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1.
J Cell Biochem ; 114(3): 599-605, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22991171

RESUMO

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-ß-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II.


Assuntos
Clonagem Molecular , Coriolaceae/enzimologia , Lacase , Sequência de Aminoácidos , Cromatografia Líquida , Dicroísmo Circular , Coriolaceae/genética , Ensaios Enzimáticos , Escherichia coli/enzimologia , Escherichia coli/genética , Amplificação de Genes , Hidrazonas/química , Isopropiltiogalactosídeo/genética , Óperon Lac , Lacase/química , Lacase/genética , Lacase/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
2.
Biosystems ; 94(3): 228-32, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18721850

RESUMO

Proteins represent versatile building blocks for realization of nanostructured materials of unique structure-function relationship to be applied in nanobiotechnology. Following a recent work [Bruzzese, D., Pastorino, L., Pechkova, E., Sivozhelezov, Nicolini, C., Increase of catalytic activity of lipase towards olive oil by Langmuir-Film Immobilization of Lipase, Enzyme and Microbial Technology, submitted for publication.], the Langmuir-Blodgett technique was utilized to develop nanostructured crystal materials based on enzymes interfacially activated with olive oil as substrate. Particularly, thin films of lipase from both Mucor miehei and Candida rugosa were fabricated and characterized by UV-vis spectroscopy, Atomic force microscopy and biochemical assays. As the first step the M. miehei protein films were studied at the air-water interface and then transferred onto a solid support for further characterization of the enzymatic activity also versus surface pressure, proving that Langmuir-Blodgett film provides a better catalytic effect in lipase than a mere oil-water boundary. Moreover, improvement of lipase catalytic performance was achieved for the M. miehei versus the C. rugosa, despite its almost random distribution of hydrophobic patches and the low purity of its preparation.


Assuntos
Biotecnologia/métodos , Candida/enzimologia , Lipase/química , Modelos Moleculares , Mucor/enzimologia , Nanoestruturas , Relação Estrutura-Atividade , Eletroforese em Gel de Poliacrilamida , Azeite de Oliva , Óleos de Plantas , Espectrofotometria Ultravioleta
3.
J Biol Chem ; 283(36): 24460-8, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18606819

RESUMO

Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 microm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , ADP-Ribosil Ciclase 1/genética , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/química , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Hemostáticos/farmacologia , Humanos , Glicoproteínas de Membrana/genética , Células Musculares/metabolismo , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/metabolismo , Vesículas Secretórias/química , Vesículas Secretórias/metabolismo , Estereoisomerismo , Trombina/farmacologia
4.
Biosens Bioelectron ; 23(5): 655-60, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17766101

RESUMO

In this work, to improve the mechanical stability of electrodes based on P450scc for LDL-cholesterol detection and measure, anodic porous alumina (APA) was used. This inorganic matrix, which pores can be tuned in diameter modifying the synthesis parameters, was realized with cavities 275 nm wide and 160 microm deep (as demonstrated with AFM and SEM measurement), to allow the immobilization of P450scc macromolecules preserving their electronic sensitivity to its native substrate, cholesterol. Even if the sensitivity of the APA+P450scc system was slightly reduced with respect to the pure P450scc system, the readout was stable for a much longer period of time, and the measures remained reproducible inside a proper confidentiality band, as demonstrated with several cyclic voltammetry measures. To optimize the adhesion of P450scc to APA, a layer of poly-L-lysine, a poly-cathion, was successfully implemented as intermediate organic structure.


Assuntos
Óxido de Alumínio , Técnicas Biossensoriais/instrumentação , LDL-Colesterol/análise , Eletrodos , Sistema Enzimático do Citocromo P-450 , Eletroquímica , Porosidade
5.
J Med Chem ; 50(12): 2886-95, 2007 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-17500510

RESUMO

The synthesis and in vitro antiplatelet activity significant data of coumarin derivatives 5i-x and quinolin-2(1H)-one derivatives 22a,b, as well as the corresponding structure-activity relationships are described. The recently reported 8-methyl-4-(1-piperazinyl)-7-(3-pyridylmethoxy)coumarin 5f and its potent 7-(2-morpholinoethoxy)-substituted new analogue 5u were notably more effective inhibitors of pure human platelet PDE3 than milrinone and cilostazol: these data were related, through a molecular modeling study, with the molecular interactions of the four compounds with the human PDE3A catalytic site.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Cumarínicos/síntese química , Morfolinas/síntese química , Inibidores de Fosfodiesterase/síntese química , Piperazinas/síntese química , Inibidores da Agregação Plaquetária/síntese química , 3',5'-AMP Cíclico Fosfodiesterases/sangue , 3',5'-AMP Cíclico Fosfodiesterases/química , Domínio Catalítico , Cumarínicos/química , Cumarínicos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Humanos , Técnicas In Vitro , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacologia , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia
6.
Thromb Haemost ; 97(2): 218-27, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17264950

RESUMO

Retinal vein occlusion (RVO) is the most common retinal vascular disorder second to diabetic retinopathy. The main risk factors in patients with RVO are hypertension, diabetes, hyperlipidemia, increased blood viscosity and glaucoma. The pathogenesis of RVO has not yet been clarified. In these events platelets could play a very important role. In the present study the platelet response to collagen was deeply investigated. Experiments were carried out on a selected group of RVO patients, which were compared to a group of healthy subjects matched for age, sex, clinical and metabolic characteristics. In resting and activated platelets of both groups of subjects p72syk phosphorylation, phospholipase Cgamma2 phosphorylation, protein kinase C activation, intra-cellular calcium levels and nitric oxide formation were measured. Results show that platelets of patients were more responsive to collagen or ADP than healthy subjects and that the response was significantly different (p < 0.0005) at low concentrations of these agonists. In platelets of patients stimulated with collagen increased phosphorylation of p72syk and phospholipase Cgamma2 was found. Also protein kinase C was more activated in patients. In addition intracellular calcium rise induced by collagen was significantly higher in patients than in healthy subjects. RVO patients showed a lower basal level of nitric oxide both in resting and stimulated platelets compared to healthy subjects. Altogether these results suggest that the platelet hyperaggregability described in patients might be an important factor in the development of RVO contributing to the thrombogenic effects.


Assuntos
Plaquetas/efeitos dos fármacos , Colágeno/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Oclusão da Veia Retiniana/sangue , Difosfato de Adenosina , Idoso , Plaquetas/metabolismo , Cálcio/metabolismo , Estudos de Casos e Controles , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Oclusão da Veia Retiniana/tratamento farmacológico , Oclusão da Veia Retiniana/metabolismo , Quinase Syk , Fatores de Tempo
7.
J Cell Biochem ; 100(5): 1255-65, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17063483

RESUMO

The aim of this study was to examine the homocysteine effect on phospholipase Cgamma2 (PLCgamma2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCgamma2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor-mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N-acetyl-L-cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCgamma2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCgamma2, suggesting that thromboxane A(2) could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N-acetyl-L-cysteine of 35% or 50% respectively, while the PLCgamma2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCgamma2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A(2).


Assuntos
Homocisteína/farmacologia , Fosfolipase C gama/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Cálcio/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Estresse Oxidativo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Quinase Syk , Tirosina/metabolismo
8.
Biochem Pharmacol ; 69(2): 289-96, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15627481

RESUMO

Nitric oxide production, L-arginine transport and intracellular [Ca2+] changes in human platelets stimulated without stirring by low doses of collagen have been evaluated. Collagen decreased in a dose-dependent manner the nitric oxide formation. A reduction of about 30% of the basal level was produced by 5 microg/mL. Aspirin did not change the collagen effect. The inhibition was reversed by EGTA. Moreover collagen reduced L-arginine uptake. The exposure of platelets to 5 microg/mL collagen diminished of about 30% L-arginine transport. The specific involvement of the system y+ is suggested. In addition in FURA 2-loaded platelets collagen induced a dose-dependent slow sustained [Ca2+] rise that was almost completely cancelled by EGTA. Finally the treatment of whole platelets with collagen affected in a dose-dependent manner the maximal nitric oxide formation, suggesting a direct effect at the level of nitric oxide synthase enzyme. The phosphorylation of specific serine/threonine residues regulated by protein kinase C could be involved. In conclusion during the early phases of platelet stimulation with collagen nitric oxide formation is diminished. This reduction can be due to a lower availability of L-arginine for cytosolic nitric oxide synthase and/or to a decreased activity related to modifications of the enzyme.


Assuntos
Arginina/metabolismo , Plaquetas/efeitos dos fármacos , Colágeno/farmacologia , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia
9.
Biochem Pharmacol ; 67(5): 911-8, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15104244

RESUMO

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca2+]i. The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 +/- 0.12 microM, 0.98 +/- 0.36 microM and 1.00 +/- 0.15 microM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50 = 1.73 +/- 0.35 microM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.


Assuntos
Cálcio/metabolismo , Cromonas/farmacologia , AMP Cíclico/metabolismo , Piperazinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Arginina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Cromonas/química , GMP Cíclico/metabolismo , Interações Medicamentosas , Humanos , Técnicas In Vitro , Óxido Nítrico/metabolismo , Piperazinas/química , Proteína Quinase C/metabolismo , Trombina/farmacologia
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