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1.
Metabolism ; 104: 154051, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31874143

RESUMO

BACKGROUND: Identifying changes in serum metabolites before the occurrence of acute myocardial infarction (AMI) is an important approach for finding novel biomarkers of AMI. METHODS: In this prospective cohort study, serum samples obtained from patients at risk of AMI (n = 112) and non-risk controls (n = 89) were tested using high-resolution metabolomics (HRM). Partial least-squares discriminant analysis (PLS-DA), along with univariate analysis using a false discovery rate (FDR) of q = 0.05 were performed to discriminate metabolic profiles and to determine significantly different metabolites between healthy control and AMI risk groups. RESULTS: PLS-DA significantly separated the AMI risk sera from control sera. The metabolites associated with amino acid biosynthesis, 2-oxocarboxylic acid, tryptophan, and amino sugar and nucleotide sugar metabolism pathways were mainly elevated in patients at risk of AMI. Further validation and quantification by MS/MS showed that tryptophan, carnitine, L-homocysteine sulfinic acid (L-HCSA), and cysteic acid (CA) were upregulated, while L-cysteine and L-cysteine sulfinic acid (L-CSA) were downregulated, specifically among AMI risk sera. Additionally, these discriminant metabolic profiles were not related to hypertension, smoking or alcoholism. CONCLUSION: In conclusion, detecting upregulated L-HCSA and CA along with carnitine among patients at risk for AMI could serve as promising non-invasive biomarkers for early AMI detection.


Assuntos
Carnitina/sangue , Ácido Cisteico/sangue , Homocisteína/análogos & derivados , Metabolômica , Infarto do Miocárdio/metabolismo , Idoso , Aminoácidos/metabolismo , Biomarcadores/sangue , Estudos de Coortes , Feminino , Homocisteína/sangue , Humanos , Análise dos Mínimos Quadrados , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Estudos Prospectivos , Medição de Risco , Fatores de Risco
2.
Anal Bioanal Chem ; 411(29): 7771-7781, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31667563

RESUMO

While the targeted analysis of mercapturic acid (MA) metabolites in human urine is used to assess exposure to selected chemicals, this compound class has only rarely been addressed in non-target screening utilizing diagnostic neutral loss liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, this type of analysis is severely affected by matrix effects (MEs) causing poor comparability of samples and distortion of signal intensities. However, MEs have been neglected in urinary MA non-target screening so far. Therefore, we developed a non-target screening method relying on neutral loss scanning for MAs using post column infusion of an isotope-labelled standard. For signal correction, we synthesized a structural analogue to MAs, N-acetyl-S-methyl-homocysteine-D3, lacking the characteristic neutral loss of the MAs. For method development, 16 structurally different model MA compounds and 20 spiked urine samples were used. Twelve out of the 16 model compounds could be analysed by the developed method. We found severe matrix effects (largely signal suppression) for the spiked model compounds, with only 34% of all peaks' intensities changing by less than a factor of two. This could be compensated by the post column internal standard infusion with now 68% of all peaks' intensities changing by less than a factor of two. For three compounds, an over-compensation was observed resulting in an increase of signal of up to a factor of 16. In the 20 urine samples, altogether 558 native MAs (between 74 and 175 per sample) could be detected after ME compensation. These results indicate that a large number of so far uncharacterized MAs are present in urine, which yield a potential for biomarker discovery and pattern characterisation. Graphical Abstract.


Assuntos
Acetilcisteína/urina , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Acetilcisteína/normas , Homocisteína/análogos & derivados , Homocisteína/urina , Humanos , Padrões de Referência
3.
Ann Clin Lab Sci ; 49(4): 425-438, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471331

RESUMO

The purpose of this review is to elucidate how low blood cholesterol promotes mitochondrial dysfunction and mortality by the loss of thioretinaco ozonide from opening of the mitochondrial permeability transition pore (mPTP). Mortality from infections and cancer are both inversely associated with blood cholesterol, as determined by multiple cohort studies from 10 to 30 years earlier. Moreover, low-density lipoprotein (LDL) is inversely related to all-cause and/or cardiovascular mortality, as determined by followup study of elderly cohorts. LDL adheres to and inactivates most microorganisms and their toxins, causing aggregation of LDL and homocysteinylated autoantibodies which obstruct vasa vasorum and produce intimal microabscesses, the vulnerable atherosclerotic plaques. The active site of mitochondrial oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis is proposed to consist of thioretinaco, a complex of two molecules of thioretinamide with cobalamin, oxidized to the disulfonium thioretinaco ozonide and complexed with oxygen, nicotinamide adenine dinucleotide (NAD+), phosphate, and ATP. Loss of the active site complex from mitochondria results from the opening of the mPTP and from decomposition of the disulfonium active site by electrophilic carcinogens, oncogenic viruses, microbes, and by reactive oxygen radicals from ionizing and non-ionizing radiation. Suppression of innate immunity is caused by the depletion of adenosyl methionine because of increased polyamine biosynthesis, resulting in inhibition of nitric oxide and peroxynitrite biosynthesis. Opening of the mPTP produces a loss of thioretinaco ozonide from mitochondria. This loss impairs ATP biosynthesis and causes the mitochondrial dysfunction observed in carcinogenesis, atherosclerosis, aging and dementia. Cholesterol inhibits the opening of the mPTP by preventing integration of the pro-apoptotic Bcl-2-associated X protein (BAX) in the outer mitochondrial membrane. This inhibition explains how elevated LDL reduces mitochondrial dysfunction by preventing loss of the active site of oxidative phosphorylation from mitochondria.


Assuntos
Colesterol/toxicidade , Homocisteína/análogos & derivados , Homocisteína/toxicidade , Mitocôndrias/patologia , Mortalidade , Vitamina B 12/análogos & derivados , Humanos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Vitamina B 12/toxicidade
4.
J Pharm Biomed Anal ; 174: 578-587, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31261039

RESUMO

The role of homocysteic acid (HCA) in severe diseases like Alzheimer's disease is under discussion and some recent studies correlate elevated HCA concentrations with the diagnosis of Alzheimer's. However, non-selective and insufficiently sensitive methods have been used to quantitate HCA and results of different studies show large differences in the determined HCA concentration in samples from patients and controls, and therefore non-comparable results. An accurate and precise quantitation method for the determination of HCA in human serum, urine and CSF has been developed by using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC-MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. The developed method has been fully validated in accordance with the guidelines provided by the US Food and Drug administration FDA and the European Medicines Agency EMA. Furthermore, the method has demonstrated its ability to determine the endogenous HCA concentration in serum and urine samples from healthy volunteers.


Assuntos
Cromatografia Líquida/métodos , Homocisteína/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Algoritmos , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/urina , Líquidos Corporais , Calibragem , Feminino , Voluntários Saudáveis , Homocisteína/sangue , Homocisteína/líquido cefalorraquidiano , Homocisteína/urina , Humanos , Masculino , Oxigênio/química , Controle de Qualidade , Reprodutibilidade dos Testes , Extração em Fase Sólida
5.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 592-604, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205021

RESUMO

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.


Assuntos
Homocisteína/análogos & derivados , Monoéster Fosfórico Hidrolases/química , Fosfosserina/química , Serina/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X/métodos , Escherichia coli , Homocisteína/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Fosfosserina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Serina/metabolismo
6.
PLoS One ; 14(5): e0216899, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086404

RESUMO

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 µg/mL oxLDL, 500 µM Hcy, 500 nM HCTL, 100 µg/mL AGE, 200 µM H2O2 and 200 µM H2O2 with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-κB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-κB activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Homocisteína/análogos & derivados , Lipoproteínas LDL/metabolismo , Neovascularização Patológica/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Células Cultivadas , Citocinas/metabolismo , Homocisteína/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Neovascularização Patológica/patologia , Estresse Oxidativo , Epitélio Pigmentado da Retina/patologia
7.
Ann Hepatol ; 18(4): 633-639, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31078441

RESUMO

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.


Assuntos
Circulação Colateral/efeitos dos fármacos , Ácido Fólico/farmacologia , Homocisteína/análogos & derivados , Cirrose Hepática/fisiopatologia , Neovascularização Patológica/induzido quimicamente , Sistema Porta/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacos , Complexo Vitamínico B/farmacologia , Animais , Ducto Colédoco , Hemodinâmica/efeitos dos fármacos , Homocisteína/farmacologia , Ligadura , Cirrose Hepática/complicações , Neovascularização Patológica/etiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Sistema Porta/patologia , Ratos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Biochim Biophys Acta Gen Subj ; 1863(5): 941-949, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30853337

RESUMO

BACKGROUND: Elevated homocysteine is epidemiologically related to insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling. However, the effect of homocysteine on PTP1B remains unclear. METHODS: S-homocysteinylated PTP1B was identified by LC-ESI-MS/MS. The ability of thioredoxin system to recover active PTP1B from S-homocysteinylated PTP1B was confirmed by RNA interference. To address the mechanism for homocysteine to affect PTP1B activity, we performed 5-IAF insertion, activity assays, Western blotting, co-immunoprecipitation and glucose uptake experiments. RESULTS: The thiol-containing form of homocysteine (HcySH) suppressed phosphorylation of insulin receptor-ß subunit, but enhanced PTP1B activity. This phenomenon was partially related to the fact that HcySH promoted PTP1B expression. Although the disulfide-bonded form of homocysteine (HSSH) modified PTP1B to form an inactive S-homocysteinylated PTP1B, HcySH-induced increase in the activities of cellular thioredoxin and thioredoxin reductase, components of thioredoxin system, could recover active PTP1B from S-homocysteinylated PTP1B. Thioredoxin system transferred electrons from NADPH to S-homocysteinylated PTP1B, regenerating active PTP1B in vitro and in hepatocytes. The actions of HcySH were also related with decrease in hepatic glucose uptake. CONCLUSIONS: The effect of HcySH/HSSH on PTP1B activity depends, at least partially, on the ratio of active PTP1B and S-homocysteinylated PTP1B. High HcySH-induced an increase in thioredoxin system activity is beneficial to de-S-homocysteinylation and is good for PTP1B activity. GENERAL SIGNIFICANCE: Our data provide a novel insight into post-translational regulation of PTP1B, and expand the biological functions of thioredoxin system.


Assuntos
Hepatócitos/química , Homocisteína/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Homocisteína/análogos & derivados , Homocisteína/química , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 1/química
9.
Chem Commun (Camb) ; 55(25): 3654-3657, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30855056

RESUMO

Herein, we present a serendipitously discovered chemoselective labelling of protein N-homocysteinylation with bioorthogonal azide probes. The reaction proceeds rapidly under alkaline and heating conditions. Our experiments suggest that azides can be converted to aldehydes in situ catalyzed by heme(ii), followed by a condensation with protein N-homocysteinylation to afford stable 1,3-thiazines.


Assuntos
Azidas/química , Heme/química , Homocisteína/análogos & derivados , Proteínas/química , Animais , Biotina/química , Catálise , Bovinos , Homocisteína/química , Concentração de Íons de Hidrogênio , Mioglobina/química , Mioglobina/metabolismo , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Temperatura
10.
Proteins ; 87(8): 625-634, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30869815

RESUMO

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys34 of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys34 of albumin and homocysteine attached to lysine residues.


Assuntos
Cisteína/metabolismo , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Albumina Sérica Humana/metabolismo , Humanos , Hidrólise , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
11.
Methods Mol Biol ; 1866: 285-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725425

RESUMO

The objective of the proposed clinical interventional trial is to demonstrate the efficacy of a novel therapeutic strategy in subjects with cancer and hyperhomocysteinemia. Following discovery of abnormal homocysteine thiolactone metabolism in cultured malignant cells, thioretinamide, the amide synthesized from retinoic acid and homocysteine thiolactone, and thioretinaco, the complex formed from cobalamin and thioretinamide, were demonstrated to have antineoplastic, anticarcinogenic, and anti-atherogenic properties in animal models. Retinol, ascorbate, and homocysteine thiolactone are necessary for biosynthesis of thioretinamide and thioretinaco by cystathionine synthase and for formation of thioretinaco ozonide from thioretinamide, cobalamin, and ozone. Thioretinaco ozonide is required for prevention of abnormal oxidative metabolism, aerobic glycolysis, suppressed immunity, and hyperhomocysteinemia in cancer.The pancreatic enzyme therapy of cancer promotes catabolism of proteins, nucleic acids, and glycosaminoglycans with excess homocysteinylated amino groups resulting from abnormal accumulation of homocysteine thiolactone in malignant cells. Dietary deficiencies of pyridoxal, folate, cobalamin, and nitriloside contribute to hyperhomocysteinemia in cancer, and in protein energy malnutrition. A deficiency of dietary sulfur amino acids downregulates cystathionine synthase, causing hyperhomocysteinemia.The organic sulfur compound diallyl trisulfide increases hydrogen sulfide production from homocysteine in animal models, inhibits Stat3 signaling in cancer stem cells, and produces apoptosis of malignant cells. The furanonaphthoquinone compound napabucasin inhibits Stat3 signaling and causes mitochondrial dysfunction, decreased oxidative phosphorylation, and apoptosis of malignant cells. The protocol of the proposed clinical trial in subjects with myelodysplasia consists of thioretinamide and cobalamin as precursors of thioretinaco ozonide, combined with pancreatic enzyme extracts, diallyl trisulfide, napabucasin, nutritional modification to minimize processed foods, vitamin supplements, essential amino acids, and beneficial dietary fats and proteins.


Assuntos
Envelhecimento/fisiologia , Homocisteína/análogos & derivados , Homocisteína/uso terapêutico , Neoplasias/tratamento farmacológico , Fosforilação Oxidativa , Vitamina B 12/análogos & derivados , Adulto , Idoso , Drogas em Investigação/farmacologia , Drogas em Investigação/uso terapêutico , Homocisteína/farmacologia , Humanos , Licenciamento , Pessoa de Meia-Idade , Fosforilação Oxidativa/efeitos dos fármacos , Vitamina B 12/uso terapêutico
12.
ACS Chem Biol ; 14(2): 186-191, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30668907

RESUMO

The RhlR quorum sensing (QS) receptor in the pathogen Pseudomonas aeruginosa plays a prominent role in infection, and both antagonism and agonism of RhlR have been shown to negatively regulate important virulence phenotypes. Non-native lactone ligands are known to modulate RhlR activity, but their utility as chemical probes is relatively limited due to hydrolytic instability. Herein, we report our design and biological evaluation of a suite of hybrid AHL analogs with structures merging (1) features of reported lead RhlR ligands and (2) head groups with improved hydrolytic stabilities. The most promising compounds identified were N-acyl l-homocysteine thiolactones, which displayed enhanced stabilities relative to lactones. Moreover, they were highly selective for RhlR over another key QS receptor in P. aeruginosa, LasR. These compounds are among the most potent RhlR modulators known and represent robust chemical tools to dissect the complex roles of RhlR in the P. aeruginosa QS circuitry.


Assuntos
Proteínas de Bactérias/metabolismo , Homocisteína/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Relação Dose-Resposta a Droga , Homocisteína/administração & dosagem , Homocisteína/farmacologia , Pseudomonas aeruginosa/fisiologia
13.
Physiol Res ; 68(Suppl 3): S361-S366, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31928054

RESUMO

Mechanism of ictogenesis of D- and L-stereroisomers of homocysteic acid was studied in 12-day-old rats by means of antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. There was no qualitative difference between the two stereoisomers in generation of emprosthotonic (flexion) as well as generalized tonic-clonic seizures. Moderate differences were observed in the first, nonconvulsive effects of the two isomers. As generation of the two types of seizures is concerned, NMDA and AMPA participate in generalized tonic-clonic seizures whereas NMDA receptors play a dominant role in generation of flexion seizures.


Assuntos
Homocisteína/análogos & derivados , Convulsões/induzido quimicamente , 2-Amino-5-fosfonovalerato/análogos & derivados , Animais , Benzodiazepinas , Maleato de Dizocilpina , Homocisteína/química , Homocisteína/toxicidade , Masculino , Quinoxalinas , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Estereoisomerismo
14.
J Intern Med ; 285(2): 232-244, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30193001

RESUMO

OBJECTIVES: No individual homocysteine (Hcy) metabolite has been studied as a risk marker for coronary artery disease (CAD). Our objective was to examine Hcy-thiolactone, a chemically reactive metabolite generated by methionyl-tRNA synthetase and cleared by the kidney, as a risk predictor of incident acute myocardial infarction (AMI) in the Western Norway B-Vitamin Intervention Trial. DESIGN: Single centre, prospective double-blind clinical intervention study, randomized in a 2 × 2 factorial design. SUBJECTS AND METHODS: Patients with suspected CAD (n = 2049, 69.8% men; 61.2-year-old) were randomized to groups receiving daily (i) folic acid (0.8 mg)/vitamin B12 (0.4 mg)/vitamin B6 (40 mg); (ii) folic acid/vitamin B12 ; (iii) vitamin B6 or (iv) placebo. Urinary Hcy-thiolactone was quantified at baseline, 12 and 38 months. RESULTS: Baseline urinary Hcy-thiolactone/creatinine was significantly associated with plasma tHcy, ApoA1, glomerular filtration rate, potassium and pyridoxal 5'-phosphate (positively) and with age, hypertension, smoking, urinary creatinine, plasma bilirubin and kynurenine (negatively). During median 4.7-years, 183 patients (8.9%) suffered an AMI. In Cox regression analysis, Hcy-thiolactone/creatinine was associated with AMI risk (hazard ratio = 1.58, 95% confidence interval = 1.10-2.26, P = 0.012 for trend; adjusted for age, gender, tHcy). This association was confined to patients with pyridoxic acid below median (adjusted HR = 2.72, 95% CI = 1.47-5.03, P = 0.0001; Pinteraction = 0.020). B-vitamin/folate treatments did not affect Hcy-thiolactone/creatinine and its AMI risk association. CONCLUSIONS: Hcy-thiolactone/creatinine ratio is a novel AMI risk predictor in patients with suspected CAD, independent of traditional risk factors and tHcy, but modified by vitamin B6 catabolism. These findings lend a support to the hypothesis that Hcy-thiolactone is mechanistically involved in cardiovascular disease.


Assuntos
Doença da Artéria Coronariana/urina , Ácido Fólico/administração & dosagem , Homocisteína/análogos & derivados , Infarto do Miocárdio/etiologia , Vitamina B 12/administração & dosagem , Vitamina B 6/administração & dosagem , Biomarcadores/urina , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/tratamento farmacológico , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Seguimentos , Homocisteína/urina , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/urina , Prognóstico , Estudos Prospectivos , Complexo Vitamínico B/administração & dosagem
15.
Neurobiol Dis ; 121: 106-119, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30266286

RESUMO

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.


Assuntos
Disfunção Cognitiva/tratamento farmacológico , Giro Denteado/efeitos dos fármacos , Córtex Entorrinal/efeitos dos fármacos , Homocisteína/análogos & derivados , Hiperalgesia/tratamento farmacológico , Potenciação de Longa Duração/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Animais , Disfunção Cognitiva/etiologia , Homocisteína/administração & dosagem , Camundongos Endogâmicos C57BL , Vias Neurais/efeitos dos fármacos , Neuralgia/complicações , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Traumatismos dos Nervos Periféricos/complicações , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/ultraestrutura , Receptores de AMPA/metabolismo , Nervo Isquiático/lesões
16.
Bioorg Med Chem ; 27(1): 36-42, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30473360

RESUMO

Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Liases de Carbono-Enxofre/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Homocisteína/análogos & derivados , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/química , Liases de Carbono-Enxofre/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Homocisteína/síntese química , Homocisteína/farmacologia , Percepção de Quorum/efeitos dos fármacos
17.
Exp Eye Res ; 178: 228-237, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29608906

RESUMO

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller cells were exposed to L-Hcy-thiolactone [50µM-10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 µM - 1 mM]. Moreover, when exposed to elevated Hcy, Müller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Müller cells harvested from Nrf2-/- mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2-/- Müller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Müller cells.


Assuntos
Células Ependimogliais/efeitos dos fármacos , Homocisteína/análogos & derivados , Fator 2 Relacionado a NF-E2/metabolismo , Protetores contra Radiação/farmacologia , Animais , Elementos de Resposta Antioxidante/fisiologia , Sobrevivência Celular , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Glutationa/metabolismo , Homocisteína/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima
18.
FEBS Lett ; 592(20): 3399-3413, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30194685

RESUMO

Glutamate racemases (GR) catalyze the racemization of d- and l-glutamate and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d-homocysteic acid (d-HCA), while l-HCA is a poor substrate. This enantioselectivity arises because l-HCA perturbs FnGR's monomer-dimer equilibrium toward inactive monomer. The inhibitory effect of l-HCA may be overcome by increasing the total FnGR concentration or by adding glutamate, but not by blocking access to the active site through site-directed mutagenesis, suggesting that l-HCA binds at an allosteric site. This phenomenon is also exhibited by GR from Bacillus subtilis, suggesting that enantiospecific, "substrate"-induced dissociation of oligomers to form inactive monomers may furnish a new inhibition strategy.


Assuntos
Isomerases de Aminoácido/química , Proteínas de Bactérias/química , Homocisteína/análogos & derivados , Estrutura Quaternária de Proteína , Sítio Alostérico , Isomerases de Aminoácido/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Fusobacterium nucleatum/enzimologia , Homocisteína/química , Homocisteína/metabolismo , Cinética , Estereoisomerismo , Especificidade por Substrato
19.
Artigo em Inglês | MEDLINE | ID: mdl-30241070

RESUMO

It is well established that homocysteine thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC-MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min-1 and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01-0.20 nmol mL-1 urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL-1, respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from

Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Homocisteína/análogos & derivados , Formiatos/química , Homocisteína/isolamento & purificação , Homocisteína/urina , Humanos , Limite de Detecção , Modelos Lineares , Extração Líquido-Líquido , Reprodutibilidade dos Testes
20.
Tumour Biol ; 40(9): 1010428318797869, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30178714

RESUMO

Paraoxonase 1 plays an important role in protection from oxidative stress and also decomposes homocysteine thiolactone, the toxic metabolite of homocysteine. A limited number of reports evaluated the role of paraoxonase 1 in women affected by female genital tract neoplasms, including endometrial cancer. This study aimed to analyze the paraoxonase activity in the group of endometrial cancer patients (n = 48) who underwent primary surgery and to compare the data available with a well-matched control group (n = 30). Due to the role of paraoxonase 1 in the metabolism of homocysteine (Hcy) thiolactone, the amount of Hcy-thiolactone as well as total serum Hcy concentrations was also measured. Serum paraoxonase 1 activity toward synthetic substrates, paraoxon and phenyl acetate, in the study group was significantly lower compared to the control one. The mean paraoxonase 1 activity toward homocysteine thiolactone tended to be lower in the endometrial cancer group but this difference was not significant. There was no relationship between endometrial cancer and Q192R polymorphism of PON1 assessed by the dual substrate method. No differences in paraoxonase 1 activity between endometrial cancer subgroups according to clinico-pathological features were detected. Total serum homocysteine and protein-bound homocysteine thiolactone did not differ between control and cancer groups. In conclusion, reduced paraoxonase 1 activity suggests diminished important antioxidant mechanisms during the development of primary endometrial cancers in humans. PON1 Q192R polymorphism is not associated with the risk of endometrial cancer. Despite lower paraoxonase 1 activity, homocysteine concentration, and protein N-homocysteinylation in endometrial cancers do not differ from matched controls.


Assuntos
Arildialquilfosfatase/metabolismo , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/patologia , Homocisteína/análogos & derivados , Processamento de Proteína Pós-Traducional , Idoso , Estudos de Casos e Controles , Neoplasias do Endométrio/cirurgia , Feminino , Homocisteína/metabolismo , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico
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