PHOSPHODIESTERASE-4 INHIBITION REDUCES PROTEOLYSIS AND ATROGENES EXPRESSION IN RAT SKELETAL MUSCLES

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371-381, 2011

TIME COURSE OF SKELETAL MUSCLE LOSS AND OXIDATIVE STRESS IN RATS WITH WALKER 256 SOLID TUMOR

Reactive oxygen species oxidize proteins and modulate the proteasomal system in muscle-wasting cancer cachexia. On day 5 (D5), day 10 (D10), and day 14 (D14) after tumor implantation, skeletal muscle was evaluated. Carbonylated proteins and thiobarbituric acid reactive substances were measured. Chemiluminescence was employed for lipid hydroperoxide estimation. Glutathione, superoxide dismutase, and total radical antioxidant capacity were evaluated. The proteasomal system was assessed by mRNA atrogin-1 expression. Increased muscle wasting, lipid hydroperoxide, and superoxide dismutase, and decreased glutathione levels and total radical antioxidant capacity, were found on D5 in accordance with increased mRNA atrogin-1 expression. All parameters were significantly modified in animals treated with alpha-tocopherol. The elevation in aldehylde levels and carbonylated proteins observed on D10 were reversed by cc-tocopherol treatment. Oxidative stress may trigger signal transduction of the proteasomal system and cause protein oxidation. These pathways may be associated with the mechanism of muscle wasting that occurs in cancer cachexia. Muscle Nerve 42: 950-958, 2010

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by > 60%, but also the level of myosin heavy chains (MHCs) by > 40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Preliminary analysis of miRNA pathway in Schistosoma mansoni

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational at-rest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. in this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma Mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 Putative proteins in the parasite. In addition, the transcript levels of SinDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro Cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni. (C) 2008 Published by Elsevier Ireland Ltd.

Angiotensin processing is partially carried out by carboxypeptidases in the rat mesenteric arterial bed perfusate

Here we investigated the possible association between the carboxypeptidase A (CPA)-like activity of the rat mesenteric arterial bed (MAB) perfusate and the ability of this fluid of forming angiotensin (Ang) 1-9 and Ang 1-7 upon incubation with Ang I and Ang II, respectively. Initially, we observed that anion exchange chromatography of the perfusate would consistently split the characteristic Z-Val-Phe-hydrolyzing activity of CPA-like enzymes into five distinct peaks, whose proteolytic activities were then determined using also Ang I and Ang II as substrates. The resulting proteolytic profile for each peak indicated that rat MAB perfusate contains a complex mixture of carboxypeptidases; tentatively, five carboxypeptidases were distinguished based on their substrate preferences toward Z-Val-Phe. Ang I and Ang II. The respective reactions, namely, Z-Val-Phe cleavage, Ang I to Ang 1-9 conversion and Ang II to Ang 1-7 conversion, were inhibited by 1,10-phenanthroline and nearly fully blocked by potato carboxypeptidase inhibitor. Also, all the CPA-like activity peaks prepared by anion exchange chromatography were tested negative for contaminating Ang I-converting enzyme-2, cathepsin A and prolylcarboxypeptidase. Overall, our results indicate that rat MAB perfusate contains a multiplicity of Ang I and Ang II-processing CPA-like enzymes whose proteolytic specificities suggest they might perform peculiar regulatory roles in the local resin-angiotensin system. (C) 2008 Elsevier B.V. All rights reserved.

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats

The present work investigated the role of the sympathetic nervous system (SINS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0,007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-depenclent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.

Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins

The sumoylation pathway is a post-translational modification of nuclear proteins widespread among several organisms. SMT3C is the main protein involved in this process and it is covalently conjugated to a diverse assortment of nuclear protein targets. To date, 3 SUMO paralogues (SMT3C, A/B) have been characterized in mammals and plants. In this work we characterized two SUMO related genes, named SMT3B and SMT3C throughout Schistosoma mansoni life cycle. The SmSMTB/C encodes for proteins sharing significant amino acid homology with SMT3. Phylogenetical analyses revealed that both SmSMT3B/C are distinct proteins. Additionally, SmSMT3B and C are expressed in cercariae, adult worms, eggs and schistosomula however SinSMT3C gene showed an expression level 7 to 9 fold higher than SmSMT3B in eggs, schistosomula and adult worms. The comparison between the SmSMT3C genomic and cDNA sequences established that the encoding sequence is interrupted by 3 introns of 70, 37 and 36 bp. Western Blot has shown SMT3 conjugates are present in nuclear and total protein fractions of adults and cercariae. Therefore our results suggest a functional sumoylation pathway, and the presence of two paralogues also suggests the specificity of substrates for SMT3 in S. mansoni. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

Coxsackievirus 135 induced apoptosis of HeLa cells: Effects on p53 and SUMO

Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system. (C) 2009 Elsevier Inc. All rights reserved.

Determination of Kininogens Levels and Kallikrein/Kininase II Activities in Patients with Thromboangiitis Obliterans

Some components of the kinin system such as plasma kallikrein levels, the activities of tissue kallikrein (including saliva) and kininase II and the concentrations of kininogen fractions (low-molecular weight/LKg and high-molecular weight/HKg) were evaluated in the plasma of patients with thromboangiitis obliterans (TAO) presenting clinical symptoms of the condition. Twenty TAO were diagnosed by means of the traditional Shionoya and Olin criteria and later classified into non-smokers (n = 11) and active smokers (n = 9). Fifty-three normal, non-smoking/smoking individuals (control) were also studied. Kininogen levels were determined by ELISA; the activities of kallikreins and kininase II were determined using selective substrates. The levels of enzymes (kallikreins and kininase II) and protein (kininogens) were significantly higher in patients with TAO who were active smokers compared to the control groups (no matter whether control individuals were active smokers or non-smokers, P < 0.001 for all comparisons). Interestingly, regardless of the time of disease onset, a significant increase in the levels of these components of the kinin system was also observed in patients when TAO active smokers were compared with TAO ex-smokers (P < 0.01 for all analysed parameters). Activation of the kinin system in patients with TAO may indicate the involvement of vasodilatation in an attempt to control vascular changes, thereby favouring the deposition of immune complexes at the vascular level because of nicotine stimulation. Moreover, our results corroborate the idea that TAO can be an autoimmune disorder with specific mechanisms.

Use of alprostadil, a stable prostaglandin E(1) analogue, for the attenuation of rat skeletal muscle ischemia and reperfusion injury

Aim. Some stable prostaglandin analogues such as alprostadil have been used to attenuate the deleterious effects of ischemia and reperfusion injury. The aim of this paper was to test if alprostadil can decrease the ischemia- reperfusion injury in rat skeletal muscle using muscular enzymes as markers, such as aspartate aminotransferase (AST), creatine kinase (CPK), lactate dehydrogenase (LDH); degeneration products of cell membrane-malondialdehyde (MDA) and muscle glycogen storage. Methods. Thirty male Wistar rats were used in a model of hind limb ischemia achieved by infrarenal aortic cross-clamping. The animals were randomized into three equal groups (N=10) submitted to 5 hours of ischemia followed by one hour of reperfusion. The first group (control) received continuous intravenous infusion of saline solution and the second group (preischemia, GPI) received continuous intravenous infusion of alprostadil throughout the experiment starting 20 minutes before the aortic cross-clamping. The third group, prereperfusion (GPR), received alprostadil only during the reperfusion period, with intravenous infusion being started 10 min before the clamp release. Results. There was no difference in CPK, LDH, AST or tissue glycogen values between groups. However, a significant elevation in MDA was observed in the GPI and GPR groups compared to the control group, with no difference between the GPI and GPR. Conclusion. Under conditions of partial skeletal muscle ischemia, alprostadil did not reduce the release of muscular enzymes, the consumption of tissue glycogen or the effects of ischemia and reperfusion on the cell membrane, characterized by lipid peroxidation.

Influence of quinidine, fluvoxamine, and ketoconazole on the enantioselective pharmacokinetics of citalopram in rats

Citalopram (CITA) is available as a racemic mixture or as (+)-(S)-CITA. In humans, CITA is metabolized to demethylcitalopram (DCITA) by CYP2C19, CYP2D6, and CYP3A and to didemethylcitalopram by CYP2D6. There are no data regarding the enzymes involved in CITA and DCITA metabolism in rats. The present study investigated the influence of CYP inhibitors on the enantioselective metabolism of CITA in rats. Male Wistar rats (n = 6) received a single dose of 20 mg.kg(-1) CITA after pretreatment with 80 mg.kg(-1) quinidine, 10 mg.kg(-1) fluvoxamine, 50 mg.kg(-1) ketoconazole, or vehicle (control). Blood samples were collected up to 20 h after CITA administration. The CITA and DCITA enantiomers were analyzed by LC-MS/MS using a Chiralcel OD-R column. The kinetic disposition of CITA was enantioselective in rats (AUC(S/R) ratio = 0.4). Coadministration with quinidine resulted in non-enantioselective inhibition of the metabolism of CITA. Coadministration with fluvoxamine or ketoconazole, however, inhibited only the metabolism of (+)-(S)-CITA, but not of (-)-(R)-CITA when the racemic drug was administered to rats.

Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

Genetic polymorphisms involved in folate metabolism and concentrations of methylmalonic acid and folate on plasma homocysteine and risk of coronary artery disease

Objectives Alterations in the enzymes involved in homocysteine (Hcy) metabolism or vitamin deficiency could play a role in coronary artery disease (CAD) development. This study investigated the influence of MTHFR and MTR gene polymorphisms, plasma folate and MMA on Hcy concentrations and CAD development. MMA and folate concentrations were also investigated according to the polymorphisms. Methods Two hundred and eighty-three unrelated Caucasian individuals undergoing coronary angiography (175 with CAD and 108 non-CAD) were assessed in a case-control study. Plasma Hcy and MMA were measured by liquid chromatography/tandem mass spectrometry. Plasma folate was measured by competitive immunoassay. Dietary intake was evaluated using a nutritional questionnaire. Polymorphisms MTHFR and MTR were investigated by polymerase chain reaction (PCR) followed by enzyme digestion or allele-specific PCR. Results Hcy mean concentrations were higher in CAD patients compared to controls, but below statistical significance (P = 0.246). Increased MMA mean concentrations were frequently observed in the CAD group (P = 0.048). Individuals with MMA concentrations > 0.5 mu mol/l (vitamin B(12) deficiency) were found only in the CAD group (P = 0.004). A positive correlation between MMA and Hcy mean concentrations was observed in both groups, CAD (P = 0.001) and non-CAD (P = 0.020). MMA mean concentrations were significantly higher in patients with hyperhomocysteinemia in both groups, CAD and non-CAD (P = 0.0063 and P = 0.013, respectively). Folate mean concentration was significantly lower in carriers of the wild-type MTHFR 1298AA genotype (P = 0.010). Conclusion Our results suggest a correlation between the MTHFR A1298C polymorphism and plasma folate concentration. Vitamin B(12) deficiency, reflected by increased MMA concentration, is an important risk factor for the development both of hyperhomocysteinemia and CAD.