The study on mechanism of the modified Chinese herbal compound, jianpijiedu, on a mouse model of hepatic carcinoma cachexia.

Various studies have investigated hepatic carcinoma cachexia, however, there is little published information regarding the effect of Chinese Medicine carcinoma cachexia. The present study was performed to investigate the effect of modified Chinese herbal compound jianpijiedu (MJPJD) on a mouse model of ascites­induced hepatic carcinoma cachexia. C57BL/6 mice were randomized to five groups: Control (Group A); xenograft tumor (Group B); low concentration of MJPJD (Group C); high concentration of MJPJD (Group D) and medroxyprogesterone (MPA) combined with indometacin (IND; Group E). The mouse model of ascites­induced hepatic carcinoma cachexia was established by abdominal injection of H22 hepatic carcinoma cells. Subsequently, the body weight, food intake and gastrocnemius weight were recorded, and the levels of interleukin (IL)­lα, IL­6, tumor necrosis factor­α (TNF­α) in ascites were detected by enzyme­linked immunosorbent assay. The protein expression levels of muscle RING­finger protein­1 (MU­RF1) and atrogin 1 were detected by western blotting and immunohistochemistry, and the mRNA levels in gastrocnemius were detected by reverse transcription­quantitative polymerase chain reaction. Compared with the xenograft tumor group, the administration of MJPJD inhibited the increase in body weight and the volume of ascites, the consumption of gastrocnemius was reduced, the net weight of ascites was maintained, the food intake was enhanced and the levels of the cytokines IL­lα, IL­6, TNF­α in ascites and the levels of MU­RF1 and atrogin 1 proteins were reduced. These results indicated that MJPJD delays the pathological process of ascites­induced hepatic carcinoma cachexia, and the mechanism of action may be correlated with a reduction in the levels of IL­lα, IL­6, TNF­α and inhibiting the activation of the ubiquitin proteosome pathway.

Morphological Alterations in Gastrocnemius and Soleus Muscles in Male and Female Mice in a Fibromyalgia Model.

BACKGROUND: Fibromyalgia (FM) is a chronic musculoskeletal pain disorder, characterized by chronic widespread pain and bodily tenderness and is often accompanied by affective disturbances, however often with unknown etiology. According to recent reports, physical and psychological stress trigger FM. To develop new treatments for FM, experimental animal models for FM are needed to be development and characterized. Using a mouse model for FM including intermittent cold stress (ICS), we hypothesized that ICS leads to morphological alterations in skeletal muscles in mice. METHODS: Male and female ICS mice were kept under alternating temperature (4 °C/room temperature [22 °C]); mice constantly kept at room temperature served as control. After scarification, gastrocnemius and soleus muscles were removed and snap-frozen in liquid nitrogen-cooled isopentane or fixed for electron microscopy. RESULTS: In gastrocnemius/soleus muscles of male ICS mice, we found a 21.6% and 33.2% decrease of fiber cross sectional area (FCSA), which in soleus muscle concerns the loss of type IIa and IIx FCSA. This phenomenon was not seen in muscles of female ICS mice. However, this loss in male ICS mice was associated with an increase in gastrocnemius of the density of MIF+ (8.6%)-, MuRF+ (14.7%)-, Fbxo32+ (17.8%)-cells, a 12.1% loss of capillary contacts/muscle fiber as well as a 30.7% increase of damaged mitochondria in comparison with male control mice. Moreover, significant positive correlations exist among densities (n/mm(2)) of MIF+, MuRF+, Fbxo32+-cells in gastrocnemius/ soleus muscles of male ICS mice; these cell densities inversely correlate with FCSA especially in gastrocnemius muscle of male ICS mice. CONCLUSION: The ICS-induced decrease of FCSA mainly concerns gastrocnemius muscle of male mice due to an increase of inflammatory and atrogenic cells. In soleus muscle of male ICS and soleus/gastrocnemius muscles of female ICS mice morphological alterations seem to occur not at all or delayed. The sex-specificity of findings, which is not easily reconciled with the epidemiology of FM (female predominance), implicate that gastrocnemius muscle of male ICS mice should preferentially be used for future investigations with FM. Moreover, we suggest to investigate morphological and/or molecular alterations at different time-points (up to two weeks) after ICS.

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

MAPK signaling in the quadriceps of patients with chronic obstructive pulmonary disease.

Muscle atrophy in chronic obstructive pulmonary disease (COPD) is associated with reduced exercise tolerance, muscle strength, and survival. The molecular mechanisms leading to muscle atrophy in COPD remain elusive. The mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK 1/2 can increase levels of MAFbx/Atrogin and MuRF1, which are specifically involved in muscle protein degradation and atrophy. Our aim was to investigate the level of activation of p38 MAPK, ERK 1/2, and JNK in the quadriceps of patients with COPD. A biopsy of the quadriceps was obtained in 18 patients with COPD as well as in 9 healthy controls. We evaluated the phosphorylated as well as total protein levels of p38 MAPK, ERK 1/2, and JNK as well as MAFbx/Atrogin and MuRF1 in these muscle samples. The corresponding mRNA expression was also assessed by RT-PCR. Ratios of phosphorylated to total level of p38 MAPK (P = 0.02) and ERK 1/2 (P = 0.01) were significantly elevated in patients with COPD compared with controls. Moreover, protein levels of MAFbx/Atrogin showed a tendency to be greater in patients with COPD (P = 0.08). mRNA expression of p38 MAPK (P = 0.03), ERK 1/2 (P = 0.02), and MAFbx/Atrogin (P = 0.04) were significantly elevated in patients with COPD. In addition, phosphorylated-to-total p38 MAPK ratio (Pearson's r = -0.45; P < 0.05) and phosphorylated-to-total ERK 1/2 ratio (Pearson's r = -0.47; P < 0.05) were negatively associated with the mid-thigh muscle cross-sectional area. These data support the hypothesis that the MAPKs might play a role in the development of muscle atrophy in COPD.

Selective diaphragm muscle weakness after contractile inactivity during thoracic surgery.

RATIONALE: Postoperative pulmonary complications are significant contributors to morbidity in patients who have undergone upper abdominal, thoracic, or cardiac surgery. The pathophysiology of these complications might involve postoperative inspiratory muscle weakness. The nature of postoperative inspiratory muscle weakness is unknown. OBJECTIVE: To investigate the effect of surgery on the functioning of the diaphragm, the main muscle of inspiration. METHODS: Serial biopsies from the diaphragm and the latissimus dorsi muscle were obtained from 6 patients during thoracotomy for resection of a tumor in the right lung. Biopsies were taken as soon as the diaphragm had been exposed (t(0)) and again after 2 hours (t(2)). The contractile performance of demembranated muscle fibers, as well as fiber morphology and markers for proteolysis, was determined. RESULTS: In all patients, the force-generating capacity of diaphragm muscle fibers at t(2) was significantly reduced (~35%) compared with that at t(0), with a more pronounced force loss in type 2 fibers compared with type 1 fibers. Diaphragm weakness was not part of a generalized muscle weakness as contractile performance of latissimus dorsi fibers was preserved at t(2). Diaphragm fiber size and myofibrillar structure were not different at t(2) compared with t0, but myosin heavy chain type 2 was significantly reduced at t(2) and MuRF-1 mRNA and protein levels were elevated at t(2). CONCLUSIONS: Only 2 hours of thoracic surgery causes marked, and selective, diaphragm muscle fiber weakness.

Nucleolar targeting of the fbw7 ubiquitin ligase by a pseudosubstrate and glycogen synthase kinase 3.

E3 ubiquitin ligases catalyze protein degradation by the ubiquitin-proteasome system, and their activity is tightly controlled. One level of regulation involves subcellular localization, and the Fbw7 tumor suppressor exemplifies this type of control. Fbw7 is the substrate-binding component of an SCF ubiquitin ligase that degrades critical oncoproteins. Alternative splicing produces three Fbw7 protein isoforms that occupy distinct compartments: Fbw7α is nucleoplasmic, Fbw7ß is cytoplasmic, and Fbw7γ is nucleolar. We found that cancer-associated Fbw7 mutations that disrupt substrate binding prevent Fbw7γ nucleolar localization, implicating a substrate-like interaction in nucleolar targeting. We identified EBNA1-binding protein 2 (Ebp2) as the critical nucleolar factor that directly mediates Fbw7 nucleolar targeting. Ebp2 binds to Fbw7 like a substrate, and this is mediated by an Ebp2 degron that is phosphorylated by glycogen synthase kinase 3. However, despite these canonical substrate-like interactions, Fbw7 binding is largely uncoupled from Ebp2 turnover in vivo. Ebp2 thus acts like a pseudosubstrate that directly recruits Fbw7 to nucleoli.

An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis.

Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30-conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30-conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30-conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis.

Effects of exercise training on atrophy gene expression in skeletal muscle of mice with chronic allergic lung inflammation

We evaluated the effects of chronic allergic airway inflammation and of treadmill training (12 weeks) of low and moderate intensity on muscle fiber cross-sectional area and mRNA levels of atrogin-1 and MuRF1 in the mouse tibialis anterior muscle. Six 4-month-old male BALB/c mice (28.5 ± 0.8 g) per group were examined: 1) control, non-sensitized and non-trained (C); 2) ovalbumin sensitized (OA, 20 µg per mouse); 3) non-sensitized and trained at 50 percent maximum speed _ low intensity (PT50 percent); 4) non-sensitized and trained at 75 percent maximum speed _ moderate intensity (PT75 percent); 5) OA-sensitized and trained at 50 percent (OA+PT50 percent), 6) OA-sensitized and trained at 75 percent (OA+PT75 percent). There was no difference in muscle fiber cross-sectional area among groups and no difference in atrogin-1 and MuRF1 expression between C and OA groups. All exercised groups showed significantly decreased expression of atrogin-1 compared to C (1.01 ± 0.2-fold): PT50 percent = 0.71 ± 0.12-fold; OA+PT50 percent = 0.74 ± 0.03-fold; PT75 percent = 0.71 ± 0.09-fold; OA+PT75 percent = 0.74 ± 0.09-fold. Similarly significant results were obtained regarding MuRF1 gene expression compared to C (1.01 ± 0.23-fold): PT50 percent = 0.53 ± 0.20-fold; OA+PT50 percent = 0.55 ± 0.11-fold; PT75 percent = 0.35 ± 0.15-fold; OA+PT75 percent = 0.37 ± 0.08-fold. A short period of OA did not induce skeletal muscle atrophy in the mouse tibialis anterior muscle and aerobic training at low and moderate intensity negatively regulates the atrophy pathway in skeletal muscle of healthy mice or mice with allergic lung inflammation.

Effects of exercise training on atrophy gene expression in skeletal muscle of mice with chronic allergic lung inflammation.

We evaluated the effects of chronic allergic airway inflammation and of treadmill training (12 weeks) of low and moderate intensity on muscle fiber cross-sectional area and mRNA levels of atrogin-1 and MuRF1 in the mouse tibialis anterior muscle. Six 4-month-old male BALB/c mice (28.5 +/- 0.8 g) per group were examined: 1) control, non-sensitized and non-trained (C); 2) ovalbumin sensitized (OA, 20 microg per mouse); 3) non-sensitized and trained at 50% maximum speed _ low intensity (PT50%); 4) non-sensitized and trained at 75% maximum speed _ moderate intensity (PT75%); 5) OA-sensitized and trained at 50% (OA+PT50%), 6) OA-sensitized and trained at 75% (OA+PT75%). There was no difference in muscle fiber cross-sectional area among groups and no difference in atrogin-1 and MuRF1 expression between C and OA groups. All exercised groups showed significantly decreased expression of atrogin-1 compared to C (1.01 +/- 0.2-fold): PT50% = 0.71 +/- 0.12-fold; OA+PT50% = 0.74 +/- 0.03-fold; PT75% = 0.71 +/- 0.09-fold; OA+PT75% = 0.74 +/- 0.09-fold. Similarly significant results were obtained regarding MuRF1 gene expression compared to C (1.01 +/- 0.23-fold): PT50% = 0.53 +/- 0.20-fold; OA+PT50% = 0.55 +/- 0.11-fold; PT75% = 0.35 +/- 0.15-fold; OA+PT75% = 0.37 +/- 0.08-fold. A short period of OA did not induce skeletal muscle atrophy in the mouse tibialis anterior muscle and aerobic training at low and moderate intensity negatively regulates the atrophy pathway in skeletal muscle of healthy mice or mice with allergic lung inflammation.

The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Myocardial expression of Murf-1 and MAFbx after induction of chronic heart failure: Effect on myocardial contractility.

OBJECTIVE: In chronic heart failure (CHF) the myocardial expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), which is thought to contribute to myocardial remodeling, was found to be increased. However, it is unknown whether the E3-ubiquitin ligases MAFbx and Murf-1 are involved in this remodeling process and whether their expression is regulated by TNF-alpha. METHODS: Rats underwent ligation of the left coronary artery to induce CHF or were sham-operated. The expression of MAFbx/Murf-1 and troponin I was analyzed by RT-PCR and Western blotting in the non-infarcted area of the left ventricle. In cell culture experiments the potency of TNF-alpha to stimulate Murf-1/MAFbx expression, the intracellular signaling pathway, and the involvement of the E3-ligases for the impairment of contractility were assessed. RESULTS: In CHF the myocardial expression of TNF-alpha was elevated 3.1-fold as compared to control. This was associated with a 4.5-fold and 2.7-fold increase in MAFbx and Murf-1 expression, respectively. A positive correlation between TNF-alpha and the expression of MAFbx or Murf-1 was evident. In neonatal rat cardiomyocytes, TNF-alpha induced the expression of MAFbx through p38MAPK-dependent pathways, whereas the induction of Murf-1 required the activation of the p42/44 MAPK pathway. Exposure of cardiomyocytes to TNF-alpha resulted in troponin I ubiquitinylation, subsequent degradation, and a decline in contractility. This was completely abrogated by siRNAs against Murf-1/MAFbx. CONCLUSION: TNF-alpha, which is increasingly expressed in CHF, induces troponin I degradation through a MAFbx/Murf-1-dependent pathway. This was associated with an impairment of contractility and might be one mechanism involved in the adverse remodeling process in CHF.

Phosphorylated IkappaBalpha is a component of Lewy body of Parkinson's disease.

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.

Assist-control mechanical ventilation attenuates ventilator-induced diaphragmatic dysfunction.

Controlled mechanical ventilation induced a profound diaphragm muscle dysfunction and atrophy. The effects of diaphragmatic contractions with assisted mechanical ventilation on diaphragmatic isometric, isotonic contractile properties, or the expression of muscle atrophy factor-box (MAF-box), the gene responsible for muscle atrophy, are unknown. We hypothesize that assisted mechanical ventilation will preserve diaphragmatic force and prevent overexpression of MAF-box. Studying sedated rabbits randomized equally into control animals, those with 3 days of assisted ventilation, and those with controlled ventilation, we assessed in vitro diaphragmatic isometric and isotonic contractile function. The concentrations of contractile proteins, myosin heavy chain isoform, and MAF-box mRNA were measured. Tetanic force decreased by 14% with assisted ventilation and 48% with controlled ventilation. Maximum shortening velocity tended to increase with controlled compared with assisted ventilation and control. Peak power output decreased 20% with assisted ventilation and 41% with controlled ventilation. Contractile proteins were unchanged with either modes of ventilation; myosin heavy chain 2X mRNA tended to increase and that of 2A to decrease with controlled ventilation. MAF-box gene was overexpressed with controlled ventilation. We conclude that preserving diaphragmatic contractions during mechanical ventilation attenuates the force loss induced by complete inactivity and maintains MAF-box gene expression in control.