Control of protein degradation pathways by BAG proteins and changes during aging

Many age-related neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and polyglutamine disorders, including Huntington’s disease, are associated with the aberrant formation of protein aggregates. These protein aggregates and/or their precursors are believed to be causally linked to the pathogenesis of such protein conformation disorders, also referred to as proteinopathies. The accumulation of protein aggregates, frequently under conditions of an age-related increase in oxidative stress, implies the failure of protein quality control and the resulting proteome instability as an upstream event of proteinopathies. As aging is a main risk factor of many proteinopathies, potential alterations of protein quality control pathways that accompany the biological aging process could be a crucial factor for the onset of these disorders.rnrnThe focus of this dissertation lies on age-related alterations of protein quality control mechanisms that are regulated by the co-chaperones of the BAG (Bcl-2-associated athanogene) family. BAG proteins are thought to promote nucleotide exchange on Hsc/Hsp70 and to couple the release of chaperone-bound substrates to distinct down-stream cellular processes. The present study demonstrates that BAG1 and BAG3 are reciprocally regulated during aging leading to an increased BAG3 to BAG1 ratio in cellular models of replicative senescence as well as in neurons of the aging rodent brain. Furthermore, BAG1 and BAG3 were identified as key regulators of protein degradation pathways. BAG1 was found to be essential for effective degradation of polyubiquitinated proteins by the ubiquitin/proteasome system, possibly by promoting Hsc/Hsp70 substrate transfer to the 26S proteasome. In contrast, BAG3 was identified to stimulate the turnover of polyubiquitinated proteins by macroautophagy, a catabolic process mediated by lysosomal hydrolases. BAG3-regulated protein degradation was found to depend on the function of the ubiquitin-receptor protein SQSTM1 which is known to sequester polyubiquitinated proteins for macroautophagic degradation. It could be further demonstrated that SQSTM1 expression is tightly coupled to BAG3 expression and that BAG3 can physically interact with SQSTM1. Moreover, immunofluorescence-based microscopic analyses revealed that BAG3 co-localizes with SQSTM1 in protein sequestration structures suggesting a direct role of BAG3 in substrate delivery to SQSTM1 for macroautophagic degradation. Consistent with these findings, the age-related switch from BAG1 to BAG3 was found to determine that aged cells use the macroautophagic system more intensely for the turnover of polyubiquitinated proteins, in particular of insoluble, aggregated quality control substrates. Finally, in vivo expression analysis of macroautophagy markers in young and old mice as well as analysis of the lysosomal enzymatic activity strongly indicated that the macroautophagy pathway is also recruited in the nervous system during the organismal aging process.rnrnTogether these findings suggest that protein turnover by macroautophagy is gaining importance during the aging process as insoluble quality control substrates are increasingly produced that cannot be degraded by the proteasomal system. For this reason, a switch from the proteasome regulator BAG1 to the macroautophagy stimulator BAG3 occurs during cell aging. Hence, it can be concluded that the BAG3-mediated recruitment of the macroauto-phagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging. Future studies will explore whether an impairment of this adaptation process may contribute to age-related proteinopathies.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model. © 2008 Yano et al; licensee BioMed Central Ltd.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

High glucose mediates prooxidant and antioxidant enzyme activities in coronary endothelial cells

Objective: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O2-)are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMEC) exposed to hyperglycaemia. Methods: CMEC were cultured under normal (5.5 mmol/L) or high glucose (22 mmol/L)concentrations for 7 days. The activity and expression (protein level) of eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses,respectively while the effects of hyperglycaemia on nitrite and O2 - generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Results: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p<0.05) and increased the protein expressions and/or activities of p22-phox, a membrane-bound component of pro-oxidant NAD(P)H oxidase and antioxidant enzymes (p<0.05). Free radical-scavengers, namely, Tiron and MPG (0.1-1 mol/L) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p<0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced prooxidant enzyme activity and diminished generation of 3 antioxidant glutathione. By increasing the antioxidant enzyme capacity CMEC may protect themselves against free radical-induced cell damage in diabetic conditions. The definitive version is available at http://www.blackwell-synergy.com

Nanotubos de carbono de parede simples funcionalizados com polietilenoglicol: avaliação de parâmetros in vivo e in vitro

Os nanomateriais apresentam uma escala na qual ao menos uma das dimensões varia entre 1 e 100 nm e possuem propriedades químicas, físicas ou biológicas dependentes da nanoestrutura e que lhes confere características funcionais de interesse para fins comerciais ou aplicações na área médica. Dentre os nanomateriais mais estudados e utilizados, destacam-se os de carbono, que incluem os fulerenos e os nanotubos de carbono (NT). Uma potencial utilização dos nanomateriais de carbono é na área biomédica, já que estes podem interagir com os sistemas biológicos em nível molecular e supramolecular com alto grau de especificidade. Em contrapartida, é importante considerar que os nanotubos de carbono podem exercer efeitos tóxicos, tendo como possível mecanismo o estresse oxidativo. Sendo assim, o objetivo desse trabalho foi investigar a ação dos nanotubos de carbono de parede única funcionalizados com polietilenoglicol (SWNT-PEG) em Danio rerio “zebrafish” (Teleostei, Cyprinidae). Avaliaram-se parâmetros bioquímicos, histológicos, comportamentais e de biodistribuição para entender como esse material se comporta in vitro e in vivo. Foi observado que o tipo de funcionalização é determinante para a ação desse material em meio biológico. No experimento in vitro o SWNT-PEG não mostrou efeito pró-oxidante nas avaliações de peroxidação lipídica, capacidade antioxidante total, conteúdo de GSH e atividade de GCL. Na exposição intraperitoneal em zebrafish constatou-se a agregação e geração de processo inflamatório, o que sugere que a cadeia de PEG utilizada para a funcionalização dos NT possui um tamanho inadequado e/ou uma funcionalização ineficiente para manter a estabilidade do material em meio biológico e evitar uma resposta inflamatória por parte do organismo exposto. Possivelmente devido a esta característica do nanomaterial, nas análises de biodistribuição, através de espectroscopia Raman, não se observou distribuição de SWNT-PEG no sistema nervoso central de zebrafish. No entanto, através da análise histológica foi observado processo inflamatório no tecido nervoso central, bem como alterações comportamentais avaliadas na tarefa de campo aberto.

Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood–brain barrier hyperpermeability

Aims: Hyperglycaemia (HG), in stroke patients, is associated with worse neurological outcome by compromising endothelial cell function and the blood–brain barrier (BBB) integrity. We have studied the contribution of HG-mediated generation of oxidative stress to these pathologies and examined whether antioxidants as well as normalization of glucose levels following hyperglycaemic insult reverse these phenomena. Methods: Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures were used to simulate the human BBB. The integrity of the BBB was measured by transendothelial electrical resistance using STX electrodes and an EVOM resistance meter, while enzyme activities were measured by specific spectrophotometric assays. Results: After 5 days of hyperglycaemic insult, there was a significant increase in BBB permeability that was reversed by glucose normalization. Co-treatment of cells with HG and a number of antioxidants including vitamin C, free radical scavengers and antioxidant enzymes including catalase and superoxide dismutase mimetics attenuated the detrimental effects of HG. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C but not phosphoinositide 3 kinase (PI3 kinase) also reversed HG-induced BBB hyperpermeability. In HBMEC, HG enhanced pro-oxidant (NAD(P)H oxidase) enzyme activity and expression that were normalized by reverting to normoglycaemia. Conclusions: HG impairs brain microvascular endothelial function through involvements of oxidative stress and several signal transduction pathways.

Key Endothelial Cell Angiogenic Mechanisms Are Stimulated By The Circulating Milieu In Sickle Cell Disease And Attenuated By Hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to sickle cell disease manifestations, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from steady-state sickle cell anemia patients presented elevated concentrations of pro-angiogenic factors (Angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly augmenting endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice, compared with non-sickle cell disease mice, consistent with an upregulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy displayed a pro-angiogenic profile and had more significant effects on endothelial cell proliferation and capillary formation than plasma of patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factor profile, in association with an inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, sickle cell anemia and retinopathic hemoglobin SC individuals present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy for preventing progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.

Cracking formation on the surface of extruded photodegraded polypropylene plates

The cracking formation during the photodegradation of polypropylene (PP) plates (1 mm thickness), with (PPOx) and without pro-oxidant [PP), has been investigated. The plates were produced by extrusion in an industrial production line and were exposed to ultraviolet radiation in the laboratory for periods of up to 480 hr. The samples were investigated by infrared spectroscopy- FTIR, optical light microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The results showed that the extension of photodegradation process is more intense for PPOx than for PP samples. For both samples, cracks were formed at the surface perpendicularly to the flow-lines. However the cracks frequency was different for both samples and sides of sample. The crack frequency was correlated with chain orientation, A(110); it was shown that lower degrees of orientation resulted in lower crack frequency. POLYM. ENG. SCI., 48:365-372, 2008. (c) 2007 Society of Plastics Engineers.

Curcumin could prevent methemoglobinemia induced by dapsone in rats

The curcumin`s effect given orally by gavage in single- or multiple-dose regimens on methemoglobinemia induced by dapsone (DDS) was investigated in male Wistar rats. In the single-dose regimen, groups of 10 rats received either vehicle alone, or curcumin at 0.1, 1.0, 10, or 30 mg/kg body weight (bw), or curcumin at 0.02, 0.1, 1, 10, or 30 mg/kg bw plus DDS at 40 mg/kg bw, intraperitoneally (i.p.), 2 hours after. In the multiple-dose regimen, groups of 10 rats received either vehicle alone, or curcumin at 0.1, 1.0, 10, or 30 mg/kg bw for 5 days, with or without DDS (40 mg/kg bw, i.p.) 2 hours after on the fifth day. In both regimens, further groups of 10 rats were given DDS alone (positive controls) or normal saline (negative controls) i.p. Single-dose treatment with curcumin at 0.02 and 0.1 mg/kg bw significantly reduced DDS-induced methemoglobin formation, while the higher doses showed a pro-oxidant effect, significantly increasing DDS-induced methemoglobinemia. In the multiple-dose regimen, treatment with curcumin at 0.1 mg/kg bw significantly reduced DDS-induced methemoglobin formation, but the higher doses were without significant effect compared to DDS alone. It is concluded that curcumin at low doses mitigates methemoglobinemia induced by dapsone in rats, both in single- and multiple-dose regimens. (C) 2011 Elsevier Ltd. All rights reserved.

Imatinib mesylate ameliorates the dystrophic phenotype in exercised mdx mice

Myofiber degeneration, inflammation, and fibrosis are remarkable features of Duchenne muscular dystrophy. We hypothesized that the administration of imatinib mesylate, an inhibitor of tyrosine kinase and TGF-beta pro-fibrogenic activity, could improve the muscular conditions in mdx mice. Four-week old mdx mice were treated and exercised for 6 weeks. Gastrocnemius and diaphragm histopathology, strength, creatine kinase, and cytokine levels were evaluated. The treated group presented increased muscular strength and decreased CK levels, injured myofibers, and inflammatory infiltrates. Pro-inflammatory cytokines and TGF-beta were also reduced, while IL-10 was increased, suggesting an immunomodulatory effect of imatinib, which can ameliorate the dystrophic phenotype in mdx mice. (C) 2009 Elsevier B.V. All rights reserved.

Vascular permeability, neutrophil migration and edematogenic effects induced by the latex of Cryptostegia grandiflora

Inflammatory responses have been described as occurring after exposure to some latex materials. In this study pro-inflammatory activity in the latex of Cryptostegia grandiflora was investigated. The soluble proteins of the latex (CgLP) were isolated from the whole latex and evaluated by in vivo assays. CgLP induced strong inflammatory activity mediated by neutrophil migration, enlarging vascular permeability and increasing myeloperoxidase activity locally in rats. CgLP-induced inflammation was observed in peritonitis, paw edema and air push models. In addition, CgLP caused hyperemia in a healing model. The peritonitis effect was lost when CgLP was previously boiled suggesting the involvement of proinflammatory proteins. Thioglycollate increased the neutrophil migration induced by CgLP, but not by fMLP Mast cell depletion provoked by 40/80 compound did not modify the course of inflammation triggered by CgLP, being similar to fMLP, which suggested that neutrophil migration was induced by direct mechanism mediated by macrophages. Neutrophil migration stimulated by CgLP was strongly inhibited by Dexamethasone and to a lesser extent by Thalidomide, indicating the involvement of cytokines in mediating neutrophil infiltration. Celecoxib and Indomethacin were inhibitory suggesting the involvement of prostaglandins. Cimetidine was effective only in the initial phase of edema. PCA 4248 was ineffective. It is concluded that the latex of C. grandiflora is a potent inflammatory fluid, and also that laticifer proteins may be implicated in this process. (c) 2008 Elsevier Ltd. All rights reserved.

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 mu M beta-mercaptoethanol (beta-ME) and 50 mu M cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.