Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia.

Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia.

Interleukin-1ß triggers muscle-derived extracellular superoxide dismutase expression and protects muscles from doxorubicin-induced atrophy.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1ß (IL-1ß) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1ß treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1ß treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1ß signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1ß (IL-1ß) expression in cultured myotubes and skeletal muscle, whereas transient IL-1ß treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1ß signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

Regular exercise stimulates endothelium autophagy via IL-1 signaling in ApoE deficient mice.

Regular exercise maintains arterial endothelial cell homeostasis and protects the arteries from vascular disease, such as peripheral artery disease and atherosclerosis. Autophagy, which is a cellular process that degrades misfolded or aggregate proteins and damaged organelles, plays an important role in maintaining organ and cellular homeostasis. However, it is unknown whether regular exercise stimulates autophagy in aorta endothelial cells of mice prone to atherosclerosis independently of their circulating lipid profile. Here, we observed that 16 weeks of voluntary exercise reduced high-fat diet-induced atherosclerotic plaque formation in the aortic root of ApoE deficient mice, and that this protection occurred without changes in circulating triglycerides, total cholesterol, and lipoproteins. Immunofluorescence analysis indicated that voluntary exercise increased levels of the autophagy protein LC3 in aortic endothelial cells. Interestingly, human umbilical vein endothelial cells (HUVECs) exposed to serum from voluntarily exercised mice displayed significantly increased LC3-I and LC3-II protein levels. Analysis of circulating cytokines demonstrated that voluntary exercise caused changes directly relevant to IL-1 signaling (ie, decreased interleukin-1 receptor antagonist [IL-1ra] while also increasing IL-1α). HUVECs exposed to IL-1α and IL-1ß recombinant protein significantly increased LC3 mRNA expression, LC3-I and LC3-II protein levels, and autophagy flux. Collectively, these results suggest that regular exercise protects arteries from ApoE deficient mice against atherosclerosis at least in part by stimulating endothelial cell autophagy via enhanced IL-1 signaling.

p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

Increased muscle contractile activity, as observed with regular exercise, prevents oxidative stress-induced muscle wasting, at least partially, by improving the antioxidant defense system. Phosphorylated p62/sequestosome1 competitively binds to the Kelch-like ECH-associated protein 1, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which stimulates transcription of antioxidant/electrophile responsive elements. However, it remains to be determined if this process is activated by regular exercise in skeletal muscle. Here, we demonstrate that muscle contractile activity increases antioxidants, Nrf2 translocation into nuclei, and Nrf2 DNA-binding activity in association with increased p62 phosphorylation (Ser351) in mouse oxidative skeletal muscle. Skeletal muscle-specific loss of Nrf2 [i.e., Nrf2 muscle-specific knockout (mKO) mice] abolished the expression of the Nrf2 target antioxidant gene NAD(P)H-quinone oxidoreductase 1 (NQO1) in both glycolytic and oxidative muscles but reduced exercise-mediated increases of antioxidants (i.e., copper/zinc superoxide dismutase (SOD) and extracellular SOD only in oxidative muscle. Interestingly, skeletal muscle-specific loss of p62 (i.e., p62 mKO mice) also abolished the expression of NQO1 and reduced exercise-mediated increases of the same antioxidants in soleus muscle. Collectively, these findings indicate that p62 and Nrf2 cooperatively regulate the exercise-mediated increase of antioxidants in oxidative muscle.-Yamada, M., Iwata, M., Warabi, E., Oishi, H., Lira, V. A., Okutsu, M. p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

Muscle-derived SDF-1α/CXCL12 modulates endothelial cell proliferation but not exercise training-induced angiogenesis.

Chemokines are critical mediators of angiogenesis in several physiological and pathological conditions; however, a potential role for muscle-derived chemokines in exercise-stimulated angiogenesis in skeletal muscle remains poorly understood. Here, we postulated that the chemokine stromal cell-derived factor-1 (SDF-1α/C-X-C motif chemokine ligand 12: CXCL12), shown to promote neovascularization in several organs, contributes to angiogenesis in skeletal muscle. We found that CXCL12 is abundantly expressed in capillary-rich oxidative soleus and exercise-trained plantaris muscles. CXCL12 mRNA and protein were also abundantly expressed in muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α transgenic mice, which have a high proportion of oxidative muscle fibers and capillaries when compared with wild-type littermates. We then generated CXCL12 muscle-specific knockout mice but observed normal baseline capillary density and normal angiogenesis in these mice when they were exercise trained. To get further insight into a potential CXCL12 role in a myofiber-endothelial cell crosstalk, we first mechanically stretched C2C12 myotubes, a model known to induce stretch-related chemokine release, and observed increased CXCL12 mRNA and protein. Human umbilical vein endothelial cells (HUVECs) exposed to conditioned medium from cyclically stretched C2C12 myotubes displayed increased proliferation, which was dependent on CXCL12-mediated signaling through the CXCR4 receptor. However, HUVEC migration and tube formation were unaltered under these conditions. Collectively, our findings indicate that increased muscle contractile activity enhances CXCL12 production and release from muscle, potentially contributing to endothelial cell proliferation. However, redundant signals from other angiogenic factors are likely sufficient to sustain normal endothelial cell migration and tube formation activity, thereby preserving baseline capillary density and exercise training-mediated angiogenesis in muscles lacking CXCL12.

Veno-venous extracorporeal membrane oxygenation (ECMO) for acute respiratory failure caused by liver abscess.

Liver abscess remains a life-threatening disease, particularly when it results in systemic organ failure necessitating intensive care. Only few cases of respiratory failure caused by liver abscess and treated with veno-venous extracorporeal membrane oxygenation (ECMO) have been reported. Here we present a case of liver abscess with rapid progression of multiple organ dysfunction, including severe acute respiratory failure on admission to the intensive care unit (ICU). Upon admission, we immediately initiated artificial organ support systems, including ventilator, continuous renal replacement therapy, and cardiovascular drug infusion for septic multiple organ failure and source control. Despite this initial management, respiratory failure deteriorated and V-V ECMO was introduced. The case developed abdominal compartment syndrome, for which we performed a bedside decompressive laparotomy in the ICU. The case gradually recovered from multiple organ failure and was discharged from the ICU on day 22 and from the hospital on day 53. Since liver abscess is potentially lethal and respiratory failure on admission is an additional risk factor of mortality, V-V ECMO may serve as an adjunctive choice of artificial organ support for cases of severe acute respiratory failure caused by liver abscess.

Role of nuclear localization of PSMB1 in transcriptional activation.

The production of retinol binding protein 4 (RBP4) is higher in adipose tissue in type 2 diabetes. We have found that proteasome subunit beta type 1 (PSMB1) is a transcriptional activator of Rbp4. In the present study, the putative tyrosine phosphorylation site in PSMB1 was mutated to phenylalanine. The mutated form of PSMB1 displayed increased nuclear translocation, resulting in activation of transcription in adipocytes.

Measurement of angular distribution of soft X-ray radiation from thin targets in the tabletop storage ring MIRRORCLE-20SX.

The only available tabletop electron storage rings are the machines from the MIRRORCLE series. The electrons are accelerated in a microtron and injected into the storage ring. During its circulation, each electron passes through a tiny target many times, emitting a photon beam. Both the spectrum and the angular distribution of the radiation depend on the material, the thickness and the shape of the target. In this paper measured angular distributions of the radiation from several different targets in the magnetic field of the 20 MeV storage ring MIRRORCLE-20SX are presented. The detector comprises a 3 mm × 3 mm × 8.5 µm plastic scintillator (PS) coupled to a photomultiplier by a bundle of optical fibers. The output of the photomultiplier is digitized by an IF converter. This detector is sensitive mostly to soft X-ray radiation, and its PS is moved by a mechanical system in a plane perpendicular to the radiation axis. The measured angular distributions for Mo and Sn targets contain an annulus which is attributed to transition radiation. The angular distributions for Al, carbon nanotube and diamond-like carbon (DLC) targets show some suppression of the radiation along the magnetic field. This is the first evidence of observation of the angular distribution of synchrotron Cherenkov radiation, which represents Cherenkov radiation in a magnetic field. The power radiated from the DLC target is estimated.

Estimation of surface structure and carbon monoxide oxidation site of shape-controlled Pt nanoparticles.

Surface structures of shape-controlled Pt nanoparticles have been estimated using cyclic voltammetry (CV) and infrared reflection absorption spectroscopy (IRAS). Cubic and cuboctahedral Pt nanoparticles are prepared using a capping polymer. These nanoparticles give CVs similar to those of single crystal electrodes of Pt in sulfuric acid solution. The CV of cubic nanoparticles is similar to that of the Pt(510) [=5(100)-(110)] electrode, while the CV of cuboctahedral nanoparticles is reproduced well with the convolution of Pt(766) [=13(111)-(100)] and Pt(17 1 1) [=9(100)-(111)] electrodes. These results suggest that the planes of the cubic and cuboctahedral nanoparticles are composed of step-terrace and atomically flat terraces, respectively. Adsorbed carbon monoxide (CO) on the shape-controlled nanoparticles gives the IR bands that are assigned to on-top and bridged CO. The band of on-top CO is deconvoluted to two bands: the higher and the lower frequency bands are assigned to the CO on the plane and the edges of the nanoparticles, respectively. On-top CO adsorbed on the edges is oxidized at more negative potential than that on the planes. Edge sites of the nanoparticles promote CO oxidation.

Fabrication of a tubular FeCo bimetallic nanostructure using a cellulose-cobalt hexacyanoferrate composite as a precursor.

A tubular fibre structure with a ca. 9-11 microm outer diameter comprised of FeCo nanoparticle assemblies with an amorphous carbon core was fabricated via reductive thermal decomposition of a cellulose-cobalt hexacyanoferrate (Fe-CN-Co) composite material.

Maternal exercise training attenuates endotoxin-induced sepsis in mice offspring.

Regular exercise during pregnancy can prevent offspring from several diseases, such as cardiovascular diseases, obesity, and type II diabetes during adulthood. However, little information is available about whether maternal exercises during pregnancy protect the offspring from infectious diseases, such as sepsis and multiple organ dysfunction syndrome (MODS). This study aimed to investigate whether maternal exercise training protects the offspring from endotoxin-induced septic shock in mice. Female C57BL/6 mice performed voluntary wheel exercises during pregnancy. All dams and offspring were fed normal chow with sedentary activity during lactation and after weaning. At 10-week-old, mice were intraperitoneally injected a lethal (30 mg/kg) or nonlethal (15 mg/kg) dose of lipopolysaccharide (LPS), following which the survival of mice that were administered a lethal dose was monitored for 60 h. Plasma, lung, and liver samples were collected 18 h after the injection to evaluate the cytokine concentration or mRNA expression from those administered a nonlethal dose. Although maternal exercise training could not prevent lethality during an LPS-induced septic shock, it significantly inhibited the LPS-induced loss of body weight in female offspring. Regular maternal exercise significantly inhibited the mRNA expression of the LPS-induced inflammatory cytokines, such as interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), in the plasma and liver. Thus, maternal exercise inhibited the LPS-induced inflammatory response in female offspring, suggesting that regular exercise during pregnancy could be a potential candidate of the onset of sepsis and MODS in offspring.

Well-leg compartment syndrome after laparoscopic low anterior resection for lower rectal cancer in the lithotomy position: A case report.

A 64-year-old man underwent laparoscopic low anterior resection for lower rectal cancer. Because he was overweight (BMI, 28.1 kg/m2 ) with rich visceral fat and a narrow pelvic cavity, the operation was technically difficult and the operation time was 686 min. Postoperatively, the patient immediately complained of pain and swelling of the left lower limb. Laboratory examination showed that serum creatinine kinase was markedly increased and urine myoglobin was positive on postoperative day 1. He was diagnosed with well-leg compartment syndrome and was transported to the trauma and critical care center for emergency fasciotomy. After initial treatment, he was transferred to our hospital on postoperative day 7. He completely recovered after 2 months. Although well-leg compartment syndrome is rare, it is necessary to consider it as a potential complication when performing a long colorectal cancer surgery with the patient in the lithotomy position, particularly when laparoscopic surgery is planned.

Cytotoxic effect of spermine on retinal pigment epithelial cells.

PURPOSE: A prior study showed inactivation of ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells by a specific irreversible inhibitor (5-fluoromethylornithine; 5-FMO) leading to cell death, in an in vitro model of gyrate atrophy (GA) of the choroid and retina. In the present study, the cytotoxicity of metabolites of ornithine, especially spermine, in RPE cells was investigated, to clarify the mechanism of ornithine cytotoxicity in RPE cells. METHODS: RPE cells were incubated with ornithine or compounds involved in ornithine metabolic pathways. The effects on RPE cell viability and proliferative activity were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and [(3)H]thymidine incorporation assays. Incorporation of spermine into RPE cells was examined by using [(14)C]spermine and dansyl-spermine. To assess spermine-induced RPE cell death, cells were double stained with annexin V and propidium iodide and subjected to flow cytometry. RESULTS: Ornithine, arginine, glutamate, proline, creatine, glycine, and putrescine exhibited no effects on the viability and proliferative activities of RPE cells, whereas spermidine and spermine (10 mM) inhibited [(3)H]thymidine incorporation by 13% and 89%, respectively. The inhibition of [(3)H]thymidine incorporation by spermine was dose dependent and was observed as early as 4 hours after addition. Further, spermine was incorporated and accumulated in the perinuclear region of RPE cells. Apoptotic RPE cell death was induced by spermine in a dose-dependent manner. CONCLUSIONS: The present results demonstrated that excessive spermine is cytotoxic to RPE cells and suggest that metabolites of ornithine, especially spermine, may be involved in the mechanism of RPE degeneration in GA.

Preparation of single-crystalline platinum nanowires with small diameters under mild conditions.

We report a convenient method to synthesize single-crystalline platinum nanowires with high aspect ratio of ca. 2.0 nm diameter by sophisticated and precise control of Pt(0) nuclei and their growth.

Temporal evolution of composition and crystal structure of cobalt hexacyanoferrate nano-polymers synthesized in reversed micelles.

Nanometer-size metal coordination polymers are fascinating to explore, since their unique properties are controlled by a large ratio of surface atoms, which is an entirely different effect from that in a bulk crystal. In this report, we have demonstrated the reaction time-induced structural conversion of nanometer-sized metal coordination nano-polymers (MCNPs). The MCNP selected here was a Prussian blue analogue, cobalt hexacyanoferrate (Fe-CN-Co) with ca. 3 nm. When Fe-CN-Co MCNPs were synthesized in reverse micelles of cationic surfactants, cetyltrimethylammonium halides [CTAX, X=B (bromide), C (chloride)], their color dramatically changed from red to green with increasing the reaction time. We investigated the mechanism of this characteristic color change using XRD, FT-IR, UV-vis spectra, CHN elemental analyses, ICP, and TGA, which indicated that the coordination geometry of Co(II) ions was changed from a 6-coordinate octahedral (Oh) to a 4-coordinate tetrahedral (Td) with clear crystal distortion. The magnetic behavior of the prepared Fe-CN-Co MCNPs was also reaction-time dependent, as illustrated by SQUID and 57Fe Mössbauer spectra.

Self-assembly of discotic liquid crystalline molecule-modified gold nanoparticles: control of 1D and hexagonal ordering induced by solvent polarity.

Gold nanoparticles fully coated with discotic liquid crystalline molecules of hexaalkoxy-substituted triphenylene (Au-TP) have been synthesised, the self-assembled structure of which could be controlled (hexagonal or 1D nanochain) just by altering the ratio of methanol to toluene in the solvent.

Novel synthetic approach to creating PtCo alloy nanoparticles by reduction of metal coordination nano-polymers.

PtCo alloy nanoparticles with different metal elemental ratios (Pt/Co = 0.9, 1.6, 2.9, and 3.6) were prepared by a novel synthetic approach, using the transformation reaction of platinum(IV)/cobalt tetracyanoplatinate metal coordination nano-polymers (Pt(II)-CN-Pt(IV)/Co)via a H2 gas-phase reduction.

Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.

PURPOSE: To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. METHODS: RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine. RESULTS: An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells. CONCLUSIONS: The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.