MDSCs use a complex molecular network to suppress T-cell immunity in a pulmonary model of fungal infection.

Background: Paracoccidioidomycosis (PCM) is a systemic endemic fungal disease prevalent in Latin America. Previous studies revealed that host immunity against PCM is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), regulatory T-cells (Tregs), and through the recruitment and activation of myeloid-derived suppressor cells (MDSCs). We have recently shown that Dectin-1, TLR2, and TLR4 signaling influence the IDO-1-mediated suppression caused by MDSCs. However, the contribution of these receptors in the production of important immunosuppressive molecules used by MDSCs has not yet been explored in pulmonary PCM. Methods: We evaluated the expression of PD-L1, IL-10, as well as nitrotyrosine by MDSCs after anti-Dectin-1, anti-TLR2, and anti-TLR4 antibody treatment followed by P. brasiliensis yeasts challenge in vitro. We also investigated the influence of PD-L1, IL-10, and nitrotyrosine in the suppressive activity of lung-infiltrating MDSCs of C57BL/6-WT, Dectin-1KO, TLR2KO, and TLR4KO mice after in vivo fungal infection. The suppressive activity of MDSCs was evaluated in cocultures of isolated MDSCs with activated T-cells. Results: A reduced expression of IL-10 and nitrotyrosine was observed after in vitro anti-Dectin-1 treatment of MDSCs challenged with fungal cells. This finding was further confirmed in vitro and in vivo by using Dectin-1KO mice. Furthermore, MDSCs derived from Dectin-1KO mice showed a significantly reduced immunosuppressive activity on the proliferation of CD4+ and CD8+ T lymphocytes. Blocking of TLR2 and TLR4 by mAbs and using MDSCs from TLR2KO and TLR4KO mice also reduced the production of suppressive molecules induced by fungal challenge. In vitro, MDSCs from TLR4KO mice presented a reduced suppressive capacity over the proliferation of CD4+ T-cells. Conclusion: We showed that the pathogen recognition receptors (PRRs) Dectin-1, TLR2, and TLR4 contribute to the suppressive activity of MDSCs by inducing the expression of several immunosuppressive molecules such as PD-L1, IL-10, and nitrotyrosine. This is the first demonstration of a complex network of PRRs signaling in the induction of several suppressive molecules by MDSCs and its contribution to the immunosuppressive mechanisms that control immunity and severity of pulmonary PCM.

The Mechanism of Action of L-Tyrosine Derivatives against Chikungunya Virus Infection In Vitro Depends on Structural Changes.

Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.

Oral Exposure to Titanium Dioxide E171 and Zinc Oxide Nanoparticles Induces Multi-Organ Damage in Rats: Role of Ceramide.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are common food additives for human consumption. We examined multi-organ toxicity of both compounds on Wistar rats orally exposed for 90 days. Rats were divided into three groups: (1) control (saline solution), (2) E171-exposed, and (3) ZnO NPs-exposed. Histological examination was performed with hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Ceramide (Cer), 3-nitrotyrosine (NT), and lysosome-associated membrane protein 2 (LAMP-2) were detected by immunofluorescence. Relevant histological changes were observed: disorganization, inflammatory cell infiltration, and mitochondrial damage. Increased levels of Cer, NT, and LAMP-2 were observed in the liver, kidney, and brain of E171- and ZnO NPs-exposed rats, and in rat hearts exposed to ZnO NPs. E171 up-regulated Cer and NT levels in the aorta and heart, while ZnO NPs up-regulated them in the aorta. Both NPs increased LAMP-2 expression in the intestine. In conclusion, chronic oral exposure to metallic NPs causes multi-organ injury, reflecting how these food additives pose a threat to human health. Our results suggest how complex interplay between ROS, Cer, LAMP-2, and NT may modulate organ function during NP damage.

In Vitro and In Silico Anti-Arboviral Activities of Dihalogenated Phenolic Derivates of L-Tyrosine.

Despite the serious public health problem represented by the diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses, there are still no specific licensed antivirals available for their treatment. Here, we examined the potential anti-arbovirus activity of ten di-halogenated compounds derived from L-tyrosine with modifications in amine and carboxyl groups. The activity of compounds on VERO cell line infection and the possible mechanism of action of the most promising compounds were evaluated. Finally, molecular docking between the compounds and viral and cellular proteins was evaluated in silico with Autodock Vina®, and the molecular dynamic with Gromacs®. Only two compounds (TDC-2M-ME and TDB-2M-ME) inhibited both ZIKV and CHIKV. Within the possible mechanism, in CHIKV, the two compounds decreased the number of genome copies and in the pre-treatment strategy the infectious viral particles. In the ZIKV model, only TDB-2M-ME inhibited the viral protein and demonstrate a virucidal effect. Moreover, in the U937 cell line infected with CHIKV, both compounds inhibited the viral protein and TDB-2M-ME inhibited the viral genome too. Finally, the in silico results showed a favorable binding energy between the compounds and the helicases of both viral models, the NSP3 of CHIKV and cellular proteins DDC and ß2 adrenoreceptor.

Global changes in nitration levels and DNA binding profile of Trypanosoma cruzi histones induced by incubation with host extracellular matrix.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.

3-Nitrotyrosine and related derivatives in proteins: precursors, radical intermediates and impact in function.

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical-radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO-), the product of the reaction of superoxide radical (O2•-) with •NO, but ONOO--independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.

Role of aspirin-triggered lipoxin A4, aspirin, and salicylic acid in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia.

PROBLEM: Oxidative stress and inflammation are key events leading to pre-eclampsia, involved in several maternal deaths. Low doses of acetylsalicylic acid (ASA) are used in the prevention and treatment of pre-eclampsia. The synthesis of aspirin-triggered lipoxin (ATL) by cyclooxygenase-2 acetylation is an alternative mechanism of ASA, which could explain the effectiveness of ASA treatments. The aim of this study was to evaluate the role of ASA, salicylates, and ATL in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia. METHOD OF STUDY: Plasma from 14 women with pre-eclampsia and 17 normotensive pregnant women was probed for inducing oxidative and inflammatory responses on endothelial cells and U937 promonocytes. The role of ATL, ASA, and salicylic acid (SA) on these events was evaluated. RESULTS: Plasma from women with pre-eclampsia induced TBARS and nitrotyrosine production on endothelial and U937 cells. Pre-treatment with both ATL and ASA decreased the TBARS production, while ATL decreased the nitrotyrosine. Pre-eclamptic plasma augmented the translocation of NF-kB on U937 cells, which decreased by a high dose of ASA and SA. Finally, the pre-eclamptic plasma increased the adhesion of leukocytes-PMN and monocytes-to endothelium, and we were able to determine a state of resolution of inflammation, since ATL decreased the PMN adhesion, and conversely, it increased the monocytes adhesion to endothelium. CONCLUSION: Together, these results suggest that ATL could explain the beneficial actions of ASA and support further research on mechanisms, real efficacy, and rational use of ASA in pre-eclampsia.

Mimosine increases the expressions of tyrosine phosphorylated protein in mouse seminal vesicles / La mimosina aumenta la expresión de la proteína tirosina fosforilada en las vesículas seminales del ratón

Acute effect of purified mimosine (MiMo) extracted from Leucaena leucocephala on testicular histopathology has been documented with seminal vesicle (SV) atrophy. Since protein phosphorylation and seminal secretions play important roles in sperm physiology, this study aimed to study the alteration of substances including tyrosine phosphorylated (TyrPho) proteins in seminal vesicle treated with MiMo. Male mice were divided into a control and experimental groups treated with purified MiMo at 3 doses of 15, 30, and 60 mg/KgBW, respectively for 35 consecutive days. The morphology and weights of SV were compared among groups. The levels of magnesium and fructosamine in SV fluid were assayed. The profiles of equally SV total proteins were compared using SDS-PAGE. The expression of seminal TyrPho proteins was detected by western blotting. Recent results showed the decreased weights of SV in MiMo treated mice compared to control. However MiMo in all doses did not affect the levels of magnesium and fructosamine in SV fluid. The SV protein expression of 130 and 55 kDas was obviously decreased in a high dose MiMo. In dose-dependent response, the expressions of 72 and 55 kDas TyrPho proteins of SV were increased. In conclusion, MiMo could affect SV morphological size and protein secretions especially TyrPho proteins.

El efecto agudo de la mimosina purificada (MiMo) extraída de Leucaena leucocephala en la histopatología testicular se ha documentado con atrofia de vesícula seminal (VS). Debido a que la fosforilación de proteínas y las secreciones seminales tienen un papel importante en la fisiología de los espermatozoides, este estudio tuvo como objetivo estudiar la alteración de sustancias como la proteína tirosina fosforilada (TyrPho) en vesículas seminales tratadas con MiMo. Los ratones se dividieron en un grupo control y un grupo experimental y se trataron con MiMo purificado en 3 dosis de 15, 30 y 60 mg / KgBW, respectivamente, durante 35 días seguidos. La morfología y los pesos de VS se compararon entre los grupos. Fueron analizados los niveles de magnesio y fructosamina en el fluido VS. Los perfiles de las proteínas totales de VS se compararon utilizando SDS-PAGE. La expresión de la proteína TyrPho en las vesículas seminales se detectó mediante transferencia de Western blot. Los resultados recientes muestran la disminución del peso de las VS en ratones tratados con MiMo, en comparación con el grupo control. Sin embargo, en ninguna de las dosis se vieron afectados por mimosina purificada los niveles de magnesio y fructosamina en el líquido de las VS. La expresión de la proteína en VS de 130 y 55 kDas disminuyó notablemente en una dosis alta de MiMo. En la respuesta dependiente de la dosis, aumentaron las expresiones de 72 y 55 kDas de las proteínas TyrPho en las VS. En conclusión, la mimosina purificada podría afectar el tamaño morfológico de las VS y la expresión de proteínas, especialmente las proteínas TyrPho.

The importance of inducible nitric oxide synthase and nitrotyrosine as prognostic markers for oral squamous cell carcinoma.

BACKGROUND: The prognosis of human cancer depends on the deregulations of many molecular patterns. In recent years, a great interest in the intracellular signaling mechanisms related to nitric oxide (NO)-induced carcinogenesis has appeared, as one of the most preeminent prognostic markers for many types of neoplasms. In this study, we identify the levels of iNOS and nitrotyrosine in the sample of normal oral mucosa (NOM), oral leukoplakia (OL), and oral squamous cell carcinoma (OSCC). METHODS: Quantitative polymerase chain reactions (qPCRs) were utilized to detect the NOS2 levels in fresh-frozen tissue samples of NOM (n = 6), OL (n = 20), and OSCC (n = 15). Moreover, the immunohistochemical method was used to examine the levels of iNOS and nitrotyrosine in 85 cases of OSCC (39 cases without metastases and 46 with metastases), 42 cases of OL, and 16 cases of NOM. RESULTS: There are rising tendencies in the iNOS mRNA and protein levels during human oral carcinogenesis. Similar findings were obtained in the nitrotyrosine staining. Furthermore, iNOS and nitrotyrosine immunostaining are associated with several clinical-pathological features of OSCC (site, presence of metastasis, staging, recidivism, and survival). CONCLUSIONS: The NO-signaling pathway plays a vital role in the development and progression of human oral dysplastic and neoplastic diseases. Nitrotyrosine was a significant marker for the discrimination of OSCC prognosis and survival.

Halogenated Tyrosine Derivatives from the Tropical Eastern Pacific Zoantharians Antipathozoanthus hickmani and Parazoanthus darwini.

In the search for bioactive marine natural products from zoantharians of the Tropical Eastern Pacific, four new tyrosine dipeptides, named valdiviamides A-D (1-4), were isolated from Antipathozoanthus hickmani, and two new tyramine derivatives, 5 and 6, from Parazoanthus darwini. The phenols of all six tyrosine derivatives are substituted by bromine and/or iodine atoms at the ortho positions of the hydroxyl. The planar structures of these aromatic alkaloids were elucidated from 1D and 2D NMR experiments in combination with HRESIMS data, and the absolute configurations of 1-4 were deduced from comparison between experimental and calculated electronic circular dichroism spectra. As halogenated tyrosine derivatives could represent chemotaxonomic markers of these genera, we decided to undertake the first chemical investigation of another species, Terrazoanthus cf. patagonichus. As expected, no halogenated metabolite was evidenced in the species, but we report herein the identification of two new zoanthoxanthin derivatives, named zoamides E (7) and F (8), from this species. Antimicrobial and cytotoxicity bioassays revealed that valdiviamide B (2) displayed moderate cytotoxicity against the HepG2 cell line with an IC50 value of 7.8 µM.

Carnosic Acid Pretreatment Attenuates Mitochondrial Dysfunction in SH-SY5Y Cells in an Experimental Model of Glutamate-Induced Excitotoxicity.

Mitochondria are the major site of adenosine triphosphate (ATP) production in mammalian cells. Moreover, mitochondria produce most of the reactive oxygen species (ROS) in nucleated cells. Redox and bioenergetic abnormalities have been seen in mitochondria during the onset and progression of neurodegenerative diseases. In that context, excitotoxicity induced by glutamate (GLU) plays an important role in mediating neurotoxicity. Several drugs have been used in the treatment of diseases involving excitotoxicity. Nonetheless, some patients (20-30%) present drug resistance. Thus, it is necessary to find chemicals able to attenuate mitochondrial dysfunction in the case of excitotoxicity. In this work, we treated the human neuroblastoma SH-SY5Y cell line with the diterpene carnosic acid (CA) at 1 µM for 12 h prior to the exposure to GLU for further 24 h. We found that CA prevented the GLU-induced mitochondrion-related redox impairment and bioenergetic decline in SH-SY5Y cells. CA also downregulated the pro-apoptotic stimulus elicited by GLU in this experimental model. CA exerted mitochondrial protection by a mechanism associated with the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since silencing of this protein with small interfering RNA (siRNA) suppressed the CA-induced protective effects. Future directions include investigating whether CA would be able to modulate mitochondrial function and/or dynamics in in vivo experimental models of excitotoxicity.

Indole-3-glyoxyl tyrosine: synthesis and antimalarial activity against Plasmodium falciparum.

AIM: More than 40% of the world's population, across 105 countries, live in malaria endemic areas. It is estimated that about 500 million cases of malaria and half a million deaths occur per year. RESULTS: Herein, we demonstrate the biological activity of indole-3-glyoxyl tyrosine against Plasmodium falciparum, which is the causal agent of the most virulent form of malaria in humans. We developed an efficient synthesis of indole-3-glyoxyl tyrosine derivatives, which were then used as key intermediates in the synthesis of functionalized indole-3-glyoxyl biphenyl tyrosines. CONCLUSION: In biological testing, the compounds exhibited a parasite growth inhibition of over 85%. A cell viability assay showed low cytotoxicity against human cells, with no significant changes in cell viability, making these compounds potential antimalarials.

The prognostic value of nitrotyrosine levels in coronary heart disease: long-term evaluation in the Acute Coronary Syndrome Registry Strategy (ERICO study).

INTRODUCTION: We aimed to analyze the association of nitrotyrosine (N-TYR) levels and long-term survival in an ongoing coronary heart disease (CHD) prospective cohort, the Acute Coronary Syndrome Registry Strategy (ERICO study). METHODS: N-TYR levels collected during acute and subacute phase from onset of acute coronary syndrome (ACS) symptoms (myocardial infarction and unstable angina) were evaluated in 342 patients. We calculated case-fatality rates (180-days, 1 year, 2 years and 4 years) and survival analyses up to 4 years using Kaplan-Meier curves and Cox regression with respective cumulative hazard ratios (95% confidence interval; 95%CI), according to N-TYR tertiles up to 4 years of follow-up. Models are presented as crude, age and sex-adjusted and further adjusted for lipids and other confounders. RESULTS: Overall, median level of N-TYR was 208.33 nmol/l (range: 3.09 to 1500 nmol/l), regardless ACS subtype. During follow-up of 4 years, we observed 44 (12.9%) deaths. Overall survival rate was 298 (87.1%) (Survival days: 1353, 95%CI: 1320-1387 days). N-TYR levels did not associate with mortality / survival rates up to 4 years. CONCLUSIONS: No relationship was found between N-TYR levels and mortality rates after ACS during 4-year follow-up in the ERICO study.

Signs of damage in pelvic floor muscles at the end of pregnancy in rabbits.

INTRODUCTION AND HYPOTHESIS: Temporary effects to pelvic floor muscles are linked to impairments in micturition, particularly stress urinary incontinence (SUI), during pregnancy. We hypothesize that bulbospongiosus (Bsm) and pubococcygeus (Pcm) are differently damaged in primigravid and primiparous rabbits. METHODS: Twenty-four rabbits allocated evenly (n = 6) into nulliparous, pregnant, and primiparous groups on postpartum days 3 (P3) and 20 (P20) were used to evaluate the myofiber cross-sectional area (CSA), ß-glucuronidase activity, and anti-3-nitrotyrosine (anti-3-NTyr) immunoreactivity in Bsm and Pcm muscles. Appropriate statistical tests were done to determine significant differences among groups (P ≤ 0.05). RESULTS: The average CSA of Bsm was not significantly different, albeit a high percentage of myofibers was enlarged in late-pregnant and primiparous rabbits on P3; ß-glucuronidase activity and indirect parameter of muscle damage was also higher. These variables did not change in the Pcm muscle during the different reproductive stages. In contrast, the 3-NTyr immunoreactivity, an indicator of oxidative damage, was increased on P3 for Pcm myofibers and P20 for myofibers of both muscles. CONCLUSIONS: Our findings demonstrate reliable signs of damage to Bsm and Pcm muscles in young female rabbits passing different reproductive stages. Damage to the Bsm muscles as detected at the end of pregnancy persisted after delivery. This was not the case for Pcm muscles, in which damage seems to appear after delivery.

Inhibition of GSK-3ß on Behavioral Changes and Oxidative Stress in an Animal Model of Mania.

The present study evaluated the effects of AR-A014418 on behavioral and oxidative stress parameters of rats submitted to the animal model of mania induced by ouabain (OUA). Wistar rats were submitted to stereotaxic surgery and received a single intracerebroventricular (ICV) injection of artificial cerebrospinal fluid (aCSF), OUA, or AR-A014418. After 7 days, the animals were submitted to open-field test. After behavioral analysis, the brains were dissected in frontal cortex and hippocampus to the evaluation of oxidative stress. The OUA induced manic-like behavior in rats, which was reversed by AR-A014418 treatment. The ICV administration of OUA increases the levels of superoxide in submitochondrial particles, lipid hydroperoxide (LPH), 4-hydroxynonenal (4-HNE), 8-isoprostane, protein carbonyl, 3-nitrotyrosine, and activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) in both structures evaluated. In general, the treatment with AR-A014418 reversed these effects of OUA on the submitochondrial particles, LPH, 4-HNE, 8-isoprostane, protein carbonyl, 3-nitrotyrosine levels, and SOD activity. Furthermore, the injection of OUA decreased the catalase activity, and AR-A014418 promoted an increase in activity of this enzyme in the brain structures. These results suggest that GSK-3ß inhibition can modulate manic-like behaviors. Also, it can be suggested that inhibition of GSK-3ß can be effective against oxidative stress. However, more studies are needed to better elucidate these mechanisms. Graphical Abstract The effects of AR-A014418 on the behavioral and oxidative stress parameters in the animal model of mania induced by ouabain. Superoxide = superoxide production in submitochondrial particles; LPH = lipid hydroperoxide; 4-HNE = 4-hydroxynonenal; SOD = superoxide dismutase; GPx = glutathione peroxidase; GR = glutathione reductase.

Redox regulation and NO/cGMP plus K+ channel activation contributes to cardiorenal protection induced by Cuphea carthagenensis (Jacq.) J.F. Macbr. in ovariectomized hypertensive rats.

BACKGROUND: One of the medicinal plants widely used by the population in the treatment of hypertension, atherosclerosis and circulatory disorders is Cuphea carthagenensis (Jacq.) J.F. Macbr. (Lythraceae), popularly known as 'sete sangrias', being found in Brazil, Hawaii and in South Pacific Islands. Despite the widespread use of this species by the population, its long-term antihypertensive and cardioprotective activities have not yet been scientifically evaluated. PURPOSE: To evaluate the possible cardioprotective effects of an ethanol-soluble fraction obtained from C. carthagenensis (ESCC) using ovariectomized hypertensive rats to simulate a broad part of the female population over 50 years of age affected by hypertension. In addition, the molecular mechanism that may be responsible for its cardiorenal protective effects was also explored. METHODS: Female Wistar rats were submitted to surgical procedures of bilateral ovariectomy and induction of renovascular hypertension (two-kidneys, one-clip model). The sham-operated group was used as negative control. ESCC was obtained and a detailed phytochemical investigation about its main secondary metabolites was performed. ESCC was orally administered at doses of 30, 100 and 300  mg/kg, daily, for 28 days, 5 weeks after surgery. Enalapril (15  mg/kg) was used as standard antihypertensive drug. Renal function was evaluated on days 1, 7, 14, 21 and 28. At the end of the experimental period, systolic, diastolic, mean arterial pressure and heart rate were recorded. The activity of the tissue enzymatic antioxidant system, thiobarbituric acid reactive substances, nitrotyrosine, nitrite, aldosterone and vasopressin levels, in addition to the activity of the angiotensin-converting enzyme were also evaluated. Additionally, vascular reactivity to acetylcholine, sodium nitroprusside, and phenylephrine, and the role of nitric oxide, prostaglandins, and K+ channels in the vasodilator response of ESCC on the mesenteric vascular bed were also investigated. RESULTS: ESCC-treatment induced an important cardiorenal protective response, preserving renal function and preventing elevation of blood pressure and heart rate in ovariectomized hypertensive rats. In addition, prolonged treatment with ESCC recovered mesenteric vascular reactivity at all doses used. This effect was associated with an important modulation of the antioxidant defense system with a possible increase in NO bioavailability. Additionally, NO/cGMP activation and K+ channel opening-dependent vasodilator effect was observed on the mesenteric vascular bed, indicating a potential mechanism for the cardiovascular effects of ESCC. CONCLUSION: A 28-days ESCC treatment reduces the progression of the cardiorenal disease in ovariectomized hypertensive rats. These effects seem to be involved with an attenuation of oxidative and nitrosative stress, affecting endothelial nitric oxide production and K+ channel opening in smooth muscle cells.

SYNTHESIS OF THE NEW 2-(3,4-DIHYDRO-3-OXO-2H-[1,2,4]TRIAZINO[4,3-C]QUINAZOLIN-4-YL) ACETIC ACID DERIVATIVES AND ANALYSIS OF THEIR ANTIOXIDANT ACTIVITY IN NITROSATIVE STRESS MODELS.

Today we know that NO· and ONOO- are clue pathophysiological factors for progression some ischemic diseases of the central nervous system. So investigation of the antioxidants which will be able to decrease NO· and ONOO- toxicity seems to be very of current interest. The six esters and three amides of 2-(3,4-dihydro-3-oxo-2H-[1,2,4]triazino[4,3-c]quinazolin-4-yl)acetic acid were synthesized for this study, and we showed evidence of antioxidant activity of these new original derivatives. We studied the effect of 2-(3,4-dihydro-3-oxo-2H-[1,2,4]triazino[4,3-c]quinazolin-4-yl)acetic acid derivatives on superoxide dismutase activity under the condition of excessive NO· and ONOO- production. NO· induction was performed by the action of light on sodium nitroprusside Na2[Fe(NO)(CN)5]×2H2O in vitro. Also, the investigation of the substances was carried out in the brain supernatant obtained from the white Wistar rats in vivo. For nitrosative stress modeling dinitrozolic complex of Fe2+ and cysteine were utilized. Our data showed that 2-(3,4-dihydro-3-oxo-2H-[1,2,4]triazino[4,3-c]quinazolin-4-yl)acetic acid is not active compound while its esters and amides have antioxidant activity. Compound benzyl ester of this acid revealed the most effective antioxidant activity.

Regulation of aldose reductase activity by tubulin and phenolic acid derivates.

In this work we demonstrate that aldose reductase (AR) interacts directly with tubulin and, was subjected to microtubule formation conditions, enzymatic AR activity increased more than sixfold. Since AR interacts mainly with tubulin that has 3-nitro-tyrosine in its carboxy-terminal, we evaluated whether tyrosine and other phenolic acid derivatives could prevent the interaction tubulin/AR and the enzymatic activation. The drugs evaluated have two characteristics in common: the presence of an aromatic ring and a carboxylic substituent. The 9 drugs tested were able to prevent both the interaction tubulin/AR and the enzymatic activation. In addition, we found that the induction of microtubule formation by high concentrations of glucose and the consequent activation of AR in cultured cells can be inhibited by phenolic acid derivates that prevent the interaction tubulin/AR. These results suggest that tubulin regulates the activation of AR through a direct interaction which can be controlled with phenolic derivates of carboxylic acids.

INCREASED OXIDATIVE STRESS IN THE BLOOD OF OSTOMY PATIENTS.

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.