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1.
Analyst ; 148(23): 5991-6000, 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37876282

RESUMO

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, pKa, viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.


Assuntos
D-Aminoácido Oxidase , Ácido Cinurênico , L-Aminoácido Oxidase , Ácido Cinurênico/química , Corantes Fluorescentes , Enguias , Animais , L-Aminoácido Oxidase/análise , D-Aminoácido Oxidase/análise , Bioensaio , Fluorescência , Cinética , Fígado/enzimologia , Rim/enzimologia
2.
J Pharm Biomed Anal ; 116: 94-100, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25749303

RESUMO

D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.


Assuntos
D-Aminoácido Oxidase/análise , D-Aminoácido Oxidase/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel Bidimensional/métodos , Animais , D-Aminoácido Oxidase/genética , Humanos , Células LLC-PK1 , Espectrometria de Massas/métodos , Ligação Proteica/fisiologia , Suínos
3.
Breast ; 14(3): 236-41, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15927833

RESUMO

The aim of this study was to evaluate the concentration of histamine (HA) and the activities of their enzymes, namely histidine decarboxylase (HDC) and diaminooxydase (DAO) in 95 women with ductal breast cancer and in healthy women. The control group comprised 60 women without any pathological changes in their breasts, in whom mammoplasties were performed. In women with breast cancer the concentration of HA in serum was significantly higher than in healthy controls (9.1+/-3.2 vs. 5.9+/-3.1 nmol/l; P<0.001). The concentration of HA was significantly higher in neoplasmatic tissues of women with breast cancers than in unchanged tissues of healthy subjects in the control group (14.2+/-5.1 vs. 6.3+/-9.1 nmol/g; P<0.001). HDC activity was significantly elevated in cancerous tissues of women with breast cancer relative to unchanged tissues of healthy subjects (54.7+/-17.1 vs. 39.3+/-26.9 pmol/min per mg; P<0.01). However, the activity of DAO was significantly lower (14.0+/-0.4 vs. 36.1+/-9.7 pmol/min per mg; P<0.001) in neoplasmatic tissues than in normal tissues of healthy women. The adjacent healthy tissue of cancer revealed higher concentrations of HA than were found in unchanged tissues of healthy subjects (6.3+/-9.1 vs. 7.5+/-5.4 pmol/min per mg), but this difference did not reach statistical significance. The activity of HDC did not show any significant difference between the healthy tissues adjacent to cancer foci of women with breast cancer and normal tissues obtained from healthy subjects (39.3+/-26.9 vs. 34.5+/-24.3 pmol/min per mg). However, the activity of DAO was markedly lower than in unchanged tissues of healthy women in the control group (36.1+/-9.7 vs. 14.4+/-10.9 pmol/min per mg; P<0.001). The concentration of HA in cancerous tissues was significantly higher than in adjacent healthy tissues (14.2+/-5.1 vs. 7.5+/-5.4 nmol/g; P<0.001). The activity of HDC was significantly higher in cancerous tissues than in adjacent healthy tissues (54.7+/-17.1 vs. 34.5+/-24.3 pmol/min per mg; P<0.001), but there was no difference in the activity of DAO (14.0+/-6.4 vs. 14.4+/-10.9 pmol/min per mg). The significant elevation of HA concentration in cancerous tissues of women with the ductal breast cancers is caused by the increased synthesis and decreased inactivation of HA.


Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Carcinoma Intraductal não Infiltrante/química , Carcinoma Intraductal não Infiltrante/enzimologia , Histamina/análise , Histamina/sangue , Adulto , Idoso , Estudos de Casos e Controles , D-Aminoácido Oxidase/análise , D-Aminoácido Oxidase/metabolismo , Feminino , Histamina/metabolismo , Histidina Descarboxilase/análise , Histidina Descarboxilase/metabolismo , Humanos , Pessoa de Meia-Idade
4.
Bull Cancer ; 91(4): E61-79, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15562560

RESUMO

We have previously shown that the addition of 4-methylthio-2-oxobutanoate (MTOB) to cultures of methionine dependent neoplastic cells which lack endogenous MTOB restores their capacity to grow in the absence of exogenous methionine. Transition state inhibitors of the MTOB transaminase,responsible for the transamination of MTOB to methionine, had also been designed and selected for their capacity to inhibit the proliferation of methionine dependent neoplastic cells but not that of normal cells in culture. We now show that the transition state analogue : L-methionine ethyl esterpyridoxal(MEEP) with a structure corresponding to the oxo acid receptor covalently linked to pyridoxamine and the amine donor analogue: D-aspartate beta hydroxamate (D-AH) are efficient inhibitors of MTOB transaminase. [3H] MEEP uptake into transformed HeLa cells is similar to that in normal MRC5 cells, yet growth inhibition is seem in the transformed but not in the normal cells.MEEP irreversibly inhibits the activity of this enzyme when added to HeLa cells in culture but not that of the purified rat liver enzyme, probably due to pyridoxal phosphate already bound in the active site. On the contrary, D-AH is a noncompetitive reversible inhibitor of the purified rat liver enzyme in vitro and also inhibits intracellular HeLa MTOB transaminase. Furthermore, in HeLa cells both inhibitors induce DNA strand breaks typical of apoptotic cell death. These results provide evidence that MTOB transaminase is a potential target for antiproliferative agents which could selectively affect methionine-dependent neoplastic cells. The transition state intermediale : MEEP as an amine acceptor analogue was found to be 20 fold more effective than D-AH as the amine donor analogue in inducing apoptosis.


Assuntos
Antineoplásicos/farmacologia , Asparagina/análogos & derivados , Asparagina/farmacologia , Metionina/análogos & derivados , Metionina/metabolismo , Metionina/farmacologia , Piridoxal/análogos & derivados , Piridoxal/farmacologia , Piridoxamina/análogos & derivados , Piridoxamina/farmacologia , Transaminases/antagonistas & inibidores , Animais , Apoptose/genética , Asparagina/metabolismo , Proliferação de Células/efeitos dos fármacos , D-Aminoácido Oxidase/análise , D-Aminoácido Oxidase/metabolismo , Fragmentação do DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glutamina/metabolismo , Células HeLa/efeitos dos fármacos , Células HeLa/enzimologia , Humanos , Fígado/enzimologia , Piridoxal/metabolismo , Ratos , Transaminases/análise
5.
EXS ; 85: 109-18, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9949871

RESUMO

In 1939 Kögl and Erxleben [1-4] reported that tumor proteins contain appreciable amounts of D-amino acids, specifically glutamic acid, valine, leucine and lysine, implying that both the initiation and autonomous character of tumors depends on the formation and maintenance of these D-amino acids in the cell proteins. This postulate remained highly controversial for over 10 years, during which time several papers both supporting and refuting this hypothesis were published. The dispute existed almost entirely between Kögl, a vigorous and able protagonist at the University of Utrecht, Netherlands, and an impressive array of equally vigorous and able dissenters in the United Kingdom and Germany. An excellent review of both sides of this controversy was written by Miller in 1950 [5]. After many years and much effort the controversy then seemed to be put to rest. However, more than 40 years later the development of much more refined analytical techniques for the resolution and detection of amino acid enantiomers provided more definitive evidence that D-amino acids are not common to all tumor tissues and probably are not integral to the cancer process. This is not surprising when one considers that a tumor consists of fast-growing cells. Thus, there would not be sufficient time for any L-amino acid to racemize to the D isomer. Some D-amino acids may originate in foods consumed, but it is uncertain whether enzyme systems are able to incorporate D-amino acids into tumor proteins during growth. Nevertheless, if significant levels of D-amino acids were to be found in tumor proteins, the implications could be far-reaching. Confirmation of the presence of D-amino acids at any concentration in tumors would provide new insights into the mechanism for autogenesis and maintenance of tumors.


Assuntos
Envelhecimento/metabolismo , Aminoácidos/metabolismo , Proteínas de Neoplasias/química , D-Aminoácido Oxidase/análise , Neoplasias/enzimologia , Estereoisomerismo
6.
Cell Biol Int ; 21(9): 575-84, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9570892

RESUMO

Various tissues of the marine bivalve Mytilus galloprovincialis were analysed histochemically for oxidases capable of generating reactive oxygen species (ROS) using the cerium-DAB technique. Incubations were performed on unfixed cryostat sections using polyvinyl alcohol and semipermeable membranes. High xanthine oxidoreductase and D-amino acid oxidase (DAOX) activities were observed in kidney epithelial cells of mussels. DAOX also presented a strong activity in all the digestive epithelia. No xanthine oxidase activity was observed in any of the mussel tissues tested suggesting the presence of an enzyme only showing dehydrogenase activity. Mannitol oxidase, associated with special organelles called 'mannosomes' of terrestrial gastropods, presented a weak activity in the stomach epithelium and a strong specific activity in the haemocytes. Only DAOX presented a discrete granular distribution compatible with a peroxisomal compartmentalization. No urate oxidase activity could be demonstrated in tissues of mussels. These observations suggest a role for peroxisomes in ROS generation and determine the tissues capable of producing oxygen radicals in the digestive gland. This study raises the question of the behaviour of these enzymes in conditions in which ROS-generating organic xenobiotics are accumulated in the digestive gland of molluscs.


Assuntos
Bivalves/enzimologia , D-Aminoácido Oxidase/análise , Xantina Desidrogenase/análise , Xantina Oxidase/análise , Animais , Sistema Digestório/citologia , Sistema Digestório/enzimologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Brânquias/citologia , Brânquias/enzimologia , Hemócitos/citologia , Hemócitos/enzimologia , Histocitoquímica , Rim/citologia , Rim/enzimologia , Especificidade de Órgãos , Organelas/enzimologia , Organelas/ultraestrutura
7.
Artigo em Inglês | MEDLINE | ID: mdl-1359694

RESUMO

The presence of peroxisomes and their enzymic content were investigated and compared in healthy and neoplastic human colon epithelial cells using cytochemical studies at the ultrastructural level as well as biochemical analyses. Catalase-positive organelles were found to be more numerous in normal than in colonic neoplastic cells. Biochemical assays revealed that no D-aminoacid oxidase or L-alpha-hydroxyacid oxidase activity was detected in normal or tumor tissues. The specific activities of catalase, fatty-acyl CoA oxidase and enoyl-CoA hydratase/3 hydroxyacyl-CoA dehydrogenase (the so-called peroxisomal bifunctional enzyme of the beta-oxidation system) were found to be diminished in carcinoma cells compared with the control tissue. The fall in catalase activity correlated well with tumor stage according to Dukes, suggesting that this peroxisomal enzyme could be used as a potential prognostic marker.


Assuntos
Adenocarcinoma/ultraestrutura , Neoplasias do Colo/ultraestrutura , Microcorpos/enzimologia , Proteínas de Neoplasias/análise , Neoplasias Retais/ultraestrutura , 3-Hidroxiacil-CoA Desidrogenases/análise , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Oxirredutases do Álcool/análise , Catalase/análise , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , D-Aminoácido Oxidase/análise , Enoil-CoA Hidratase/análise , Humanos , Isomerases/análise , Pessoa de Meia-Idade , Complexos Multienzimáticos/análise , Estadiamento de Neoplasias , Enzima Bifuncional do Peroxissomo , Prognóstico , Neoplasias Retais/enzimologia , Neoplasias Retais/patologia
9.
J Biol Chem ; 255(13): 6044-6, 1980 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-6104662

RESUMO

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.


Assuntos
D-Aminoácido Oxidase/análise , Peptídeos/análise , Tirosina/análise , Sequência de Aminoácidos , Aminoácidos/análise , Sítios de Ligação , Cloro/metabolismo , Quimotripsina/metabolismo , D-Aminoácido Oxidase/metabolismo
10.
Acta Neuropathol ; 51(2): 113-7, 1980.
Artigo em Inglês | MEDLINE | ID: mdl-6108044

RESUMO

Morphologically and cytochemically defined peroxisomes (microbodies) were demonstrated in a series of 11 human glial tumors. The cytochemical methods used were diaminobenzidine method for catalase and a tetrazolium salt-phenozine methosulfate method for D-amino-acid oxidase. Ultrastructurally, the peroxisomes were found as single membrane limited organelles with a granular matrix. Marginal plates as well as continuities with the smooth endoplasmic reticulum could be demonstrated. Peroxisomes were found most abundantly in subependymal giant cell astrocytomas, to a lesser number in astrocytomas and least abundantly in glioblastomas.


Assuntos
Neoplasias Encefálicas/ultraestrutura , Glioma/ultraestrutura , Microcorpos/ultraestrutura , Organoides/ultraestrutura , Adolescente , Adulto , Idoso , Astrocitoma/enzimologia , Astrocitoma/ultraestrutura , Neoplasias Encefálicas/enzimologia , Catalase/análise , D-Aminoácido Oxidase/análise , Feminino , Glioma/enzimologia , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade
11.
Biochim Biophys Acta ; 527(2): 497-500, 1978 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-31918

RESUMO

A rapid and sensitive method has been developed to determine lysopine dehydrogenase (EC 1.5.1-) and nopaline dehydrogenase activities in crown gall tumour tissues. By this method, enzyme activities as low as 0.2 micrometerol octopine or nopaline per h per g fresh weight tumour tissue can still be detected. In non-infected young pea seedlings, no lysopine dehydrogenase activity was detected.


Assuntos
D-Aminoácido Oxidase/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/análise , Tumores de Planta/enzimologia , Rhizobium , Arginina/análogos & derivados , Glutaratos , Lisina/análogos & derivados , Microquímica/métodos
12.
Biochem Genet ; 15(11-12): 1023-35, 1977 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23758

RESUMO

The use of L-glutamate dehydrogenase (GLUD) as a reagent in staining mixtures to detect the isozymes of enzymes which catalyze the production of ammonia has been investigated. Methods have been devised for the electrophoresis and detection, using GLUD, of seven enzymes: cytidine deaminase, adenosine deaminase, adenosine monophosphate deaminase, arginase, argininosuccinase, D-amino acid oxidase, and D-aspartate oxidase. GLUD-linked staining methods appear to be sensitive, specific, and of general application.


Assuntos
Amônia/metabolismo , Eletroforese em Gel de Amido/métodos , Glutamato Desidrogenase , AMP Desaminase/análise , Adenosina Desaminase/análise , Aminoácido Oxirredutases/análise , Arginase/análise , Argininossuccinato Liase/análise , Citidina Desaminase/análise , D-Aminoácido Oxidase/análise , Humanos , Indicadores e Reagentes , Leucócitos/enzimologia , Fenótipo
13.
Biochem J ; 142(1): 1-6, 1974 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-4155288

RESUMO

1. A rat kidney lysosomal fraction was prepared by the method of Maunsbach (1966) and characterized by its content of representative marker enzymes for lysosomes, mitochondria, peroxisomes and endoplasmic reticulum. 2. It was shown that both pH-dependent and progesterone-induced lysis lead to a decrease in the E(520) of suspensions of this preparation. This decrease parallels quantitatively and temporally the release of soluble acid phosphatase. 3. It is suggested that E(520) measurements are a valid method for the continuous measurement of changes in lysosomal integrity. 4. As an example, results are included which demonstrate the ability of Zn(2+) to stabilize lysosomes against spontaneous and progesterone-induced lysis.


Assuntos
Rim/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Progesterona/farmacologia , Fosfatase Ácida/análise , Animais , Fracionamento Celular , D-Aminoácido Oxidase/análise , Retículo Endoplasmático/análise , Galactosidases/análise , Glucose-6-Fosfatase/análise , Glutamato Desidrogenase/análise , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Rim/análise , Mitocôndrias/análise , Organoides/análise , Ratos , Soroalbumina Bovina , Espectrofotometria Atômica , Sulfatases/análise , Ultrafiltração , Zinco/farmacologia
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