roles and acting mechanisms of differentially expressed proteins (DEPs) by bioinformatics analysis of glomeruli proteomic profiles during the course of anti-Thy1 nephritis. In total, 108 DEPs were found by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), and 40 DEPs were identified by matrix-assisted laser desorption ionization/time of flight and liquid chromatography-MS. DEPs were classified into five clusters (Clusters 1-5), according to their expression trends using Cluster 3.0 software, involved in regulating biological processes such as the stress response, cell proliferation, apoptosis, energy metabolism, transport, and the actin cytoskeleton. The expression patterns of ten DEPs, distributed across five clusters, including AKR1A1, AGAT, ATP6V1B2, HIBADH, MDH1, MPST, NIT2, PRDX6, PSMB7, and TPI1, were validated by Western blotting. Based on Western blotting and immunohistochemistry, we also found that the DEP FHL2, which was primarily expressed in the mesangial region, was down-regulated on days 3 and 5, and up-regulated on day 10. In vitro, we found that FHL2 overexpression induced mesangial cell proliferation by increasing the number of S-phase cells and decreasing G2/M-phase cells, whereas inhibiting FHL2 had the opposite effect. This study explored novel DEPs and their expression patterns during anti-Thy1 nephritis, and elucidated FHL2's effect on mesangial cell proliferation. These results will contribute to our understanding of the pathogenesis of mesangial proliferation.

Background: Grass carp, with an annual production exceeding 4 × 10(6) t in China in 2009, has not been developed into a high-value product. In this study the antioxidant activities and functional properties of grass carp protein hydrolysates prepared with Alcalase 2.4L (HA) and papain (HP) were investigated. The hydrolysate with strongest radical-scavenging activity and reducing power was assessed further for changes in its antioxidant activity during simulated gastrointestinal digestion., Results: As the degree of hydrolysis (DH) increased, the metal-chelating activity of both HA and HP increased while their reducing power and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(•) )-scavenging activity decreased (P < 0.05). At the same DH, HP possessed higher DPPH(•) -scavenging activity and reducing power than HA (P < 0.05). The metal-chelating activity of HP with 10% DH was significantly increased after in vitro gastrointestinal metabolism (P < 0.05). Regarding their functional properties, all hydrolysates were more than 81% soluble over a wide range of pH (3-8). At the same DH, HP showed higher emulsion activity index but lower solubility and foaming capacity than HA., Conclusion: Grass carp protein hydrolysates showed high solubility over a wide pH range and could be used as natural antioxidants in food systems., (Copyright © 2011 Society of Chemical Industry.)

Background: The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including beta-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking., Methodology/principal Findings: Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in beta-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc(Delta14/+) mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy., Conclusions/significance: Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.

A novel antioxidative peptide (APVPH I, antioxidative peptides from venison protein hydrolysates I) was purified from venison by enzymatic hydrolysis, column chromatography of DEAE-Sephacel and high performance liquid chromatography. The molecular weight of the purified peptide was found to be 9,853 Da and the amino acid sequences of the purified peptide was Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly. The purpose of this study was to evaluate the effects of APVPH I against H2O2-induced neuronal cells damage in PC-12 cells. Antioxidative enzyme levels in cultured neuronal cells were increased in the presence of the peptide. In addition, APVPH I inhibited productions of nitric oxide (NO), reactive oxygen species (ROS), malondialdehyde (MDA) and cell death against H2O2-induced neuronal cells damage in PC-12 cells. It was presumed to be APVPH I involved in regulating the apoptosis-related gene expression in the cell environment. Present results indicate APVPH I substantially contribute to antioxidative properties in neuronal cells.

Background: Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins., Results: Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 degrees C and completely disappeared at 60 degrees C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre-incubated at 37 degrees for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid-oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg(-1) TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 degrees C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 degrees C., Conclusion: TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin-like proteinase, rendering an improvement in surimi gelation set at 37-40 degrees C., ((c) 2009 Society of Chemical Industry.)

Objectives: To determine if peripheral blood monocytes from patients with ankylosing spondylitis (AS) differed in protein expression compared to rheumatoid arthritis (RA) and healthy controls (HC)., Methods: Monocyte protein expression was characterised by 2D gel electrophoresis and by label-free quantitative expression profiling, using nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS(E), where (E) refers to low/high collision energy switching). Data sets were analysed using the Waters expression profiling system and Ingenuity pathway analysis (IPA)., Results: Two-dimensional gel electrophoresis showed upregulation of proteasomal constituents in AS monocytes, including the beta subunit of proteasome activator (PA)28. Monocyte expression profiling and IPA showed that significant changes in protein expression within the ubiquitin proteasome pathway (UPP) were restricted to AS monocytes. Statistically significant differences in protein expression involving the leucocyte extravasation, vascular endothelial growth factor, integrin and Toll-like receptor signalling pathways were seen in AS and RA monocytes compared to healthy controls. No evidence of upregulation of proteins involved in the endoplasmic reticulum stress response pathway was found in either AS or RA monocytes. Finally, the PA28 complex was shown to increase the generation of human leucocyte antigen (HLA)-B27 antigenic epitopes by the proteasome in vitro., Conclusions: Our proteomic analyses support the hypothesis that monocytes play an important role in the pathogenesis of AS and RA, and further suggest a specific role in AS for the UPP. Quantitative proteomic expression profiling constitutes a powerful new tool for rheumatology research.

The mechanism for the hypocholesterolemic action of a cattle heart protein hydrolysate (HPH) is clarified. The micellar solubility of cholesterol in vitro was significantly lower in the presence of HPH than in the presence of casein. The suppression of cholesterol uptake by Caco-2 cells was significantly higher in the cholesterol micelles containing HPH than in the cholesterol micelles containing casein. The serum cholesterol concentrations and atherogenic index were significantly lower in the rats fed with HPH than in those fed with casein. The cholesterol absorption measured by [(3)H]-cholesterol was significantly lower by HPH feeding than by casein feeding in rats in vivo accompanying the changes in fecal steroid excretion. Thus, the hypocholesterolemic action of HPH involved the inhibition of jejunal cholesterol absorption. The cattle heart protein hydrolysate ultra-filtrate (HPHU, MW < ca. 1,000 Da peptide fraction) derived from HPH imparted stronger hypocholesterolemic activity than HPH in rats.