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1.
Neurology ; 94(16): e1702-e1715, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32234823

RESUMO

OBJECTIVE: To characterize subclinical abnormalities in asymptomatic heterozygote NPC1 mutation carriers as markers of neurodegeneration. METHODS: Motor function, cognition, mood, sleep, and smell function were assessed in 20 first-degree heterozygous relatives of patients with Niemann-Pick disease type C (NPC) (13 male, age 52.7 ± 9.9 years). Video-oculography and abdominal ultrasound with volumetry were performed to assess oculomotor function and size of liver and spleen. NPC biomarkers in blood were analyzed. 18F-fluorodesoxyglucose PET was performed (n = 16) to detect patterns of brain hypometabolism. RESULTS: NPC heterozygotes recapitulated characteristic features of symptomatic NPC disease and demonstrated the oculomotor abnormalities typical of NPC. Hepatosplenomegaly (71%) and increased cholestantriol (33%) and plasma chitotriosidase (17%) levels were present. The patients also showed signs seen in other neurodegenerative diseases, including hyposmia (20%) or pathologic screening for REM sleep behavior disorder (24%). Cognitive function was frequently impaired, especially affecting visuoconstructive function, verbal fluency, and executive function. PET imaging revealed significantly decreased glucose metabolic rates in 50% of participants, affecting cerebellar, anterior cingulate, parieto-occipital, and temporal regions, including 1 with bilateral abnormalities. CONCLUSION: NPC heterozygosity, which has a carrier frequency of 1:200 in the general population, is associated with abnormal brain metabolism and functional consequences. Clinically silent heterozygous gene variations in NPC1 may be a risk factor for late-onset neurodegeneration, similar to the concept of heterozygous GBA mutations underlying Parkinson disease.


Assuntos
Hepatomegalia/diagnóstico por imagem , Heterozigoto , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transtornos da Motilidade Ocular/fisiopatologia , Esplenomegalia/diagnóstico por imagem , Adulto , Idoso , Colestanóis/sangue , Disfunção Cognitiva/epidemiologia , Disfunção Cognitiva/fisiopatologia , Medições dos Movimentos Oculares , Família , Feminino , Hepatomegalia/epidemiologia , Hepatomegalia/genética , Hexosaminidases/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Doença de Niemann-Pick Tipo C/diagnóstico por imagem , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/fisiopatologia , Doença de Niemann-Pick Tipo C/psicologia , Transtornos da Motilidade Ocular/epidemiologia , Transtornos da Motilidade Ocular/genética , Transtornos do Olfato/epidemiologia , Fenótipo , Tomografia por Emissão de Pósitrons , Transtorno do Comportamento do Sono REM/epidemiologia , Esplenomegalia/epidemiologia , Esplenomegalia/genética , Ultrassonografia
2.
Cell Physiol Biochem ; 53(6): 933-947, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805226

RESUMO

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 µM) and TRIOL (2, µM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect.


Assuntos
Colestanóis/farmacologia , Eriptose/efeitos dos fármacos , Cetocolesteróis/farmacologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Hemoglobinas/química , Humanos , Oxirredução , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo
3.
J Steroid Biochem Mol Biol ; 194: 105447, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31415823

RESUMO

Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.


Assuntos
Colestanóis/análise , Imidazóis/análise , Mama/metabolismo , Neoplasias da Mama/metabolismo , Colestanóis/metabolismo , Cromatografia Líquida/métodos , Feminino , Humanos , Imidazóis/metabolismo
4.
Steroids ; 151: 108472, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31400392

RESUMO

A facile novel strategy has been developed to obtain a key intermediate of squalamine, 7α, 24R -dihydroxy-5α-cholestan-3-one, starting from methyl Δ5-3ß-hydroxycholanate. The pure product was successfully synthesized and separated from the C-24 position epimers in good purity, d.e.% and yield.


Assuntos
Colestanonas/química , Colestanonas/síntese química , Técnicas de Química Sintética , Colestanóis/química , Estereoisomerismo
5.
J Steroid Biochem Mol Biol ; 192: 105390, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31170473

RESUMO

Dendrogenin A (DDA) is a tumor suppressor mammalian cholesterol-derived metabolite and a new class of ligand of the Liver X receptor (LXR), which displays tumor cell differentiation. In human MCF7 breast adenocarcinoma cells, DDA-induced cell differentiation was associated with an increased accumulation of neutral lipids and proteins found in milk indicating that DDA re-activates some functions of lactating cells. Active iodide transport occurs in the normal lactating mammary cells through the sodium/iodide symporter (NIS) and iodide (I) is secreted into milk to be used by the nursing newborn for thyroid hormones biosynthesis. In the present study, we assessed whether DDA may induce other characteristic of lactating cells such as NIS expression and iodine uptake in MCF7 breast cancer cells and extended this study to the papillary B-CPAP and undifferentiated anaplastic 8505c thyroid cancer cells. Moreover, we evaluated DDA impact on the expression of thyroid specific proteins involved in thyroid hormone biogenesis. We report here that DDA induces NIS expression in MCF7 cells and significantly increases the uptake of 131-I by acting through the LXR. In addition, DDA induces phenotypic, molecular and functional characteristics of redifferentiation in the two human thyroid carcinoma cell lines and the uptake of 131-I in the undifferentiated 8505c cells was associated with a strong expression of all the specific proteins involved in thyroid hormone biosynthesis, TSH receptor, thyroperoxidase and thyroglobulin. 131-I incorporation in the 8505c cells was stimulated by DDA as well as by the synthetic LXR ligand, GW3965. Together these data show that the re-differentiation of breast and thyroid cancer cells by DDA, is associated with the recovery of functional NIS expression and involves an LXR-dependent mechanism. These results open new avenues of research for the diagnosis of thyroid cancers as well as the development of new therapeutic approaches for radioiodine refractory thyroid cancers.


Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Colestanóis/farmacologia , Imidazóis/farmacologia , Radioisótopos do Iodo/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Apoptose , Autoantígenos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Iodeto Peroxidase/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Camundongos Nus , Receptores da Tireotropina/metabolismo , Simportadores/metabolismo , Tireoglobulina/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Elife ; 82019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31045492

RESUMO

Prey are under selection to minimize predation losses. In aquatic environments, many prey use chemical cues released by predators, which initiate predator avoidance. A prominent example of behavioral predator-avoidance constitutes diel vertical migration (DVM) in the freshwater microcrustacean Daphnia spp., which is induced by chemical cues (kairomones) released by planktivorous fish. In a bioassay-guided approach using liquid chromatography and mass spectrometry, we identified the kairomone from fish incubation water as 5α-cyprinol sulfate inducing DVM in Daphnia at picomolar concentrations. The role of 5α-cyprinol sulfate in lipid digestion in fish explains why from an evolutionary perspective fish has not stopped releasing 5α-cyprinol sulfate despite the disadvantages for the releaser. The identification of the DVM-inducing kairomone enables investigating its spatial and temporal distribution and the underlying molecular mechanism of its perception. Furthermore, it allows to test if fish-mediated inducible defenses in other aquatic invertebrates are triggered by the same compound.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestanóis/metabolismo , Daphnia/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Feromônios/metabolismo , Animais , Ácidos e Sais Biliares/isolamento & purificação , Bioensaio , Colestanóis/isolamento & purificação , Cromatografia Líquida , Peixes , Espectrometria de Massas , Feromônios/isolamento & purificação
7.
Ecotoxicol Environ Saf ; 179: 50-61, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31026750

RESUMO

In the current investigation, we studied role of castasterone (CS), (a bioactive brassinosteroid) in Brassica juncea grown under imidacloprid (IMI) stress. We observed that CS-seed treatment resulted in the recovery of seedling growth under IMI toxicity. Seed treatment with CS, significantly enhanced the contents of pigments like chlorophylls, carotenoids, anthocyanins and xanthophylls under stress. Oxidative stress generated by the production of reactive oxygen species (ROS) like hydrogen peroxide and superoxide anion, was reduced after CS treatment under IMI toxicity. Antioxidative defense system got activated after CS-seed treatment, resulting in the increased activities of enzymes. Moreover, CS-seed treatment under IMI stress also stimulated the biosynthesis of organic acids of Krebs cycle (citrate, succinate, fumarate and malate) and phenolics. We also noticed that CS is also involved in the regulation of the gene expression of some key enzymes involved in pigment metabolism (CHLASE, PSY, CHS), carbon fixation (RUBISCO), Krebs cycle (CS, SUCLG1, SDH, FH), ROS generation (RBO), antioxidative enzymes (SOD, CAT, POD, DHAR, GR, GST), phenolic biosynthesis (PAL) and pesticide detoxification system (CXE, P450, NADH). This modulated gene expression after CS-treatment activated the insecticide detoxification, leading to the reduction of IMI residues. Data analysis using multivariate statistical technique i.e. multiple linear regression, also supported the fact that CS can efficiently reduce IMI induced phytotoxicity in B. juncea.


Assuntos
Brassinosteroides/farmacologia , Colestanóis/farmacologia , Inseticidas/toxicidade , Mostardeira/efeitos dos fármacos , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Inativação Metabólica , Mostardeira/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo
8.
Biochimie ; 160: 130-140, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30844411

RESUMO

The bile alcohol 5ß-scymnol ([24R]-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol) is a therapeutic nutraceutical derived from marine sources, however very little is known about its potential for biotransformation as a xenobiotic in higher vertebrates. In this study, biotransformation products of scymnol catalysed by liver microsomes isolated from normal and streptozotocin (STZ)-treated male Wistar rats were characterised by liquid chromatography-tandem mass spectroscopy (LC-MSMS). In order of increasing polarity relative to the reversed phase sorbent, structural assignments were made for four biotransformation products, namely 3-oxoscymnol (5ß-cholestan-3-one-7α,12α,24,26,27-pentol); 7-oxoscymnol (5ß-cholestan-7-one-3α,12α,24,26,27-pentol); 3ß-scymnol (5ß-cholestan-3ß,7α,12α,24,26,27-hexol) and 6ß-hydroxyscymnol (5ß-cholestan-3α,6ß,7α,12α,24,26,27-heptol). In addition, a total of eight biotransformation products were characterised from microsomal incubations of crude oxoscymnol compounds, namely 7ß-scymnol; 3,12-dioxoscymnol; 3,7-dioxoscymnol; 7,12-dioxoscymnol; 12-oxo-3ß-scymnol; 7-oxo-3ß-scymnol; 6ß-hydroxy-12-oxoscymnol and 6ß-hydroxy-7-oxoscymnol. Collectively, the results indicate hepatic enzyme-catalysed hydroxylation, dehydrogenation and epimerisation reactions on the steroid nucleus of scymnol, and provide an insight into biotransformation pathways for scymnol use as a therapeutic nutraceutical in higher vertebrates.


Assuntos
Colestanóis/química , Colestanóis/metabolismo , Cromatografia Líquida/métodos , Cetosteroides/metabolismo , Microssomos Hepáticos/metabolismo , Esteroide Hidroxilases/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Biotransformação , Cetosteroides/química , Masculino , Ratos , Ratos Sprague-Dawley
9.
Molecules ; 24(3)2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736477

RESUMO

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on sterols with a 3ß-hydroxy-5-ene or 3ß-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3ß-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5ß-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5ß-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.


Assuntos
Ácidos e Sais Biliares/análise , Colestanóis/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Betaína/análogos & derivados , Ácidos e Sais Biliares/química , Colestanóis/química , Cromatografia Líquida , Hidroxiesteroide Desidrogenases/química , Espectrometria de Massas , Oxirredução , Esteróis/análise , Esteróis/química , Especificidade por Substrato
10.
Cancer Lett ; 449: 66-75, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30771431

RESUMO

Angiogenesis is critical for breast cancer progression. Overexpression of HER-2/neu receptors occur in 25-30% of breast cancers, and treatment with trastuzumab inhibits HER-2-overexpressing tumor growth. Notably, HER-2-mediated signaling enhances vascular endothelial growth factor (VEGF) secretion to increase tumor-associated angiogenesis. Squalamine (aminosterol compound) suppresses VEGF-induced activation of kinases in vascular endothelial cells and inhibits tumor-associated angiogenesis. We assessed antitumor effects of squalamine either alone or with trastuzumab in nude mice bearing breast tumor xenografts without (MCF-7) or with HER2-overexpression (MCF-7/HER-2). Squalamine alone inhibited progression of MCF-7 tumors lacking HER2 overexpression, and squalamine combined with trastuzumab elicited marked inhibition of MCF-7/HER2 growth exceeding that of trastuzumab alone. MCF-7/HER-2 cells secrete higher levels of VEGF than MCF-7 cells, but squalamine elicited no growth inhibition of either MCF-7/HER-2 or MCF-7 cells in vitro. However, squalamine did stop growth of human umbilical vein endothelial cells (HUVECs) and reduced VEGF-induced endothelial tube-like formations in vitro. These effects correlated with blockade of focal adhesion kinase phosphorylation and stress fiber assembly in HUVECs. Thus, squalamine effectively inhibits growth of breast cancers with or without HER-2-overexpression, an effect due in part to blockade of tumor-associated angiogenesis.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Colestanóis/administração & dosagem , Colestanóis/farmacologia , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Fosforilação/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/administração & dosagem , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Molecules ; 24(2)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642032

RESUMO

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 ± 0.03 µM), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at ≥ IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.


Assuntos
Colestanóis/farmacologia , Resistência a Medicamentos , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Colestanóis/química , Concentração Inibidora 50 , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Tripanossomicidas/química , Tripanossomíase Africana/tratamento farmacológico
12.
FASEB J ; 33(2): 2809-2822, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30303744

RESUMO

Nonalcoholic steatohepatitis (NASH) is associated with an increased risk of developing cardiovascular complications and mortality, suggesting that treatment of NASH might benefit from combined approaches that target the liver and the cardiovascular components of NASH. Using genetic and pharmacologic approaches, we show that G protein-coupled bile acid-activated receptor 1 (GPBAR1) agonism reverses liver and vascular damage in mouse models of NASH. NASH is associated with accelerated vascular inflammation representing an independent risk factor for development of cardiovascular diseases and cardiovascular-related mortality. GPBAR1, also known as TGR5, is a G protein-coupled receptor for secondary bile acids that reduces inflammation and promotes energy expenditure. Using genetic and pharmacologic approaches, we investigated whether GPBAR1 agonism by 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (BAR501) reverses liver and vascular damage induced by exposure to a diet enriched in fat and fructose (HFD-F). Treating HFD-F mice with BAR501 reversed liver injury and promoted the browning of white adipose tissue in a Gpbar1-dependent manner. Feeding HFD-F resulted in vascular damage, as shown by the increased aorta intima-media thickness and increased expression of inflammatory genes (IL-6,TNF-α, iNOS, and F4/80) and adhesion molecules (VCAM, intercellular adhesion molecule-1, and endothelial selectin) in the aorta, while reducing the expression of genes involved in NO and hydrogen sulfide generation, severely altering vasomotor activities of aortic rings in an ex vivo assay. BAR501 reversed this pattern in a Gpbar1-dependent manner, highlighting a potential role for GPBAR1 agonism in treating the liver and vascular component of NASH.-Carino, A., Marchianò, S., Biagioli, M., Bucci, M., Vellecco, V., Brancaleone, V., Fiorucci, C., Zampella, A., Monti, M. C., Distrutti, E., Fiorucci, S. Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.


Assuntos
Colestanóis/farmacologia , Modelos Animais de Doenças , Inflamação/prevenção & controle , Hepatopatias/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Receptores Acoplados a Proteínas-G/agonistas , Doenças Vasculares/prevenção & controle , Animais , Dieta Hiperlipídica/efeitos adversos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores Acoplados a Proteínas-G/fisiologia , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia
13.
Environ Pollut ; 244: 818-826, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30390455

RESUMO

Sewage pollution is a principal factor of decreasing water quality, although it has not been considered a real impact in Amazonia that is still considered a pristine environment around the world. Thus, this study aimed to assess the levels of sewage contamination in sediments from three streams crossing Manaus - a Brazilian city of 2,403,796 inhabitants in the heart of the Amazon rain forest. Cholesterol, cholestanol, brassicasterol, ergosterol, stigmasterol, ß-sitosterol, campesterol, stigmastanol, coprostanol, and epicoprostanol levels were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The fecal indicator, coprostanol, was found in high concentrations (509-12 830 ng g-1) and high relative proportions (21-54%) in all samples collected in the Mindu stream that crosses many heavily populated districts of the city, and in the Quarenta stream that crosses the Industrial District of Manaus. The sediments of the Tarumã-Açu stream also presented coprostanol; however, concentrations (

Assuntos
Monitoramento Ambiental/métodos , Rios/química , Esteróis/análise , Poluentes da Água/análise , Poluição da Água/análise , Qualidade da Água , Biomarcadores/análise , Brasil , Colestadienóis/análise , Colestanol/análise , Colestanóis/análise , Colesterol/análogos & derivados , Colesterol/análise , Cromatografia Líquida , Contaminação de Medicamentos , Fezes , Sedimentos Geológicos/química , Fitosteróis/análise , Esgotos/análise , Sitosteroides/análise , Espectrometria de Massas em Tandem
14.
Int J Antimicrob Agents ; 53(3): 337-342, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30423343

RESUMO

Squalamine is a natural polycationic aminosterol extracted from the shark Squalus acanthias. Squalamine displays remarkable efficacy against antimicrobial-resistant Gram-negative and Gram-positive bacteria. Its membranolytic activity and low cytotoxicity make squalamine one of the most promising agents to fight nosocomial pathogens such as Acinetobacter baumannii. In the context of chronic diseases and therapeutic failures associated with this pathogen, the presence of dormant cells, i.e. persisters and viable but non-culturable cells (VBNCs), highly tolerant to antimicrobial compounds is problematic. The aim of this study was to investigate the antibacterial activity of squalamine against this bacterial population of A. baumannii. Bacterial dormancy was induced by cold shock and nutrient starvation in the presence of high doses of either colistin, ciprofloxacin or squalamine. Persisters and VBNCs induced by these treatments were then challenged with 100 mg/L squalamine. The efficacy of each treatment was determined by evaluating culturability on agar medium, membrane integrity (LIVE/DEAD®BacLightTM staining) and respiratory activity (BacLightTM RedoxSensorTM CTC staining) of bacteria. A. baumannii ATCC 17978 generated persisters as well as VBNCs in the presence of high doses of ciprofloxacin but not colistin or squalamine. Squalamine at 100 mg/L (below its haemolytic concentration) was able to kill dormant cells. Squalamine did not induce persister cell or VBNC formation in A. baumannii ATCC 17978. Interestingly, squalamine was significantly active against this type of dormant population generated by ciprofloxacin, making it a very promising anti-persister agent.


Assuntos
Acinetobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Colestanóis/farmacologia , Testes de Sensibilidade Microbiana
15.
J Plant Physiol ; 232: 107-114, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30537597

RESUMO

Although structurally simple, viroids can trigger numerous changes in host plants and cause loss of yield in agronomically important crops. This study investigated changes in the endogenous status of phytohormones and antioxidant enzyme activity in Solanum tuberosum cv. Désirée in response to Potato spindle tuber viroid (PSTVd) infection. Phytohormone analysis showed that the content of endogenous jasmonic acid (JA) and its precursor cis-OPDA significantly increased in leaves, while the content of castasterone (CS) increased in both leaves and tubers of systemically infected plants compared to mock-inoculated control plants at 8 weeks post-inoculation. The indole-3-acetic acid content moderately increased only in tubers, while no differences in salicylic acid and abscisic acid content were observed between infected and control plants. Changes in endogenous phytohormone content were associated with upregulated expression of genes involved in the biosynthesis of JA and brassinosteroids, and the metabolism of auxins. Additionally, PSTVd infection provoked overproduction of hydrogen peroxide, which coincided with increased activity of guaiacol peroxidase in leaves and ascorbate peroxidase in potato tubers. The activity of catalase decreased in leaves, while superoxide dismutase activity remained steady regardless of the treatment and organ type. Total ascorbate and glutathione did not change significantly, although a shift towards oxidized forms was observed. Results suggest the existence of organ-specific differences in phytohormone and antioxidative responses in potato upon PSTVd infection. Possible effects of the observed changes on symptom development are discussed.


Assuntos
Doenças das Plantas/virologia , Reguladores de Crescimento de Planta/metabolismo , Solanum tuberosum/fisiologia , Viroides/metabolismo , Antioxidantes/metabolismo , Brassinosteroides/metabolismo , Clorofila/metabolismo , Colestanóis/metabolismo , Ciclopentanos/metabolismo , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Folhas de Planta/metabolismo , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Solanum tuberosum/virologia
16.
Sci Rep ; 8(1): 16622, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413746

RESUMO

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.


Assuntos
Cálcio/metabolismo , Membrana Celular/enzimologia , Quimiotaxia , Ciona intestinalis/fisiologia , Peptídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Espermatozoides/fisiologia , Animais , Sinalização do Cálcio , Colestanóis/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Motilidade Espermática , Ésteres do Ácido Sulfúrico/metabolismo
17.
Biochem Pharmacol ; 153: 75-81, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29409832

RESUMO

Dendrogenin A (DDA) is a mammalian cholesterol metabolite recently identified that displays tumor suppressor properties. The discovery of DDA has revealed the existence in mammals of a new metabolic branch in the cholesterol pathway centered on 5,6α-epoxycholesterol and bridging cholesterol metabolism with histamine metabolism. Metabolic studies showed a drop in DDA levels in cancer cells and tumors compared to normal cells, suggesting a link between DDA metabolism deregulation and oncogenesis. Importantly, complementation of cancer cells with DDA induced 1) cancer cell re-differentiation, 2) blockade of 6-oxo-cholestan-3ß,5α-diol (OCDO) production, an endogenous tumor promoter and 3) lethal autophagy in tumors. Importantly, by binding the liver X receptor (LXR), DDA activates the expression of genes controlling autophagy. These genes include NR4A1, NR4A3, LC3 and TFEB. The canonical LXR ligands 22(R)hydroxycholesterol, TO901317 and GW3965 did not induce these effects indicating that DDA delineates a new class of selective LXR modulator (SLiM). The induction of lethal autophagy by DDA was associated with the accumulation in cancer cells of lysosomes and of the pro-lysosomal cholesterol precursor zymostenol due to the inhibition of the 3ß-hydroxysteroid-Δ8Δ7-isomerase enzyme (D8D7I). The anti-cancer efficacy of DDA was established on different mouse and human cancers such as breast cancers, melanoma and acute myeloid leukemia, including patient derived xenografts, and did not discriminate bulk cancer cells from cancer cell progenitors. Together these data highlight that the mammalian metabolite DDA is a promising anticancer compound with a broad range of anticancer applications. In addition, DDA and LXR are new actors in the transcriptional control of autophagy and DDA being a "first in line" driver of lethal autophagy in cancers via the LXR.


Assuntos
Antineoplásicos/metabolismo , Autofagia/fisiologia , Colestanóis/metabolismo , Colesterol/metabolismo , Imidazóis/metabolismo , Receptores X do Fígado/metabolismo , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Colestanóis/farmacologia , Colestanóis/uso terapêutico , Colesterol/farmacologia , Colesterol/uso terapêutico , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neoplasias/tratamento farmacológico
18.
Biochem Biophys Res Commun ; 496(1): 95-100, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29307820

RESUMO

Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures. Cholestane-3ß, 5α, 6ß-triol (Triol) is a major metabolic oxysterol of cholesterol. Although its neuroprotective effect on ischemia-induced neuronal injury and negative modulation of voltage-gated sodium (Nav) channels were well established, the physical binding site of triol to sodium channels and its effects on neuronal hyperexcitability have not yet been explored. In this study, we utilized molecular docking and molecular dynamics simulation to investigate the interaction between triol and Nav Channels. Our results demonstrated that triol binds to the indole ring of Trp122 of the Nav Channel in silico with a high biological affinity. We further found that triol negatively modulates the action potentials bursts of hippocampal neurons by cell-attached patch recording. Moreover, triol significantly inhibits low Mg2+-induced hyperexcitability in vitro. In addition, triol attenuates pentylenetetrazole (PTZ)-induced convulsive-form behavioral deficits in vivo. Together, our results suggest that triol suppresses neuronal hyperexcitability via binding to Nav channel, indicating that triol might be an attractive lead compound for the treatment of neuronal hyperexcitability-related neurological disorders, especially epileptic seizures.


Assuntos
Potenciais de Ação/fisiologia , Colestanóis/administração & dosagem , Colestanóis/química , Epilepsia/prevenção & controle , Neurônios/fisiologia , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Sítios de Ligação , Células Cultivadas , Relação Dose-Resposta a Droga , Epilepsia/fisiopatologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento
19.
Ecotoxicol Environ Saf ; 147: 725-734, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28942275

RESUMO

The aim of the present study was to explore the effect of exogenous application of castasterone (CS) on physiologic and biochemical responses in Brassica juncea seedlings under copper (Cu) stress. Seeds were pre-soaked in different concentrations of CS and grown for 7 days under various levels of Cu. The exposure of B. juncea to higher levels of Cu led to decrease of morphologic parameters, with partial recovery of length and fresh weight in the CS pre-treated seedlings. Metal content was high in both roots and shoots under Cu exposure while the CS pre-treatment reduced the metal uptake. Accumulation of hydrogen peroxide (H2O2) and superoxide anion radical (O2-) were chosen as stress biomarker and higher levels of H2O2 (88.89%) and O2- (62.11%) showed the oxidative stress in metal treated B. juncea seedlings, however, CS pre-treatment reduced ROS accumulation in Cu-exposed seedlings. The Cu exposures lead to enhance the plant's enzymatic and non-enzymatic antioxidant system. It was observed that enzymatic activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), glutathione perxoidase (GPOX) and gultrathione-s-transferase increased while activity of monodehydroascorbate reductase (MDHAR) decreased under Cu stress. The pre-treatment with CS positively affected the activities of enzymes. RT-PCR analysis showed that mRNA transcript levels were correlated with total enzymatic activity of DHAR, GR, GST and GSH. Increase in the gene expression of DHAR (1.85 folds), GR (3.24 folds), GST-1 (2.00 folds) and GSH-S (3.18 folds) was noticed with CS pre-treatment. Overall, the present study shows that Cu exposure induced severe oxidative stress in B. juncea plants and exogenous application of CS improved antioxidative defense system by modulating the ascorbate-glutathione cycle and amino acid metabolism.


Assuntos
Antioxidantes/metabolismo , Colestanóis/farmacologia , Cobre/toxicidade , Mostardeira/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Poluentes do Solo/toxicidade , Aminoácidos/metabolismo , Cobre/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Mostardeira/enzimologia , Mostardeira/genética , Poluentes do Solo/metabolismo
20.
Int J Dev Neurosci ; 66: 18-23, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29197565

RESUMO

BACKGROUND: Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3ß,5α,6ß-triol, both oxidized cholesterol products. OBJECTIVE: In this work, we aimed to evaluate the performance of cholestane-3ß,5α,6ß-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3ß,5α,6ß-triol value as a tool for therapy monitoring. RESULTS: Considering molecular assay as golden standard, it was verified that cholestane-3ß,5α,6ß-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3ß,5α,6ß-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. CONCLUSION: Taken together, the present data show that cholestane-3ß,5α,6ß-triol analysis has a high potential to be an important NP-C screening assay, and also can be used for therapy monitorization with miglustat in NP-C patients.


Assuntos
1-Desoxinojirimicina/análogos & derivados , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Glicoproteínas de Membrana/genética , Mutação/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , 1-Desoxinojirimicina/uso terapêutico , Adolescente , Adulto , Criança , Colestanóis/sangue , Feminino , Filipina/metabolismo , Hexosaminidases/metabolismo , Humanos , Masculino , Doença de Niemann-Pick Tipo C/patologia , Pele/metabolismo , Pele/patologia , Adulto Jovem
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