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

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

The Protective Effect of Rhodobryum giganteum (Schwaegr.) Par. on Umbilical Vascular Endothelial Cells and the Influence on Secretion of NO and NOS / 中国实验方剂学杂志

Objective: To study the effect and mechanism of Rhodobryum giganteum (Schwaegr.) Par. 's anti-atherosclerotic effect. Methods:Vascular endothelial ceils were cultivated and the H2O2 were used to induce the oxidative stress injury of human umbilical vascular endothelial cells (HUVEC). Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was added to cultivated HUVEC, and the activity of the cells was carefully determined by OD value with MTF method. The Griess Reagent was used to detect the NO concentration of different groups. At the same time, the NO fluorescence analysis probe, DAF-FM DA. (3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate), was used to determine the activity of NO synthase. Results:The most suitable stimulating concentration of H2O2 on HUVEC is 12.5 mmol· L-1. Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was co-cultured with HUVEC damaged byH2O2 .The OD values indicate that 3.33,2.50 mg· mL-1 water extract groups increase activity of the cells significantly compared with the model group (P <0.05) ,and 3.33 mg·mL-1 is much better than 2.50 mg·mL-1 dose group. To determine the content of NO and NOS, the 2.50 mg·mL-1 dose has significant effect (P < 0.05). Conclusions: The H2O2 concentration of 12.5 mmol· L-1 could be used to establish the injured model of endothelial cell successfully. 2.50 and 3.33 mg·mL-1 water extract of Rhodobryum giganteum (Schwaegr.) Par. Have protective effect on ECV304 injured by H2O2. 2.50 mg· mL-1 water extract of Rhodobryurn giganteum (Schw aegr.) Par could increase the activity of NOS and promote the synthesis and secretion of NO.

Muscarinic M5 receptor subtype and its biologic characterizations / 中国药理学通报

The fifth muscarinic receptor (M5), the last one of the muscarinic receptor family to be cloned, has the same basic formation characterization as G-protein coupled receptor family. M5 transduces signals by coupling with G-proteins, which then modulate the activities of a number of effector enzymes and ion channels. As M5 also plays a variety of prominent physiological roles by regulating central transmitters NO and DA, it has been considered as a novel drug therapy target for drug addiction, dysfunction of dopamine-ergic nervous system, Alzheimers disease and cerebral ischemia.

Effect of Ligustrazine on nNOS expression in different encephalic regions after focal cerebral ischemia in adult rats / 西安交通大学学报(医学版)

0.05).In SVZ,nNOS expression in ischemic model group was reduced on days 1-14,but increased on day 21;after Ligustrazine administration,nNOS expression was obviously decreased on days 3-14 in all Ligustrazine dose groups,but began to increase on day 21.In CC,nNOS expression in ischemic model group was reduced on days 3-14,and began to increase on day 21;in the different-dose Ligustrazine groups,nNOS expression was significantly decreased on days 3-14,especially in medium-and high-dose groups,but increased on day 21.In striatum and cortex peri-infarction,nNOS expression in ischemic model group was obviously decreased on days 3 and 7,but enhanced on days 14 and 21;in various-dose Ligustrazine groups,nNOS expression was decreased on days 3-21,especially in medium-and high-dose groups,but increased slightly on day 21.In DG and CA1 areas,nNOS expression in ischemic model group was reduced on days 3 and 7,but began to increase on day 14;nNOS expression in all Ligustrazine groups were decreased during 3-21d.There were significant differences between ischemic model group and different-dose Ligustrazine groups at different time points(P

Subconvulsive electrical stimuli facilitate increased NO level and neuronal NOS expression in hippocampi of rats / 第三军医大学学报

Objective To observe the alterations of nitric oxide (NO) level and the neuronal NOS (nNOS) expression in rats with posttraumatic stress disorder (PTSD) like behavior following subconvulsive electrical stimuli. Methods After the establishment of the PTSD animal model following subconvulsive stimuli to hippocampus, the nitric oxide (NO) and NO synthase (NOS) levels and the neuronal NOS (nNOS) expression in hippocampus and frontal cortex of experimental rats were investigated by neurochemistry and Western blotting, respectively. Results The NO level in hippocampus of experimental rats with PTSD like behavior following subconvulsive stimulation increased significantly at 12 h after the last stimulation (4.65?1.22 ?mol/g protein, P

Expression of Nitric Oxide Synthase in the Olfactory Bulb of the Rat during Development / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The presence and distribution of NADPH-diaphorase activity in the olfactory bulb during development has been reported. But the precise localization of NO-synthase (NOS) in the olfactory bulb during the developmental stages has not been studied yet. Therefore, we investigated the localization of NOS-immunoreactivity in a developing rat olfactory bulb by immunohistochemistry. MATERIALS AND METHODS: A total of 32 male and female Sprague-Dawley rats. They were of several prenatal and postnatal stages, such as the following: embryonic day 16 (E16), E18, E20, postnatal day 1 (P1), P5, P7, P14 and adult. Indirect immunoperoxidase method using rabbit polyclonal anti-bNOS antibody was performed for detecting the NOS immunoreactivity. RESULTS: In the main olfactory bulb, the first NOS-immunoreactive (IR) neurons were observed in the presumptive granule cell layer (GCL) by E18, and in the glomerular layer (GL) by P1. The density of these neurons was increased as the development stage approached the adult stage. In the GCL, two types of NOS-immunoreactive neurons were observed: intensively stained large, short axon cells and weakly stained small, granule cells. The first, localized in the deeper part of the GCL, was observed in the earlier developmental stages, and the latter which increased in number to the adult period was observed by P1. In the accessory olfactory bulb, NOS-IR neurons were first detected in the GCL by P1, and increased in number to the adult period. The pattern of NOS-IR neurons in the GCL of the accessory olfactory bulb is similar to that in the main olfactory bulb. CONCLUSION: Our results demonstrated that bNOS had a characteristic temporal and spatial patterns of expression in the main and accessory olfactory bulb of the rat during development.

The effects of oxygen radicals on the activity of nitric oxide synthase and guanylate cyclase

Reactive oxygen species such as superoxides, hydrogen peroxide (H2O2) and hydroxyl radicals have been suggested to be involved in the catalytic action of nitric oxide synthase (NOS) to produce NO from L-arginine. An examination was conducted on the effects of oxygen radical scavengers and oxygen radical-generating systems on the activity of neuronal NOS and guanylate cyclase (GC) in rat brains and NOS from the activated murine macrophage cell line J774. Catalase and superoxide dismutase (SOD) showed no significant effects on NOS or GC activity. Nitroblue tetrazolium (NBT, known as a superoxide radical scavenger) and peroxidase (POD) inhibited NOS, but their inhibitory actions were removed by increasing the concentration of arginine or NADPH respectively, in the reaction mixture. NOS and NO-dependent GC were inactivated by ascorbate/FeSO4 (a metal-catalyzed oxidation system), 2'2'-azobis-amidinopropane (a peroxy radical producer), and xanthine/xanthine oxidase (a superoxide generating system). The effects of oxygen radicals or antioxidants on the two isoforms of NOS were almost similar. However, H2O2 activated GC in a dose-dependent manner from 100 microM to 1 mM without significant effects on NOS. H2O2-induced GC activation was blocked by catalase. These results suggested that oxygen radicals inhibited NOS and GC, but H2O2 could activate GC directly.

Muscarinic M5 receptor subtype and its biologic characterizations / 中国药理学通报

The fifth muscarinic receptor (M5), the last one of the mus ca rinic receptor family to be cloned, has the same basic formation characterizatio n as G-protein coupled receptor family. M5 transduces signals by coupling with G-proteins, which then modulate the activities of a number of effector enzymes and ion channels. As M5 also plays a variety of prominent physiological roles by regulating central transmitters NO and DA, it has been considered as a novel dr ug therapy target for drug addiction, dysfunction of dopamine-ergic nervous sys tem, Alzheimers disease and cerebral ischemia.