Biochemical and microscopy evidence on adverse effects of nitroxidized human serum albumin

Earlier researches have pointed about the accumulation of peroxynitrite modified proteins and their aggregates in the etiopathogenesis of many age-related neurodegenerative and several autoimmune diseases. Human serum albumin (HSA) is present in abundance in plasma and is susceptible to modification by peroxynitrite. In this study, HSA modified with peroxynitrite (nitroxidized-HSA) formed aggregate besides other gross structural changes. Aggregation or assembly of aberrant proteins is responsible for increase in production of reactive species and is often correlated with toxicity in neurodegenerative diseases. However, lack of literature on the cytotoxicity of aggregated nitroxidized-HSA led us to explore its toxicity using human peripheral blood lymphocytes. Elevated protein carbonyl coupled with decreased protein thiol, and release of antioxidant enzymes and lactate dehydrogenase (LDH) were observed upon incubation of lymphocytes with nitroxidized-HSA. Trypan blue exclusion and MTT assays indicated nitroxidized-HSA induced injury/death of lymphocytes. This may be attributed to the observed reactive oxygen species generation during the interaction of nitroxidized-HSA with lymphocytes. Moreover, the analysis of the cellular morphology by dual staining, fluorescence and confocal microscopy further confirms the cytotoxicity of nitroxidized-HSA. Since various age-related degenerative diseases are characterized by deposition of protein aggregates, the demonstrated toxicity of nitroxidized-HSA may be an important driver in the pathophysiology of neurodegenerative diseases.

Modificações pós-traducionais durante o processo de amastigogênese do Trypanosoma cruzi / Post-translational modifications during the amastigogenesis of Trypanosoma cruzi

A doença de Chagas é uma doença negligenciada causada pelo protozoário Trypanosoma cruzi constituindo-se em um problema de saúde pública em vários países da América Latina. No seu complexo ciclo de vida, o protozoário passa por quatro estágios diferentes: tripomastigota metacíclica, amastigota, tripomastigota sanguíneo e epimastigota, que permitem sua sobrevivência nos diferentes ambientes com os quais o parasita entra em contato. A diferenciação dos tripomastigotas de T. cruzi em amastigotas (amastigogênese) ocorre com grandes mudanças morfológicas, estruturais e metabólicas no parasita e pode ser reproduzido in vitro por exemplo, pela acidificação do meio extracelular. Apesar dos vários trabalhos descritos na literatura, o processo ainda não é totalmente compreendido. A participação de NO na transdução de sinal durante a amastigogênese, sugerida por dados não publicados de nosso grupo, assim como a via de sinalização dependente de AMPc, foram o foco do presente estudo. A indução da amastigogênese foi obtida por incubação de tripomastigotas em meio de cultura acidificado (pH 6,0) e os parâmetros estudados comparados com parasitas controle (meio de cultura, pH 7,4). Estudamos a variação no perfil de nucleotídios cíclicos (AMPc, GMPc), de quinases (PKA, MAPK- ERK1/2), de uma fosfatase (PP2A), assim como o perfil de proteínas fosforiladas, S-nitrosiladas e nitradas até 6 h do início da amastigogênese. O processo foi dividido nas etapas: inicial (até 60 minutos) e tardio (em torno de 3-4 h), caracterizados por um aumento de formas amastigotas na etapa tardia. Houve um aumento de aproximadamente 17 vezes no nível de AMPc nos primeiros 15 minutos da amastigogênese (meio pH 6,0), seguido por aumento discreto no nível de PKA fosforilada, utilizado como indicador de atividade enzimática, este mais evidente na etapa tardia (360 minutos). Quanto à subunidade catalítica fosforilada da MAPK (ativa), há uma aparente diminuição no nível de fosforilação na fase inicial (30 minutos) e aumento na etapa tardia (120 minutos) do processo de amastigogênese. Quanto ao perfil geral de fosforilação de proteínas, há uma diminuição de fosforilação em torno de 30 minutos, seguida de aumento de fosforilação em proteínas de aproximadamente 5 e 100 kDa, mas de maneira geral, não se observaram grandes mudanças nesse perfil com a metodologia utilizada. Quanto às modificações por NO e seus derivados, foram observadas modificações por S-nitrosilação e nitração das proteínas, além do aumento de GMPc em torno de 60 minutos. Embora essas modificações modulem a atividade biológica de uma grande diversidade de proteínas, seu papel biológico não foi explorado.8 Em resumo, nossos resultados apontam para uma variação no perfil de fosforilação, S-nitrosilação e nitração de proteínas, além do aumento de AMPc e GMPc ao longo do processo de amastigogênese in vitro, com a via de sinalização dependente de quinases/ fosfatases e de óxido nítrico ocorrendo ao longo do processo de amastigogênese

Chagas disease is a neglected disease caused by the parasite Trypanosoma cruzi and is a public health problem in several Latin American countries. In its complex life cycle, the protozoan goes through four different stages: metacyclic trypomastigote, amastigote, blood trypomastigote and epimastigote, which allow its survival in the different environments which the parasite comes into contact. The differentiation of T. cruzi trypomastigotes into amastigotes (amastigogenesis) occurs with large morphological, structural and metabolic changes in the parasite and can be reproduced in vitro by, for example, acidification of the extracellular medium. Despite the many data described in the literature, the process is not yet fully understood. The participation of NO in signal transduction during amastigogenesis, suggested by unpublished data from our group, as well as the cAMP-dependent signaling pathway, were the focus of the present study. The induction of amastigogenesis was obtained by incubating trypomastigotes in acidified culture medium (pH 6.0) and the studied parameters compared with control parasites (culture medium, pH 7.4). We studied the variation in the profile of cyclic nucleotides (cAMP, cGMP), kinases (PKA, MAPK-ERK1 / 2), phosphatase (PP2A), as well as the profile of phosphorylated, S-nitrosylated and nitrated proteins up to 6 h. onset of amastigogenesis. The process was divided into early (up to 60 minutes) and late (around 3-4 hours), characterized by an increase in amastigote forms in the late stage. There was an approximately 17-fold increase in cAMP level in the first 15 minutes of amastigogenesis (pH 6.0 medium), followed by a slight increase in phosphorylated PKA level, most evident in the late stage (360 minutes). As for the phosphorylated catalytic subunit of MAPK (active), there is an apparent decrease in the phosphorylation level in the early phase (30 minutes) and increase in the late stage (120 minutes) of the amastigogenesis process. As for the general protein phosphorylation profile, there is a decrease in phosphorylation around 30 minutes, followed by an increase in phosphorylation of proteins (approximately 5 and 100 kDa), but overall, no major changes were observed in this profile with the methodology used. As for modifications by NO and its derivatives, modifications were observed by S-nitrosylation and protein nitration, besides the increase of cGMP around 60 minutes. Although these modifications modulate the biological activity of a wide range of proteins, their biological role has not been explored. In summary, our results point to a variation in phosphorylation, S-nitrosylation and nitration profile of proteins, as well as an increase in cAMP and cGMP along the amastigogenesis process, implicating kinases / phosphatases and nitric oxide dependent signaling pathways in this differentiation

Aumento de estresse oxidativo no sangue de pacientes com ostomia / Increased oxidative stress in the blood of ostomy patients

ABSTRACT BACKGROUND: Ostomy is a surgical procedure that creates a stoma that aims to construct a new path for the output of feces or urine. The relationship of oxidative stress (OxS) markers in patients with ostomy is still poorly described. OBJECTIVE: The present study was aimed at investigating the changes in oxidative stress parameters in peripheral blood collected from ostomy patients when compared with a healthy control group. METHODS: It was evaluated 29 ostomy patients and 30 healthy control patients. The oxidative stress parameters evaluated were: lipid peroxidation [lipid hydroperoxide (LPO), 8-isoprostane (8-ISO) and 4-hydroxynonenal (4-HNE)], protein oxidation and nitration [carbonyl and 3-nitrotyrosine (3-NT)] and DNA oxidation [8-hydroxy-2'-deoxyguanosine (8-OHDG)] in serum from ostomy patients compared to health controls. RESULTS: The data showed an increase of LPO, 8-ISO, 4-HNE, 3-NT and 8-OHDG in serum collected from ostomy patients when compared to healthy controls. CONCLUSION: The findings support the hypothesis that ostomy triggers the oxidative stress observed in the blood collected from these patients.

RESUMO CONTEXTO: Ostomia é um procedimento cirúrgico que cria um estoma com objetivo de construir um novo caminho para a saída das fezes ou urina. A relação dos marcadores de estresse oxidativo em pacientes ostomizados ainda é pouco descrita. OBJETIVO: O presente estudo tem como objetivo investigar as alterações dos parâmetros de estresse oxidativo em sangue de pacientes ostomizados comparados a controles saudáveis. MÉTODOS: Foram avaliados 29 pacientes ostomizados e 30 controles saudáveis. Os parâmetros de estresse oxidativo avaliados foram: peroxidação lipídica [hidroperóxido de lipídio (LPO), 8-isoprostano (8-ISO) e 4-hidroxinonenal (4-HNE)], oxidação e nitração de proteínas [carbonila e 3-nitrotirosina (3-NT)] e oxidação do DNA [8-hidroxi-2'-desoxiguanosina (8-OHDG)] em soro de pacientes ostomizados comparados a controles saudáveis. RESULTADOS: Os dados mostraram um aumento de LPO, 8-ISO, 4-HNE, 3-NT e 8-OHDG em soro de pacientes ostomizados em comparação a controles saudáveis. CONCLUSÃO: Os achados sustentam a hipótese de que a ostomia desencadeia o estresse oxidativo observado no sangue coletado destes pacientes.

DeepNitro: Prediction of Protein Nitration and Nitrosylation Sites by Deep Learning / 基因组蛋白质组与生物信息学报·英文版

Protein nitration and nitrosylation are essential post-translational modifications (PTMs) involved in many fundamental cellular processes. Recent studies have revealed that excessive levels of nitration and nitrosylation in some critical proteins are linked to numerous chronic diseases. Therefore, the identification of substrates that undergo such modifications in a site-specific manner is an important research topic in the community and will provide candidates for targeted therapy. In this study, we aimed to develop a computational tool for predicting nitration and nitrosylation sites in proteins. We first constructed four types of encoding features, including positional amino acid distributions, sequence contextual dependencies, physicochemical properties, and position-specific scoring features, to represent the modified residues. Based on these encoding features, we established a predictor called DeepNitro using deep learning methods for predicting protein nitration and nitrosylation. Using n-fold cross-validation, our evaluation shows great AUC values for DeepNitro, 0.65 for tyrosine nitration, 0.80 for tryptophan nitration, and 0.70 for cysteine nitrosylation, respectively, demonstrating the robustness and reliability of our tool. Also, when tested in the independent dataset, DeepNitro is substantially superior to other similar tools with a 7%-42% improvement in the prediction performance. Taken together, the application of deep learning method and novel encoding schemes, especially the position-specific scoring feature, greatly improves the accuracy of nitration and nitrosylation site prediction and may facilitate the prediction of other PTM sites. DeepNitro is implemented in JAVA and PHP and is freely available for academic research at http://deepnitro.renlab.org.

Spermidine is protective against kidney ischemia and reperfusion injury through inhibiting DNA nitration and PARP1 activation / 대한해부학회지

Kidney ischemia and reperfusion injury (IRI) is associated with a high mortality rate, which is attributed to tubular oxidative and nitrative stresses; however, an effective approach to limit IRI remains elusive. Spermidine, a naturally occurring polyamine, protects yeast cells against aging through the inhibition of oxidative stress and necrosis. In the present study, spermidine supplementation markedly attenuated histological damage and kidney dysfunction during IRI. In addition, exogenous spermidine potently inhibited poly(ADP-ribose) polymerase 1 (PARP1) activation and DNA nitrative/oxidative stress following IRI. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly enhanced DNA nitration, PARP1 activation, and functional damage during IRI. Finally, in ODC knockdown kidneys, PARP1 inhibition attenuated histological and functional damage induced by IRI, but not DNA nitrative stress. In conclusion, these data suggest that spermidine protects kidneys against IRI through blocking DNA nitration and PARP1 activation and this finding provides a novel target for prevention of acute kidney injury including IRI.

Artesunate and betulilic acid block lipopolysaccharide-induced angiogenesis and hyperplasia in mice / 中国药学杂志

OBJECTIVE: To disclose the pathological mechanism of bacterial LPS-induced angiogenesis and hyperplasia in tumorlike hyperplasia and the pharmacological mechanism underlying artesunate and betulilic acid blocking tumor-like hyperplasia. METHODS: The tumor-like hyperplasia models were established for mouse hypodermis and articular synovium using LPS or LPS-containing complete Fround's adjuvant (CFA). The morphological features (inflammatory grades) were described and the histochemical signatures (angiogenesis, hyperplasia, and inflammatory infiltration) were examined. The serial concentrations of nitric oxide (NO), blood oxygen saturation (SpO2) and 3-nitrotyrosine (3NT) were quantitatively determined by biochemical, physiological and immunological procedures, and the expression levels of inducible nitric oxide synthase (iNOS), hypoxia-induced factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) were analyzed by the immunohistochemical method. RESULTS: Both LPS and CFA can lead to the inflammatory phenotypes and abnormal hyperplasia-related microscopic alterations. They enable the elevation of NO, decline of SpO2, and increase of 3NT (protein nitration). The considerable upregulation of iNOS, HIF-1α and VEGF are accompanied by angiogenesis and hyperplasia. While a NO donor compound replicates such pathogenesis, a NO synthesis inhibitor antagonizes this effect. Artesunate and betulilic acid down-regulate iNOS, HIF-1α and VEGF, decrease NO, and increase SpO2, thereby leading to the melioration or interruption of aberrant inflammatory angiogenesis and hyperplasia. CONCLUSION: LPS-triggered iNOS overexpression and potent NO burst may represent the direct drivers for angiogenesis and hyperplasia. Due to repression of the pathogenic process of tumor-like hyperplasia, artesunate and betulilic acid possess the prophylactic and therapeutic potentials.

Nitration of α-Synuclein in Parkinson disease: Recent progress / 第二军医大学学报

Parkinson disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantial nigra and the formation of intracellular Lewy body. Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of PD. Here we described the biophysical and biochemical properties of α-synuclein and nitrated α-synuclein protein and reviewed their biological consequences; finally we discussed their roles in the mechanism and prevention/treatment of PD.

Mass Spectrometry Analysis for Nitration of Proteins / 한양의대학술지

Various proteomics and immunological methods including mass spectrometry combined with both liquid and 2-D PAGE, and immunodetection have been employed to identify and characterize nitrated proteins from pathological samples. Nitrosative modifications regulate cellular signal transduction and pathogenesis of inflammatory responses and neurodegenerative diseases. Nitric oxide generates reactive nitrosative species, such as peroxynitrite (ONOO-) that may be involved in a number of diseases. ONOO- can mediate protein tyrosine nitration which causes structural changes of affected proteins and leads to their inactivation. Protein tyrosine nitration is a biomarker of oxidative stress and also influences protein structure and function. Recent advances in mass spectrometry have made it possible to identify modified proteins and specific modified amino acid residues. This review focuses on the significance of protein tyrosine nitration and the progress achieved in analytical methods. Although mass spectrometry of nitrated peptides has become a powerful tool for the analysis of nitrated peptides, the low stoichiometry of protein tyrosine nitration clearly demands the use of affinity chromatography to enrich modified proteins (or peptides).

The Study on Synthesis on 20(S)-9-nitrocamptothecin / 医学研究杂志

20(S)-9-nitrocamptothecin was synthesized from camptothecin by nitration reaction using mixed nitrate salts in sulfuric acid medium.The yield was raised from 31% to 40% after chromatographic purification treatment.The best reaction conditions was as follows:0.5 g of camptothecin;30ml of sulfuric acid;0.007mol of nitrate salts(NH4NO3/CaNO3=1/1).The reaction was completed in 24h at 20℃.The advantage of this method is high yield and easily purification.

Highly efficient synthesis of methyl bryoanthrathiophene-1-carboxylate / 第三军医大学学报

Objective To study the synthesis of methyl bryoanthrathiophene-1-carboxylate with high efficiency. Methods Target compounds were synthesized from 1,8-dihydroxy-9,10-anthraquinone via 5 steps including protecting, nitration, methyl mercaptoacetate substitution, intramolecular Aldol reaction and deprotecting. Results Target molecule was synthesized in the total yield of 45.8%. Conclusion Our synthesis of methyl bryoanthrathiophene-1-carboxylate is characterized by short synthetic route, high selectivity and high efficiency.