Role of Protein Ubiquitination in NFH-LacZ mice

In neurodegenerative diseases, one can observe deposits of degradation products that represent hallmark structures. Actually, the underlying mechanisms are not well understood, but some hypotheses claim that the ubiquitin-proteasome system is perturbed in neurodegenerative diseases. Some of the influencing factors are aging, oxidation and the formation of free radicals, as well as genetic mutations which affect the function of proteins and result in an accumulation and formation of aggresomes. The amyotrophic lateral sclerosis, in which a malfunction of the sodium dismutase perturbs the redox system, is characterized by the accumulation of elements of the cytoskeleton in motor neurons and a progressive neuronal death. We suppose that in these diseases the ubiquitin- proteasome system is deregulated and try to demonstrate this hypothesis by comparing the ubiquitination of different neurofilaments in brain and spinal cord of transgenic and control mice. These NFH-LacZ mice with a truncated NF-H protein and a ß-galactosidase marker protein induce an accumulation of NF-proteins and neurofilaments are no longer transported into axons or dendrites. The accumulation of such aggregates resembles the phenotype of amyotrophic lateral sclerosis. Beside the ubiquitination the neurofilament expression and phosphorylation state was investigated. The results cannot demonstrate a perturbation of the ubiquitin-proteasome system of neurofilaments in transgenic mice. In contrast, in accordance with the mechanism of the NFH-LacZ mice a decrease of high and medium density neurofilaments and a hypophosphorylation were found. In conclusion, to elicit the pathological mechanism of amyotrophic lateral sclerosis and to develop focused treatments, we have to review the pathological mechanism of the transgenic mice and repeat the experiments with other animal models or with human material. Other possibilities would be to focus on other degradation mechanisms, such as the endosome/lysosome system, and to define their role in the amyotrophic lateral sclerosis more clearly.

Role of peroxynitrite in the redox regulation of cell signal transduction pathways.

Peroxynitrite is a potent oxidant and nitrating species formed from the reaction between the free radicals nitric oxide and superoxide. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in multiple cardiovascular pathologies, such as myocardial infarction, heart failure and atherosclerosis. Whereas initial works focused on direct oxidative biomolecular damage as the main route of peroxynitrite toxicity, more recent evidence, mainly obtained in vitro, indicates that peroxynitrite also behaves as a potent modulator of various cell signal transduction pathways. Due to its ability to nitrate tyrosine residues, peroxynitrite affects cellular processes dependent on tyrosine phosphorylation. Peroxynitrite also exerts complex effects on the activity of various kinases and phosphatases, resulting in the up- or downregulation of signalling cascades, in a concentration- and cell-dependent manner. Such roles of peroxynitrite in the redox regulation of key signalling pathways for cardiovascular homeostasis, including protein kinase B and C, the MAP kinases, Nuclear Factor Kappa B, as well as signalling dependent on insulin and the sympatho-adrenergic system are presented in detail in this review.

NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperaemia in portal hypertensive rats.

Then, the expression of angiogenesis markers (western blotting), the formation of portosystemic collaterals (radioactive microspheres) and the production of superoxide anion (lucigenin-enhanced chemiluminescence) were determined. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow and resistance were also measured.Results: In portal hypertensive rats, NAD(P)H oxidase blockade significantly decreased portosystemic collateral formation, and superior mesenteric arterial flow. It also reduced the splanchnic expression of VEGF, VEGF receptor-2 and CD31, and attenuated the increased production of superoxide, compared with vehicle.Conclusions: NAD(P)H oxidase plays an important role in experimental portal hypertension, modulating splanchnic angiogenesis, the formation of portosystemic collaterals and the development of splanchnic hyperdynamic circulation. These results suggest that NAD(P)H oxidase may represent a new target in the treatment of portal hypertension.

Biologie oxydative et implications cliniques du peroxynitrite [Oxidative biology and clinical implications of peroxynitrite]

Peroxynitrite is a strong biological oxidant formed from the reaction between two free radicals, superoxide and nitric oxide. It inflicts serious damages to most biomolecules, including proteins, lipids and nucleic acids, either through direct oxidation or through the secondary generation of highly reactive free radicals. When such damage reaches a critical threshold, cells eventually die by necrosis or apoptosis. An excessive production of peroxynitrite is instrumental in the development of organ damage and dysfunction in conditions such as circulatory shock and ischemia-reperfusion. In such circumstances, various synthetic metalloporphyrins, able to degrade peroxynitrite, disclose important beneficial effects in animal models, and might therefore represent novel pharmacological agents in the future.

Vitamin C supplementation in the critically ill patient.

PURPOSE OF REVIEW: Vitamin C is not only an essential nutrient involved in many anabolic pathways, but also an important player of the endogenous antioxidant defense. Low plasma levels are very common in critical care patients and may reflect severe deficiency states. RECENT FINDINGS: Vitamin C scavenges reactive oxygen species such as superoxide and peroxynitrite in plasma and cells (preventing damage to proteins, lipids and DNA), prevents occludin dephosphorylation and loosening of the tight junctions. Ascorbate improves microcirculatory flow impairment by inhibiting tumor-necrosis-factor-induced intracellular adhesion molecule expression, which triggers leukocyte stickiness and slugging. Clinical trials in sepsis, trauma and major burns testing high-dose vitamin C show clinical benefit. Restoration of normal plasma levels in inflammatory patients requires the administration of 3 g/day for several days, which is 30 times the daily recommended dose. SUMMARY: The recent research on the modulation of oxidative stress and endothelial protection offer interesting therapeutic perspectives, based on the biochemical evidence, with limited or even absent side-effects.

Efeitos do flúor em folhas de plantas aquáticas de Salvinia auriculata

O objetivo deste trabalho foi avaliar os efeitos do flúor em folhas de plantas aquáticas de Salvinia auriculata Aubl., visando fornecer subsídios para a utilização dessa espécie, no monitoramento da poluição ambiental, causada por esse elemento tóxico. As plantas foram cultivadas sob condições controladas, em vasos com solução nutritiva, e submetidas à aplicação de chuva simulada contendo KF, nas concentrações de 0, 13, 26 e 39 mM, pela manhã, durante cinco dias sucessivos. Os resultados evidenciaram a ocorrência de alterações morfológicas, com o desenvolvimento de lesões nos tricomas e na porção adaxial do limbo foliar. As alterações nas atividades das enzimas peroxidase, polifenol oxidase, superóxido dismutase e catalase indicaram a ocorrência de danos oxidativos em resposta ao flúor, embora testes relacionados à peroxidação dos lipídios tenham apresentado resultados negativos. As alterações na concentração de pigmentos também direcionam para a ocorrência de estresse oxidativo, causado pelo flúor, presente na chuva simulada. Como as alterações morfológicas, enzimáticas e na composição de pigmentos, de plantas de S. auriculata, são passíveis de detecção por métodos relativamente simples, elas podem ser empregadas no biomonitoramento da poluição atmosférica, causada por esse elemento altamente reativo.

Plântulas de soja 'Tracajá' expostas ao ozônio sob condições controladas

O objetivo deste trabalho foi avaliar o crescimento inicial, acúmulo de biomassa, trocas gasosas e defesas antioxidativas de soja 'Tracajá', cultivada na Região Amazônica, exposta ao ozônio sob condições controladas. Sementes germinadas em vasos foram levadas para duas câmaras, uma com ar filtrado (AF) e outra com ar filtrado mais 30 ppb de ozônio (AF + O3). Aos 10 e 20 dias após a semeadura, as trocas gasosas, crescimento em altura e acúmulo de biomassa foram medidos; aos 20 dias após a semeadura, as defesas antioxidativas (ácido ascórbico e superóxido dismutase) foram analisadas. Aos 10 dias após a semeadura, a fotossíntese líquida, condutância estomática, transpiração, altura, área foliar e biomassa foram 16, 27, 11, 22, 29 e 18% menores, respectivamente, no tratamento AF + O3. Aos 20 dias após a semeadura, além dessas variáveis, comprimento da raiz, diâmetro do caule e razão raiz:parte aérea foram 10, 15 e 12% menores, respectivamente, apesar da concentração de ácido ascórbico e da atividade da superóxido dismutase terem aumentado. Plântulas de soja 'Tracajá' apresentam baixa tolerância à concentração de 30 ppb de ozônio.

Interplay of oxidative, nitrosative/nitrative stress, inflammation, cell death and autophagy in diabetic cardiomyopathy.

Diabetes is a recognized risk factor for cardiovascular diseases and heart failure. Diabetic cardiovascular dysfunction also underscores the development of diabetic retinopathy, nephropathy and neuropathy. Despite the broad availability of antidiabetic therapy, glycemic control still remains a major challenge in the management of diabetic patients. Hyperglycemia triggers formation of advanced glycosylation end products (AGEs), activates protein kinase C, enhances polyol pathway, glucose autoxidation, which coupled with elevated levels of free fatty acids, and leptin have been implicated in increased generation of superoxide anion by mitochondria, NADPH oxidases and xanthine oxidoreductase in diabetic vasculature and myocardium. Superoxide anion interacts with nitric oxide forming the potent toxin peroxynitrite via diffusion limited reaction, which in concert with other oxidants triggers activation of stress kinases, endoplasmic reticulum stress, mitochondrial and poly(ADP-ribose) polymerase 1-dependent cell death, dysregulates autophagy/mitophagy, inactivates key proteins involved in myocardial calcium handling/contractility and antioxidant defense, activates matrix metalloproteinases and redox-dependent pro-inflammatory transcription factors (e.g. nuclear factor kappaB) promoting inflammation, AGEs formation, eventually culminating in myocardial dysfunction, remodeling and heart failure. Understanding the complex interplay of oxidative/nitrosative stress with pro-inflammatory, metabolic and cell death pathways is critical to devise novel targeted therapies for diabetic cardiomyopathy, which will be overviewed in this brief synopsis. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Perfis proteicos e desempenho fisiológico de sementes de café submetidas a diferentes métodos de processamento e secagem

O objetivo deste trabalho foi avaliar os perfis proteicos e o desempenho fisiológico de sementes de café submetidas a diferentes métodos de processamento e secagem. Foram avaliados os processamentos por via seca e úmida, e as secagens natural, em terreiro, e artificial a 60ºC, ou a 60ºC até 30% de umidade e 40ºC até teor final de 11% (base úmida). Após serem processadas e secadas, as sementes foram avaliadas quanto ao desempenho fisiológico e submetidas a análises bioquímicas, por meio da eletroforese de proteínas resistentes ao calor LEA ("late embryogenesis abundant") e das enzimas superóxido dismutase, catalase, peroxidase, esterase, polifenoloxidase, isocitrato desidrogenase, álcool desidrogenase e malato desidrogenase. O perfil proteico de sementes de café é afetado pelo método de processamento e de secagem. Os cafés processados por via úmida apresentam maior tolerância à secagem - revelada pela maior atividade de enzimas antioxidativas e pelo melhor desempenho fisiológico - do que os processados por via seca. A atividade de proteínas resistentes ao calor e de enzimas antioxidantes é variável promissora para diferenciar a qualidade dos cafés submetidos a diferentes manejos pós-colheita.

Indução de resistência à podridão‑amarga em maçãs pelo uso de eliciadores em pós‑colheita

O objetivo deste trabalho foi avaliar o efeito dos eliciadores acibenzolar‑S‑metílico (ASM) e proteína harpina, aplicados em pós‑colheita, na indução de resistência sistêmica à podridão‑amarga em maçãs. Realizaram-se ferimentos mecânicos em maçãs 'Royal Gala' seguidos da aplicação dos eliciadores. Doze horas depois, procedeu-se à inoculação do fungo Colletotrichum gloeosporioides. Após 72 horas, realizaram-se as avaliações quanto à área lesionada e ao número de esporos, bem como a coleta de tecido dos frutos para quantificação de proteínas, açúcares totais e redutores, fenóis totais, e para determinação da atividade das enzimas fenilalanina amônia‑liase, superóxido dismutase, catalase, peroxidase e ascorbato peroxidase. A harpina e, em menor grau, o ASM proporcionaram aumento da atividade da enzima peroxidase e a consequente redução da área lesionada e da esporulação de C. gloeosporioides nas maçãs. Esses eliciadores podem ser utilizados como ferramenta de controle no manejo integrado da podridão‑amarga, em pós‑colheita de maçãs 'Royal Gala'.

Alterações isoenzimáticas em sementes de cultivares de soja em diferentes condições de armazenamento

O objetivo deste trabalho foi determinar alterações fisiológicas e isoenzimáticas em sementes de genótipos de soja, em diferentes condições de armazenamento. Utilizou-se o delineamento experimental inteiramente casualizado com quatro repetições, em arranjo fatorial com seis cultivares de soja (TMG 1176, TMG 1179, TMG 132, TMG 133, TMG 115 e GB 874) e cinco periodos de armazenamento (0, 2, 4, 6 e 8 meses), em dois ambientes de armazenamento (câmara fria e seca, a 10ºC e 50% de umidade relativa; e armazém convencional, em condições não controladas). A qualidade fisiológica foi avaliada por meio de testes de germinação, de envelhecimento acelerado e de frio. As expressões isoenzimáticas determinadas foram as de malato desidrogenase (MDH), álcool desidrogenase (ADH), esterase (EST), isocitrato liase (ICL), superóxido dismutase (SOD) e peroxidase (PO). Sementes de soja armazenadas em câmara fria e seca conservaram sua qualidade fisiológica. Após seis meses de armazenamento, em condições não controladas, a qualidade das sementes e as atividades isoenzimáticas de MDH, ADH, EST, ICL, SOD e PO diminuíram. No armazenamento em câmara fria e seca, não ocorreu alteração nas sementes. Os genótipos de soja apresentam diferentes níveis de tolerância ao armazenamento e expressões isoenzimáticas.

Etileno e peróxido de hidrogênio na formação de aerênquima em milho tolerante a alagamento intermitente

Resumo:O objetivo deste trabalho foi avaliar o papel do etileno e do peróxido de hidrogênio (H2O2) na formação do aerênquima em ciclos de seleção genética da cultivar de milho BRS 4154, sob alagamento. Plantas dos ciclos C1 e C18 foram submetidas a alagamento por 7 dias, com coleta das raízes aos 0 (controle, sem alagamento), 1 e 7 dias. Foram analisados: a expressão gênica das enzimas ACC sintase (ACS), ACC oxidase (ACO), dismutase do superóxido (SOD) e peroxidase do ascorbato (APX); a produção de etileno e o conteúdo de H2O2; a atividade da enzima ACO; e a proporção de aerênquima no córtex. Não houve expressão de ACS e ACO. Houve variação na atividade de ACO e na produção de etileno. A expressão da SOD foi maior em plantas C1 e a da APX, em C18, com redução aos 7 dias. O conteúdo de H2O2 não diferiu entre os tratamentos. A proporção de aerênquima aumentou com o tempo, tendo sido maior em plantas C18 e relacionada à taxa de formação do aerênquima. O tempo de alagamento e o nível de tolerância do ciclo de seleção influenciam a produção do etileno. A expressão da APX indica maior produção de H2O2 no início do alagamento.

Role of peroxynitrite in the cardiovascular dysfunction of septic shock.

The intense systemic inflammatory response characterizing septic shock is associated with an increased generation of free radicals by multiple cell types in cardiovascular and non cardiovascular tissues. The oxygen-centered radical superoxide anion (O2 .-) rapidly reacts with the nitrogen-centered radical nitric oxide (NO.) to form the potent oxidant species peroxynitrite. Peroxynitrite oxidizes multiple targets molecules, either directly or via the secondary generation of highly reactive radicals, resulting in significant alterations in lipids, proteins and nucleic acids, with significant cytotoxic consequences. The formation of peroxynitrite is a key pathophysiological mechanism contributing to the cardiovascular collapse of septic shock, promoting vascular contractile failure, endothelial and myocardial dysfunction, and is also implicated in the occurrence of multiple organ dysfunction in this setting. The recent development of various porphyrin-based pharmacological compounds accelerating the degradation of peroxynitrite has allowed to specifically address these pathophysiological roles of peroxynitrite in experimental septic shock. Such agents, including 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrinato iron III chloride (FeTTPs), manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP), Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin) (FP-15) and WW-85, have been shown to improve the cardiovascular and multiple organ failure in small and large animal models of septic shock. Therefore, these findings support the development of peroxynitrite decomposition catalysts as potentially useful novel therapeutic agents to restore cardiovascular function in sepsis.

Expanding view of phenotype and oxidative stress in Friedreich's ataxia patients with and without idebenone

Summary: Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia, is characterised by progressive ataxia with dysarthria of speech, loss of deep-tendon reflexes, impaired vibratory and proprioceptive sensations and corticospinal weakness with a Babinski's sign. Patients eventually also develop kyphoscoliosis, cardiomyopathy and diabetes mellitus. The disease is a GAA repeat disorder resulting in severely reduced levels of frataxin, with secondary increased sensitivity to oxidative stress. The anti-oxidative drug, idebenone, is effective against FRDA-associated cardiomyopathy. We provide detailed clinical, electrophysiological and biochemical data from 20 genetically confirmed FRDA patients and have analysed the relation-ship between phenotype, genotype and malondialdehyde (MDA), which is a marker of superoxide formation. We assessed the effects of idebenone biochemically by measuring blood M DA and clinically by serial measurements of the International Cooperative Ataxia Rating Scale (ICARS). The GAA repeat length influenced the age at onset (p <0.001), the severity of ataxia (p= 0.02), the presence of cardiomyopathy (p =0.04) and of low-frequency hearing loss (p = 0.009). Multilinear regression analysis showed (p = 0.006) that ICARS was dependent on the two variables of disease duration (p = 0.01) and size of the GAA expansion (p = 0.02). We found no correlation to bilateral palpebral ptosis visual impairment, diabetes mellitus or skeletal deformities, all of which appear to be signs of disease progression rather than severity. We discuss more thoroughly two underrecognised clinical findings: palpebral ptosis and GAA length-dependent low-frequency hearing loss. The average ICARS remained unchanged in 10 patients for whom follow-up on treatment was available (mean 2.9 years), whereas most patients treated with idebenone reported an improvement in dysarthria (63%), hand dexterity (.58%) and fatigue (47%) after taking the drug for several weeks or months. Oxidative stress analysis showed an unexpected increase in blood MDA levels in patients on idebenone (p = 0.04), and we discuss the putative underlying mechanism for this result, which could then explain the unique efficacy of idebenone in treating the FRDA-associated cardiomyopathy, as opposed to other antioxidative drugs. Indeed, idebenone is not only a powerful stimulator of complexes II and III of the respiratory chain, but also an inhibitor of complex I activity, then promoting superoxide formation. Our preliminary clinical observations are the first to date supporting an effect of idebenone in delaying neurological worsening. Our MDA results point to the dual effect of idebenone on oxidative stress and to the need for controlled studies to assess its potential toxicity at high doses on the one hand, and to revisit the exact mechanisms underlying the .physiopathology of Friedreich's ataxia on the other hand, while recent reports suggest non-oxidative pathophysiology of the disease.

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.