Specific oxidative alterations in vastus lateralis muscle of patients with the diagnosis of chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a poorly understood disease characterized by mental and physical fatigue, most often observed in young white females. Muscle pain at rest, exacerbated by exercise, is a common symptom. Although a specific defect in muscle metabolism has not been clearly defined, yet several studies report altered oxidative metabolism. In this study, we detected oxidative damage to DNA and lipids in muscle specimens of CFS patients as compared to age-matched controls, as well as increased activity of the antioxidant enzymes catalase, glutathione peroxidase, and transferase, and increases in total glutathione plasma levels. From these results we hypothesize that in CFS there is oxidative stress in muscle, which results in an increase in antioxidant defenses. Furthermore, in muscle membranes, fluidity and fatty acid composition are significantly different in specimens from CFS patients as compared to controls and to patients suffering from fibromyalgia. These data support an organic origin of CFS, in which muscle suffers oxidative damage.     

The effect of α-tocopherol transfer protein gene disruption on Trypanosoma congolense infection in mice

At present 15 to 20 million people are estimated to be infected with pathogenic trypanosome parasites worldwide, mainly in developing countries. There are a number of factors that affect the severity of trypanosomiasis, including the nutritional status of the host. However, the relationship between micronutrient levels and trypanosomiasis outcome has yet to be reported in detail. Here, we demonstrate that the inhibition of α-tocopherol transfer protein, a determinant of the vitamin E concentration in host circulation, confers resistance to Trypanosoma congolense infection, evidently owing to oxidative damage to parasite DNA. These results suggest that transient inhibition of α-tocopherol transfer gene activity could possibly be exploited as a strategy for both the prevention and the treatment of trypanosomiasis.     

Diazeniumdiolation of protamine sulfate reverses mitogenic effects on smooth muscle cells and fibroblasts

After vascular interventions, endothelial cells are typically injured or lacking, resulting in decreased NO synthesis to maintain vascular health. Moreover, inflammation as a result of the tissue injury and/or the presence of an implanted foreign polymer such as a vascular graft causes excessive generation of reactive oxygen species (ROS) (e.g., superoxide), which can react with NO. The combination of the above creates a general decline in NO bioavailability, as well as oxidative stress due to less available NO to scavenge ROS. Localized NO delivery is an attractive solution to alleviate these issues; however, NO donors typically exhibit unpredictable NO payload release when using nitrosothiols or the risk of nitrosamine formation for synthetic diazeniumdiolates. The objective of this study was therefore to synthesize an NO donor from a biological peptide that could revert to its native form upon NO release. To this effect, protamine sulfate (PS), an FDA-approved peptide with reported vasodilator and anticoagulant properties, was diazeniumdiolated to form PS/NO. PS/NO showed diazeniumdiolate-characteristic UV peaks and NO release in physiological solutions and was capable of scavenging radicals to decrease oxidative stress. Furthermore, PS/NO selectively inhibits the proliferation of smooth muscle cells and adventitial fibroblasts, thereby reversing reported mitogenic properties of PS. Endothelial cell growth, on the other hand, was promoted by PS/NO. Finally, PS retained its anticoagulant properties upon diazeniumdiolation at clinically relevant concentrations. In conclusion, we have synthesized an NO prodrug from a biological peptide, PS/NO, that selectively inhibits proliferation of smooth muscle cells and fibroblasts, retains anticoagulant properties, and reverts back to its native PS form upon NO payload release.     

The response of primary cultured adult mouse sensory neurons to ethanol,propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50’s of 600 μM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

The FBXL family of F-box proteins: variations on a theme

The ubiquitin–proteasome system (UPS) is responsible for the rapid targeting of proteins for degradation at 26S proteasomes and requires the orchestrated action of E1, E2 and E3 enzymes in a well-defined cascade. F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases that determine which proteins are ubiquitinated. To date, around 70 FBPs have been identified in humans and can be subdivided into distinct families, based on the protein-recruiting domains they possess. The FBXL subfamily is defined by the presence of multiple leucine-rich repeat (LRR) protein-binding domains. But how the 22 FBPs of the FBXL family achieve their individual specificities, despite having highly similar structural domains to recruit their substrates, is not clear. Here, we review and explore the FBXL family members in detail highlighting their structural and functional similarities and differences and how they engage their substrates through their LRRs to adopt unique interactomes.     

TNFalpha-induced cytoprotection requires the production of free radicals within mitochondria in C2C12 myotubes

We previously reported that tumour necrosis factor alpha (TNFalpha) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFalpha-induced reactive oxygen species (ROS). We hypothesized that TNFalpha cytoprotection occurs through the generation of ROS which originate within the mitochondria. C(2)C(12) myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFalpha (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFalpha-induced ROS was examined with Mitotracker Red CM-H(2)XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFalpha improved cell viability compared with the simulated ischemic control (TNFalpha: 75 +/- 1% versus 34 +/- 1% for the control: p<0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFalpha-stimulated cells to 15 +/- 1% (p<0.001 versus TNFalpha). Similar results were obtained with IPC. TNFalpha stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFalpha to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFalpha-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFalpha.     

The lipid peroxidation end-product 4-HNE induces COX-2 expression through p38MAPK activation in 3T3-L1 adipose cell

Oxidative stress and low grade chronic inflammation are increased in accumulating fat. Our objective was to test whether 4-hydroxynonenal (4-HNE), an end-product of lipid peroxidation, affects cyclooxygenases in 3T3-L1 adipose cells. 4-HNE increased COX-2 mRNA and protein expression and p38MAP-kinase phosphorylation in a dose-dependent manner. Pretreatment of 3T3-L1 cells by a selective inhibitor of p38MAPK (PD 169316) abolished 4-HNE and glucose oxidase induced COX-2 expression. Our results show that oxidative stress induces COX-2 expression through the production of 4-HNE which activates p38MAPKinase, suggesting that 4-HNE links oxidative stress and chronic inflammation through the activation of cyclooxygenase.     

Influence of crocetin on high-cholesterol diet induced atherosclerosis in rats via anti-oxidant activity together with inhibition of inflammatory response and p38 MAPK signaling pathway

The present study aimed to investigate the effect and possible mechanism of action of crocetin on the high cholesterol diet (HCD) induced atherosclerosis rat. The Wistar rats were used in the current investigation. The rats were divided into following group, Group I: control, Group II: HCD induced AS, Group III: AS + crocetin (25 mg/kg), Group IV: AS + crocetin (50 mg/kg) and Group V: AS + Simvastatin, respectively. AS was induced in the rats using the vitamin D₃ and HCD. The rats received the pre-determined treatment for the 10 weeks. After the study period, the level of lipid profile, malonaldehyde (MDA) and superoxide dismutase (SOD) were also estimated. The proinflammatory cytokines viz., tumor necrosis factor (TNF)-α and interleukin (IL)-6 were scrutinized using the ELISA kits. We also estimated the expression of phosphorylated p38 (p-p38) MAPK using the Western blot techniques. The results revealed that the AS was successfully induced in the rats. The AS control group rats showed the modulated level of lipid profile, and decreased the level of the SOD and boost the level of the MDA as compared with the normal control. However, crocetin thrived in enhancing the lipid profile toward the standard value in the normal control group rats. The crocetin and simvastatin group rats significantly inhibited the expression of the p-p38 MAPK as compared to the AS group rats. In conclusion, the current investigation revealed that the crocetin reduced the HCD induced dyslipidemia in the Wistar rats, the possible mechanism of action may be connected to the antioxidative, down regulating of p-p38 MAPK and antiinflammatory effect by crocetin.     

The prolific ATL family of RING-H2 ubiquitin ligases

An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.     

Acetaminophen attenuates glomerulosclerosis in obese Zucker rats via reactive oxygen species/p38MAPK signaling pathways

Focal segmental glomerulosclerosis is a critical pathological lesion in metabolic syndrome-associated kidney disease that, if allowed to proceed unchecked, can lead to renal failure. However, the exact mechanisms underlying glomerulosclerosis remain unclear, and effective prevention strategies against glomerulosclerosis are currently limited. Herein, we demonstrate that chronic low-dose ingestion of acetaminophen (30 mg/kg/day for 6 months) attenuates proteinuria, glomerulosclerosis, podocyte injury, and inflammation in the obese Zucker rat model of metabolic syndrome. Moreover, acetaminophen treatment attenuated renal fibrosis and the expression of profibrotic factors (fibronectin, connective tissue growth factor, transforming growth factor β), reduced inflammatory cell infiltration into the glomeruli, and decreased the expression of monocyte chemoattractant protein, glutathione (GSH) reductase, and nuclear factor erythroid 2-related factor 2, but increased the level of GSH synthetase in obese animals. Further in vivo and in vitro studies using human renal mesangial cells exposed to high glucose or hydrogen peroxide suggested that the renoprotective effects of acetaminophen are characterized by diminished renal oxidative stress and p38MAPK hyperphosphorylation.     

Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway

Capsaicin has been reported to regulate blood glucose levels and to ameliorate insulin resistance in obese mice. This study demonstrates that capsaicin increases glucose uptake directly by activating AMP-activated protein kinase (AMPK) in C2C12 muscle cells, which manifested as an attenuation of glucose uptake when compound C, an AMPK inhibitor, was co-administered with capsaicin. However, the insulin signaling molecules insulin receptor substrate-1 (IRS-1) and Akt were not affected by capsaicin. Additional results showed that p38 mitogen-activated protein kinase (MAPK) is also involved in capsaicin-induced glucose transport downstream of AMPK because capsaicin increased p38 MAPK phosphorylation significantly and its specific inhibitor SB203580 inhibited capsaicin-mediated glucose uptake. Treatment with an AMPK inhibitor reduced p38 MAPK phosphorylation, but the p38 MAPK inhibitor had no effect on AMPK. Capsaicin stimulated ROS generation in C2C12 muscle cells, and when ROS were captured using the nonspecific antioxidant NAC, the increase in both capsaicin-induced AMPK phosphorylation and capsaicin-induced glucose uptake was attenuated, suggesting that ROS function as an upstream activator of AMPK. Taken together, these results suggest that capsaicin, independent of insulin, increases glucose uptake via ROS generation and consequent AMPK and p38 MAPK activations.     

Recognition mechanism of p63 by the E3 ligase Itch: Novel strategy in the study and inhibition of this interaction

The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.     

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.     

The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2^−/− mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2^−/− red cells and reduced Prx2^−/− red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2^−/− mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2^−/− mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2^−/− mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.     

The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara

Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara.