Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 217
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146362

RESUMO

BACKGROUND: To investigate the expression of vascular adhesion protein-1 (VAP-1) in joint tissues and serum in symptomatic knee osteoarthritis (SKOA) patients and examine whether VAP-1 levels predict increased risk of disease severity in a cross-sectional study. METHODS: Baseline VAP-1 expression and soluble VAP-1 (sVAP-1) levels were assessed in the synovium synovial fluid and in the serum in cohorts of patients with tibiofemoral medial knee OA and healthy subjects. Standardized fixed-flexion poster anterior knee radiographs scored for Kellgren-Lawrence (KL) grade (0-4) and medial joint space width (JSW). KL1/2 vs. KL3/4 scores defined early and advanced radiographic severity, respectively. Biochemical markers assessed in serum or synovial fluids (SF) comprised sVAP-1, interleukin 1 receptor antagonist (IL-1Ra), interleukin 6 (IL-6), soluble receptor for advanced glycation end-products (sRAGE), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 4 (CCL4), cluster of differentiation 163 (CD163), high sensitivity C-reactive protein (hsCRP), and matrix metalloproteinases (MMPs)-1,-3,-9. Associations between biomarkers and radiographic severity KL1/2 vs. KL3/4 (logistic regression controlling for covariates) and pain (Spearman correlation) were evaluated. RESULTS: Elevated levels of sVAP-1 observed in OA synovial fluid and VAP-1 expression in synovium based on immunohistochemical, microarray, and real-time quantitative polymerase chain reaction (qRT-PCR) analyses. However, serum sVAP-1 levels in OA patients were lower than in controls and inversely correlated with pain and inflammation markers (hsCRP and soluble RAGE). Soluble VAP-1 levels in serum were also lower in radiographically advanced (KL3/4) compared with early KL1/2 knee SKOA patients. CONCLUSION: Local (synovial fluid) semicarbazide-sensitive amine oxidase (SSAO)/sVAP-1 levels were elevated in OA and correlated with radiographic severity. However, systemic (serum) sVAP-1 levels were lower in SKOA patients than normal and inversely correlated with pain and inflammation markers. Serum sVAP-1 levels were higher in early (KL1/2) compared with advanced (KL3/4) SKOA patients.


Assuntos
Amina Oxidase (contendo Cobre)/sangue , Moléculas de Adesão Celular/sangue , Osteoartrite do Joelho/metabolismo , Adulto , Idoso , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/sangue , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Radiografia , Líquido Sinovial/metabolismo
2.
Mol Med Rep ; 19(2): 959-966, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569103

RESUMO

Adipose tissue has a primary role in lipid and glucose metabolism as a storage site for fatty acids, and also functions as an endocrine organ, producing large numbers of hormones and cytokines. Adipose dysfunction triggers a number of obesity­associated health problems. The aim of the present study was, therefore, to investigate the molecular mechanisms of white adipose tissue (WAT). The GSE9954 microarray data were downloaded from the Gene Expression Omnibus. Adipose­specific genes were identified through limma package analysis, based on samples of WAT and 17 other types of non­adipose tissue obtained from mice. Process and pathway enrichment analyses were performed for these genes. Finally, protein­protein interaction (PPI) and co­expression networks were constructed and analyzed. In total, 202 adipose­specific genes were identified, which were involved in key biological processes (including fat cell differentiation and lipid metabolic process) and one key pathway [namely, the adenine monophosphate­activated protein kinase (AMPK) signaling pathway]. Construction of the PPI network and further molecular complex detection revealed the presence of 17 key genes, including acetyl­CoA carboxylase α, peroxisome proliferator­activated receptor (PPAR) γ and leptin, that were involved in the AMPK, PPAR and insulin signaling pathways. In addition, amine oxidase copper containing 3 and adrenoceptor beta 3 were communication hubs in the co­expression network of adipose­specific genes. In conclusion, the present study promotes our understanding of the underlying molecular mechanisms of WAT, and may offer an insight into the prevention and treatment of obesity­associated diseases caused by adipose dysfunction.


Assuntos
Tecido Adiposo Branco/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Metabolismo dos Lipídeos/genética , Transcriptoma , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Leptina/genética , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Especificidade de Órgãos , PPAR gama/genética , PPAR gama/metabolismo , Mapeamento de Interação de Proteínas , Receptores Adrenérgicos/genética , Receptores Adrenérgicos/metabolismo , Transdução de Sinais
3.
Biochemistry ; 57(36): 5301-5314, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30110143

RESUMO

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ∼32 Šfrom the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Amina Oxidase (contendo Cobre)/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Espécies Reativas de Oxigênio/química
4.
J Am Soc Nephrol ; 29(5): 1513-1524, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29545352

RESUMO

Background The kidneys have a central role in the generation, turnover, transport, and excretion of metabolites, and these functions can be altered in CKD. Genetic studies of metabolite concentrations can identify proteins performing these functions.Methods We conducted genome-wide association studies and aggregate rare variant tests of the concentrations of 139 serum metabolites and 41 urine metabolites, as well as their pairwise ratios and fractional excretions in up to 1168 patients with CKD.Results After correction for multiple testing, genome-wide significant associations were detected for 25 serum metabolites, two urine metabolites, and 259 serum and 14 urinary metabolite ratios. These included associations already known from population-based studies. Additional findings included an association for the uremic toxin putrescine and variants upstream of an enzyme catalyzing the oxidative deamination of polyamines (AOC1, P-min=2.4×10-12), a relatively high carrier frequency (2%) for rare deleterious missense variants in ACADM that are collectively associated with serum ratios of medium-chain acylcarnitines (P-burden=6.6×10-16), and associations of a common variant in SLC7A9 with several ratios of lysine to neutral amino acids in urine, including the lysine/glutamine ratio (P=2.2×10-23). The associations of this SLC7A9 variant with ratios of lysine to specific neutral amino acids were much stronger than the association with lysine concentration alone. This finding is consistent with SLC7A9 functioning as an exchanger of urinary cationic amino acids against specific intracellular neutral amino acids at the apical membrane of proximal tubular cells.Conclusions Metabolomic indices of specific kidney functions in genetic studies may provide insight into human renal physiology.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Transporte Biológico/genética , Túbulos Renais/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Acil-CoA Desidrogenase/genética , Adulto , Idoso , Amina Oxidase (contendo Cobre)/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Glutamina/urina , Humanos , Metabolismo dos Lipídeos/fisiologia , Lisina/urina , Masculino , Metaboloma , Pessoa de Meia-Idade , Estudos Prospectivos , Putrescina/metabolismo
5.
Jpn J Ophthalmol ; 62(2): 256-264, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29392528

RESUMO

PURPOSE: To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). STUDY DESIGN: Experimental. METHODS: Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. RESULTS: IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. CONCLUSIONS: The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.


Assuntos
Amina Oxidase (contendo Cobre)/genética , Capilares/metabolismo , Moléculas de Adesão Celular/genética , Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Vasos Retinianos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Amina Oxidase (contendo Cobre)/biossíntese , Western Blotting , Capilares/patologia , Moléculas de Adesão Celular/biossíntese , Células Cultivadas , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese
6.
PLoS Pathog ; 14(2): e1006850, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29425238

RESUMO

Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.


Assuntos
Aldeído Desidrogenase/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Compostos de Boro/metabolismo , Modelos Biológicos , Pró-Fármacos/metabolismo , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/enzimologia , Ativação Metabólica , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Aldeído Oxirredutases/antagonistas & inibidores , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/genética , Substituição de Aminoácidos , Animais , Compostos de Boro/química , Compostos de Boro/farmacologia , Resistência a Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Mutação , Filogenia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/fisiologia
7.
J Diabetes Investig ; 9(5): 1067-1074, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29364582

RESUMO

AIMS/INTRODUCTION: Vascular adhesion protein-1 (VAP-1) is a membrane-bound amine oxidase highly expressed in mature adipocytes and released into the circulation. VAP-1 has been strongly implicated in several pathological processes, including diabetes, inflammation, hypertension, hepatic steatosis and renal diseases, and is an important disease marker and therapeutic target. Here, we aimed to identify the genetic loci for circulating VAP-1 levels. MATERIALS AND METHODS: We carried out a genomic-wide linkage scan for the quantitative trait locus of circulating VAP-1 levels in 1,100 Han Chinese individuals from 398 families in the Stanford Asian Pacific Program for Hypertension and Insulin Resistance study. Regional association fine mapping was carried out using additional single-nucleotide polymorphisms. RESULTS: The estimated heritability of circulating VAP-1 levels is high (h2 = 69%). The most significant quantitative trait locus for circulating VAP-1 was located at 38 cM on chromosome 20, with a maximum empirical logarithm of odds score of 4.11 (P = 6.86 × 10-6 ) in females. Regional single-nucleotide polymorphism fine mapping within a 1-unit support region showed the strongest association signals in the MACRO domain containing 2 (MACROD2) gene in females (P = 5.38 × 10-6 ). Knockdown of MACROD2 significantly suppressed VAP-1 expression in human adipocytes, as well as the expression of key adipogenic genes. Furthermore, MACROD2 expression was found to be positively associated with VAP-1 in human visceral adipose tissue. CONCLUSION: MACROD2 is a potential genetic determinant of serum VAP-1 levels, probably through transcriptional regulation of adipogenesis.


Assuntos
Adipogenia/genética , Amina Oxidase (contendo Cobre)/genética , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Enzimas Reparadoras do DNA/genética , Regulação da Expressão Gênica , Ligação Genética , Hidrolases/genética , Feminino , Seguimentos , Humanos , Resistência à Insulina , Masculino , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Taiwan
8.
Benef Microbes ; 9(2): 247-255, 2018 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-29022381

RESUMO

Spermine (SPM) and its precursor putrescine (PUT), regulated by ornithine decarboxylase (ODC) and diamino-oxidase (DAO), are polyamines required for cell growth and proliferation. Only a few studies have investigated the anti-inflammatory and tumour inhibitory properties of probiotics on mucosal polyamine levels. We investigated the effects of a high concentration multistrain probiotic for human use on colonic polyamine biosynthesis in dogs. Histological sections (inflammatory bowel disease, n=10; polyposis, n=5) were assessed after receiving 112 to 225×109 lyophilised bacteria daily for 60 days at baseline (T0) and 30 days after treatment end (T90). Histology scores, expression of PUT, SPM, ODC and DAO, and a clinical activity index (CIBDAI) were compared at T0 and T90. In polyps, cellular proliferation (Ki-67 expression), and apoptosis (caspase-3 protein expression) were also evaluated. After treatment, in inflammatory bowel disease significant decreases were observed for CIBDAI (P=0.006) and histology scores (P<0.001); PUT, SPM and ODC expression increased (P<0.01). In polyps, a significant decrease in polyamine levels, ODC activity, and Ki-67, and a significant increase in caspase-3 positivity and DAO expression (P=0.005) was noted. Our results suggest potential anti-proliferative and anti-inflammatory effects of the probiotic mixture in polyps and inflammation, associated with reduced mucosal infiltration and up-regulation of PUT, SPM, and ODC levels.


Assuntos
Fenômenos Fisiológicos Bacterianos , Pólipos do Colo/veterinária , Doenças do Cão/tratamento farmacológico , Doenças Inflamatórias Intestinais/veterinária , Probióticos/uso terapêutico , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Colo/metabolismo , Colo/patologia , Pólipos do Colo/tratamento farmacológico , Pólipos do Colo/microbiologia , Pólipos do Colo/patologia , Doenças do Cão/microbiologia , Doenças do Cão/patologia , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Putrescina/biossíntese , Espermina/biossíntese , Resultado do Tratamento
9.
Methods Mol Biol ; 1694: 215-223, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29080170

RESUMO

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.


Assuntos
Agrobacterium/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Tabaco/enzimologia , Agrobacterium/genética , Amina Oxidase (contendo Cobre)/genética , Expressão Gênica , Genes Reporter , Redes e Vias Metabólicas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Plasmídeos/genética , Poliaminas/metabolismo , Tabaco/genética
10.
Inflamm Res ; 67(3): 245-253, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29164268

RESUMO

OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO were expressed as glutathione-S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/imunologia , DNA Complementar/genética , Epitopos , Escherichia coli/genética , Humanos , Plasmídeos , Ligação Proteica
11.
Mol Vis ; 23: 853-871, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29259392

RESUMO

Purpose: We investigated the link among the proinflammatory cytokine high-mobility group box 1 (HMGB1) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative DNA damage, the endothelial adhesion molecule and oxidase enzyme vascular adhesion protein-1 (VAP-1), and the inducible cytoprotective molecule heme oxygenase-1 (HO-1) in proliferative diabetic retinopathy (PDR). We correlated the levels of these molecules with clinical disease activity and studied the proinflammatory activities of HMGB1 on rat retinas and human retinal microvascular endothelial cells (HRMECs). Methods: Vitreous samples from 47 PDR and 19 non-diabetic patients, epiretinal membranes from 11 patients with PDR, human retinas (16 from diabetic patients and 16 from non-diabetic subjects), rat retinas, and HRMECs were studied by enzyme-linked immunosorbent assay, immunohistochemistry, western blot immunofluorescence, and RT-PCR analyses. In addition, we assessed the adherence of leukocytes to HMGB1-stimulated HRMECs. Results: HMGB1, 8-OHdG, and soluble VAP-1 (sVAP-1) levels were significantly higher in vitreous samples from PDR patients than in those from non-diabetics (p = 0.001, <0.0001, <0.0001, respectively). The HMGB1, 8-OHdG, sVAP-1, and HO-1 levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p = 0.025, <0.0001, <0.0001, 0.012, respectively). Significant positive correlations were observed between the levels of HMGB1 and the levels of 8-OHdG (r = 0.422; p = 0.001) and sVAP-1 (r = 0.354; p = 0.004) and between the levels of 8-OHdG and the levels of sVAP-1 (r = 0.598; p<0.0001). In epiretinal membranes, VAP-1 and 8-OHdG were expressed in vascular endothelial cells and stromal cells. Significant increases in the VAP-1 mRNA and protein levels were detected in the RPE, but not in the neuroretina of diabetic patients. Treatment of HRMEC with HMGB1, diabetes induction, and an intravitreal injection of HMGB1 in normal rats induced a significant upregulation of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in HRMECs and retinas. On the other hand, the expressions of vascular cell adhesion molecule-1 and VAP-1 were not affected. Oral administration of the HMGB1 inhibitor glycyrrhizin in rats attenuated the diabetes-induced upregulation of the retinal ICAM-1 expression. Treatment of HRMECs with HMGB1 increased leukocyte adhesion and induced the upregulation of 8-OHdG and HO-1 and the membranous translocation of VAP-1. Conclusions: Our results suggest a potential link among the proinflammatory cytokine HMGB1, VAP-1, oxidative stress, and HO-1 in the pathogenesis of PDR.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Moléculas de Adesão Celular/metabolismo , Desoxiguanosina/análogos & derivados , Retinopatia Diabética/metabolismo , Proteína HMGB1/metabolismo , Heme Oxigenase-1/metabolismo , Estresse Oxidativo , Adulto , Idoso , Amina Oxidase (contendo Cobre)/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Moléculas de Adesão Celular/genética , Dano ao DNA , Desoxiguanosina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Proteína HMGB1/farmacologia , Heme Oxigenase-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Corpo Vítreo/metabolismo
12.
J Exp Bot ; 68(17): 4851-4867, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28992128

RESUMO

Root system architecture depends on endogenous and environmental signals, including polar transport of the phytohormone auxin, reactive oxygen species (ROS), nutrient availability, and stresses. In our study, we describe a novel Arabidopsis thaliana peroxisome-localized copper amine oxidase ζ (CuAOζ), which is highly expressed in cortical cells, and the ROS derived from CuAOζ are essential for lateral root (LR) development. Loss of CuAOζ results in retarded auxin-induced ROS generation, PINFORMED2 (PIN2)-mediated auxin transport, and LR development in response to added indole-3-butyric acid. Auxins enhance CuAOζ protein levels and their cellular translocation toward the plasma membrane in the cortex. CuAOζ interacts physically with PEROXINS5 via an N-terminal signal tag, Ser-Lys-Leu, and is transported into the peroxisome upon this interaction, which is required for the functions of CuAOζ in the auxin response. Together, our results suggest a peroxisomal ROS-based auxin signaling pathway involving spatiotemporal-dependent CuAOζ functional regulation of PIN2 homeostasis, auxin distribution, and LR development.


Assuntos
Amina Oxidase (contendo Cobre)/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Peróxido de Hidrogênio/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Indóis/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
13.
Sci Rep ; 7(1): 2766, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28584293

RESUMO

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.


Assuntos
Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Colletotrichum/enzimologia , Colletotrichum/genética , Alcaloides/biossíntese , Amina Oxidase (contendo Cobre)/isolamento & purificação , Carboxiliases/isolamento & purificação , Cromatografia Líquida , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ordem dos Genes , Lisina/metabolismo , Plasmídeos , Proteínas Recombinantes , Sesquiterpenos
14.
Mol Med Rep ; 16(2): 1189-1199, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28627649

RESUMO

Hemorrhagic shock following major trauma results in mortality, but the function of vascular adhesion protein­1 (VAP­1), implicated in intracranial hemorrhage, remains unknown. This study aimed to determine whether expression of the AOC3 gene and its encoded protein, VAP­1, is altered by hypoxia. Rat hepatic sinusoidal endothelial cells (RHSECs) and rat intestinal microvascular endothelial cells (RIMECs) were transduced with a viral vector carrying AOC3, and AOC3 mRNA expression levels were measured by reverse transcription­quantitative polymerase chain reaction. VAP­1 protein expression levels were measured by western blot analysis and compared between normoxic and hypoxic conditions. Following this, AOC3 mRNA and VAP­1 protein expression levels in hepatic and intestinal tissues were assessed in a rat model of hemorrhagic shock with or without fluid resuscitation; and serum semicarbazide­sensitive amine oxidase (SSAO) activity was measured by fluorometric assays. The effects of 2­bromoethylamine (2­BEA) on AOC3/VAP­1 levels and 24 h survival were investigated. AOC3 mRNA and VAP­1 protein levels were increased in RHSECs and RIMECs by hypoxia, and in hepatic and intestinal tissues from rats following hemorrhagic shock. Hypoxia increased serum SSAO activity in these animals. 2­BEA reduced AOC3 mRNA and VAP­1 protein levels in hepatic and intestinal tissues from rats following hemorrhagic shock, and appeared to improve survival in animals not receiving resuscitation following hemorrhagic shock. In conclusion, hemorrhagic shock upregulates AOC3/VAP­1 expressions, and this potentially occurs via hypoxia. Therefore, inhibition of VAP­1 may be beneficial in the setting of hemorrhagic shock. Further studies are required to confirm these findings and to establish whether VAP­1 may be a valid target for the development of novel therapies for hemorrhagic shock.


Assuntos
Amina Oxidase (contendo Cobre)/genética , Moléculas de Adesão Celular/genética , Regulação da Expressão Gênica , Choque Hemorrágico/genética , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Biomarcadores , Dióxido de Carbono/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Etilaminas/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Oxigênio/metabolismo , Ratos , Ressuscitação , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/metabolismo , Choque Hemorrágico/mortalidade
15.
Eur Neuropsychopharmacol ; 27(5): 442-449, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28400155

RESUMO

Sedation is a common adverse effect of clozapine treatment, which may be partly related to clozapine binding to histamine receptors in the central nervous system. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the histaminergic system are associated with sedation in clozapine-treated patients. The study population comprised 237 clozapine-treated, Finnish, Caucasian patients that were diagnosed with schizophrenia and 176 were genotyped using Illumina HumanCoreExome-12 BeadChip. Sedation levels were assessed using self-rating questions from the Liverpool University Neuroleptic Side Effect Rating Scale (LUNSERS). The relationships between 55 different SNPs in the histaminergic system and adverse sedation effects were examined. SNPs were analyzed separately, and in groups, to formulate a genetic risk score (GRS). A permutation test was performed to avoid type I errors. Eight linked SNPs (r2 = 1) in the HNMT gene were also associated with sedation according to the GLM, adjusted for age, gender and BMI (false-discovery-rate-adjusted p = 0.013). An association on a trend level between a GRS of four different SNPs (recessive histamine N-methyltransferase HNMT rs2737385, additive histamine receptor H1 rs1552498, dominant HRH1 rs17034063 and recessive amine oxidase, copper containing 1 AOC1 rs6977381) and sedation was found (permuted p-value = 0.066) in a generalized linear model (GLM) incorporating age, gender and body mass index (BMI; adjusted R2 = 0.22). Polymorphisms in genes encoding histamine receptors or enzymes related to histamine metabolism may explain individual variation in sedative effects experienced during clozapine treatment.


Assuntos
Antipsicóticos/efeitos adversos , Clozapina/efeitos adversos , Histamina N-Metiltransferase/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores Histamínicos/genética , Esquizofrenia/tratamento farmacológico , Adulto , Amina Oxidase (contendo Cobre)/genética , Distribuição de Qui-Quadrado , Sedação Consciente , Feminino , Finlândia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome Maligna Neuroléptica/etiologia , Síndrome Maligna Neuroléptica/genética , Análise de Regressão , Esquizofrenia/genética , Índice de Gravidade de Doença
16.
FASEB J ; 31(6): 2477-2491, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28251930

RESUMO

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface-expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3-knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Moléculas de Adesão Celular/metabolismo , Fibrose Pulmonar/enzimologia , Amina Oxidase (contendo Cobre)/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Ácidos Carboxílicos , Moléculas de Adesão Celular/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Pulmão/enzimologia , Pulmão/patologia , Linfócitos/fisiologia , Camundongos , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Pirrolidinas
17.
Int J Mol Med ; 39(3): 719-724, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28112368

RESUMO

Human lysyl oxidase-like 3 (LOXL3) functions as a copper-dependent amine oxidase toward collagen and elastin. The LOXL3 protein contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus in addition to the C-terminal characteristic domains of the lysyl oxidase (LOX) family, such as a copper-binding domain, a cytokine receptor­like domain and residues for the lysyl-tyrosyl quinone cofactor. Using BLASTN searches, we identified a novel variant of LOXL3 (termed LOXL3-sv2), which lacked the sequences corresponding to exons 4 and 5 of LOXL3. The LOXL3-sv2 mRNA is at least 2,398 bp in length, encoding a 608 amino acid-long polypeptide with a calculated molecular mass of 67.4 kDa. The deletion of exons 4 and 5 do not change the open-reading frame of LOXL3 but results in deletion of the SRCR domain 2. The recombinant LOXL3-sv2 protein showed a ß-aminopropionitrile-inhibitable amine oxidase activity toward collagen type I. In RT-PCR analysis, LOXL3-sv2 was detected in all human tissues tested, along with LOXL3 and LOXL3-sv1, a previously identified variant of LOXL3. These findings indicate that the human LOXL3 gene encodes at least three variants, LOXL3, LOXL3-sv1 and LOXL3-sv2, all of which function as amine oxidases.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Aminoácido Oxirredutases/metabolismo , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/genética , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Ativação Enzimática , Éxons , Ordem dos Genes , Humanos , Íntrons , Domínios Proteicos , RNA Mensageiro/genética , Proteínas Recombinantes
18.
Biotechnol J ; 12(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27440252

RESUMO

As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.


Assuntos
Células CHO/citologia , Células CHO/ultraestrutura , Microscopia Confocal/métodos , Engenharia de Proteínas/métodos , Adalimumab/genética , Adalimumab/metabolismo , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Análise por Conglomerados , Cricetulus , Retículo Endoplasmático/ultraestrutura , Humanos , Lipídeos/química , Metais/química , Imagem Molecular/métodos , Análise de Componente Principal , Proteínas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral Raman/métodos
19.
Neurologia ; 32(8): 500-507, 2017 Oct.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-27130307

RESUMO

BACKGROUND: Low histamine metabolism has been suggested to play a role in the pathogenesis of allergy and migraine. We investigated the possible association between 2 single-nucleotide polymorphisms (SNP), C314T HNMT and C2029G DAO, and the presence and severity of migraine and migraine-related disability. MATERIALS AND METHODS: We studied the frequency of C314T HNMT and C2029G DAO allelic variants in 162 mothers of children with allergies (80 with migraine and 82 without) using a TaqMan-based qPCR Assay and a case-control model. We conducted a logistic regression analysis to examine the association between migraine and the allelic and haplotype variants. RESULTS: Mutant C2029G DAO SNP was found significantly more frequently in the group of women with migraine than in controls (OR, 1.6; 95% CI, 1.1-2.1). No significant differences were found in frequencies of genotypes or alleles in the case of C314T HNMT SNP. Both mutated alleles were associated with migraine-related disability. Coexistence of alleles for both SNPs (haplotypes) showed a strong association with migraine. Haplotypes containing both mutated alleles (either heterozygous or homozygous) were very strongly associated with MIDAS grade iv migraine (OR, 45.0; 95% CI, 5.2-358). This suggests that mutant alleles of C314T for HNMT and C2029G for DAO polymorphisms may interact in a way that increases the risk and impact of migraine. CONCLUSIONS: We suggest a synergistic association between HNMT and DAO functional polymorphisms and migraine; this hypothesis must be further confirmed by larger studies. However, the characteristics and ethnic differences between analysed populations should be considered when interpreting the results.


Assuntos
Amina Oxidase (contendo Cobre)/genética , Predisposição Genética para Doença , Histamina N-Metiltransferase/genética , Transtornos de Enxaqueca/genética , Mães , Polimorfismo de Nucleotídeo Único/genética , Adulto , Estudos de Casos e Controles , Criança , Feminino , Genótipo , Hispano-Americanos/estatística & dados numéricos , Histamina/metabolismo , Humanos , Hipersensibilidade/etiologia , México , Transtornos de Enxaqueca/diagnóstico
20.
J Alzheimers Dis ; 54(2): 427-43, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27567871

RESUMO

Stroke is a brain disease that occurs when blood flow stops, resulting in reduced oxygen supply to neurons. Stroke occurs at any time and at any age, but increases after the age of 55. It is the second leading cause of death and the third leading cause of disability-adjusted, life-years. The pathophysiology of ischemic stroke is complex and recent molecular, cellular, and animal models and postmortem brain studies have revealed that multiple cellular changes have been implicated, including oxidative stress/mitochondrial dysfunction, inflammatory responses, micro RNA alterations, and marked changes in brain proteins. These cellular changes provide new information for developing therapeutic strategies for ischemic stroke treatment. Research also revealed that stroke increases with a number of modifiable factors and most strokes can be prevented and/or controlled through pharmacological or surgical interventions and lifestyle changes. Ischemic stroke is the major risk factor for vascular dementia and Alzheimer's disease. This review summarizes the latest research findings on stroke, including causal factors and molecular links between stroke and vascular disease/Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Demência Vascular/metabolismo , Mediadores da Inflamação/metabolismo , Acidente Vascular Cerebral/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Demência Vascular/etiologia , Demência Vascular/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Humanos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fatores de Risco , Fumar/efeitos adversos , Fumar/genética , Fumar/metabolismo , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA