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1.
J Biotechnol ; 313: 11-17, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32126268

RESUMO

TLC-Bioautography is a fast and effective method for assessing the inhibitory effect of compounds present in plant extracts against microbial species. However, this method has a hidden, currently underutilized potential for evaluating the presence of inhibitory compounds against selected enzymes. The aim of this work was to design a functional TLC-Bioautography method for the evaluation of protease inhibitors present in plant extracts. The method is based on the hydrolysis of Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BApNA) by α-chymotrypsin as a representative serine protease to produce coloured para-nitroaniline (pNA). Derivatization of pNA with both sodium nitrite and N-(1-naphthyl) ethylenediamine (NPED) leads to the formation of a pink azo dye. This step improves the resolution of active compounds on the chromatogram, which appear as light spots on a pink background. The developed method was tested for the analysis of protease inhibitors in different plant materials such as grape pomace from Vitis vinifera, Picea abies bark, Hippophae rhamnoides berries, Hordeum sativum bran, Triticum aestivum bran and Avena sativa bran. Plant extracts, which could not be analysed by a commonly used spectrophotometric method due to interference, were assessed by this method.


Assuntos
Quimotripsina/antagonistas & inibidores , Hippophae/química , Picea/química , Extratos Vegetais/química , Inibidores de Serino Proteinase/isolamento & purificação , Vitis/química , Benzoilarginina Nitroanilida/metabolismo , Cromatografia , Frutas/química , Hidrólise , Casca de Planta/química , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia
2.
Enzyme Microb Technol ; 92: 18-25, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27542740

RESUMO

Before applying nanotechnologies in biomedical and environmental areas it is advised to study interactions of nanoparticles and other nanomaterials with biomacromolecule present in living system. Moreover there is scarcity of reports on interactions between nanoparticles and biomaterials. In present report a rapid, ecofriendly method of fabricating stable gold nanoparticles (AuNPs) using latex of Jatropha curcas is reported for the first time. AuNPs found to have characteristic absorption maxima centered at 540nm, multiple irregular shapes with size range from 20 to 50nm and have crystalline nature. Latex fabricated AuNPs were found to inhibit catalytic potential of trypsin (a vital enzyme responsible for digestion, insecticide resistance and in several disease conditions). The interactions between AuNPs and trypsin were analyzed by UV-vis spectrophotometry and microwave plasma-atomic emission spectrometry which suggests formation of trypsin-AuNPs complex responsible for lowering catalytic activity of trypsin. Transmission electron microscopy, Fourier transform infrared spectroscopy and particle size distribution studies further confirm complex formation between trypsin and AuNPs. Diverse interactions of metal nanoparticles with proteins such as covalent interaction, electrostatic interactions and binding to SH group of amino acid may be the reasons behind inhibition of trypsin activity. In vivo studies on serum of several vectors and agriculturally important pests supported instrumental results on AuNPs induced trypsin inhibition. This work will bring a new research direction to explore eco-friendly nanoparticle in insect control via inhibition of enzyme catalytic potential.


Assuntos
Ouro , Controle de Insetos/métodos , Nanopartículas Metálicas , Inibidores da Tripsina , Aedes/enzimologia , Animais , Benzoilarginina Nitroanilida , Inseticidas , Látex , Nanopartículas Metálicas/ultraestrutura , Nanotecnologia , Tripsina/metabolismo
3.
Electrophoresis ; 37(20): 2644-2648, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27440471

RESUMO

Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins.


Assuntos
Cisteína Proteases/química , Cisteína Proteases/metabolismo , Cisteína/química , Eletroforese/métodos , Benzoilarginina Nitroanilida/análise , Benzoilarginina Nitroanilida/química , Benzoilarginina Nitroanilida/metabolismo , Tampões (Química) , Cisteína/metabolismo , Modelos Químicos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo
4.
Rev. iberoam. micol ; 33(1): 26-33, ene.-mar. 2016. tab, ilus
Artigo em Inglês | IBECS | ID: ibc-149371

RESUMO

Background. The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen (AU)


Antecedentes. La vacuola de Saccharomyces cerevisiae está involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradación, recambio proteico, desintoxicación y protección de la célula en condiciones de estrés. Por el contrario, las proteasas vacuolares de Candida glabrata aún no han sido estudiadas por completo. Objetivos. El presente trabajo describe por primera vez la actividad proteolítica vacuolar en C. glabrata. Métodos. Los estudios bioquímicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteolíticas: aspartil proteinasa ácida, que actúa sobre sustratos como la albúmina y la hemoglobina ácida desnaturalizada; serín proteasa neutra, con actividad sobre el substrato de tipo colágeno hide powder azure, y serín carboxipeptidasa, que actúa sobre N-benzoil-tyr-pNa. Resultados. La obtención de una fracción subcelular mostró una elevada actividad enzimática específica de las tres proteasas, lo que permitió confirmar su localización vacuolar. Se realizaron análisis de la expresión de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteolíticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulación diferencial de la expresión de la proteasa, dependiendo de la fuente de nitrógeno. Conclusiones. Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrían participar en el proceso de autofagia y supervivencia de este patógeno oportunista (AU)


Assuntos
Peptídeo Hidrolases/análise , Candida glabrata , Candida glabrata/isolamento & purificação , Candida glabrata/patogenicidade , Carboxipeptidases/análise , Carboxipeptidases , Saccharomyces cerevisiae , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/patogenicidade , Vacúolos/virologia , Candida glabrata/enzimologia , Ácido Aspártico Proteases/análise , Ácido Aspártico Proteases/isolamento & purificação , Autofagia , Homeostase , Benzoilarginina Nitroanilida/análise , Infecções Oportunistas/microbiologia , Vacúolos , Vacúolos/microbiologia , Vacúolos/patologia
5.
Chemosphere ; 148: 241-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26807945

RESUMO

The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative ΔH and ΔS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs.


Assuntos
Poluentes Ambientais/toxicidade , Líquidos Iônicos/toxicidade , Tripsina/química , Animais , Benzoilarginina Nitroanilida/química , Sítios de Ligação , Bovinos , Poluentes Ambientais/química , Estabilidade Enzimática , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Líquidos Iônicos/química , Modelos Teóricos , Pâncreas/enzimologia , Espectrometria de Fluorescência , Especificidade por Substrato , Termodinâmica , Tripsina/isolamento & purificação , Tripsina/metabolismo
6.
Toxins (Basel) ; 7(8): 2872-87, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26264023

RESUMO

We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors.


Assuntos
Venenos de Aranha/química , Venenos de Aranha/farmacologia , Aedes/efeitos dos fármacos , Animais , Benzoilarginina Nitroanilida/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Hemólise , Larva/efeitos dos fármacos , Masculino , Peptídeos/análise , Fosfolipases A2/metabolismo , Proteólise , Espectrometria de Massas por Ionização por Electrospray , Aranhas , Espectrometria de Massas em Tandem
7.
Acta Trop ; 128(3): 686-91, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24076089

RESUMO

Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy.


Assuntos
Dípteros/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Animais , Benzoilarginina Nitroanilida/metabolismo , Secreções Corporais/enzimologia , Compostos Cromogênicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Larva/enzimologia , Peso Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteólise
8.
Fish Shellfish Immunol ; 35(2): 389-98, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23711467

RESUMO

Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed respectivity. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus, further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. In addition the identity of the two subunits was identified using LC/MS/MS which was similar to α-2-M of grass carp (Ctenopharyngodon idella) and bluegill sunfish (Lepomis macrochirus) on the protein hit.


Assuntos
Bass/metabolismo , Proteínas de Peixes/metabolismo , alfa-Macroglobulinas/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Cromatografia Líquida/veterinária , Eletroforese/veterinária , Proteínas de Peixes/química , Peixes/metabolismo , Soros Imunes/química , Soros Imunes/metabolismo , Immunoblotting/veterinária , Peso Molecular , Coelhos , Espectrometria de Massas em Tandem/veterinária , Tripsina/metabolismo , alfa-Macroglobulinas/química
9.
Molecules ; 18(1): 1138-49, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23325102

RESUMO

The enzymatic performance of trypsin in hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA) was improved by adsorption on Santa Barbara Amorphous (SBA)-15 mesoporous silica. The optimal immobilization conditions were screened and the properties of immobilized enzyme have also been studied. Under the optimal conditions, the immobilized trypsin displays maximum specific activity (49.8 µmol/min/g). The results also indicate that the immobilized trypsin exhibits better storage stability.


Assuntos
Benzoilarginina Nitroanilida/química , Enzimas Imobilizadas/química , Dióxido de Silício/química , Tripsina/química , Adsorção , Animais , Biocatálise , Bovinos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Pâncreas/enzimologia , Proteólise , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Ciclodextrinas/química
10.
Biofouling ; 28(7): 719-28, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22800467

RESUMO

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis.


Assuntos
Antibacterianos/química , Biofilmes , Incrustação Biológica/prevenção & controle , Química Verde , Aço Inoxidável/química , Staphylococcus epidermidis/efeitos dos fármacos , Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Carga Bacteriana , Benzoilarginina Nitroanilida/química , Biofilmes/efeitos dos fármacos , Caseínas/química , Reagentes de Ligações Cruzadas/química , Di-Hidroxifenilalanina/química , Ativação Enzimática , Fluoresceínas/química , Indóis/química , Viabilidade Microbiana , Microscopia de Fluorescência , Polímeros/química , Proteólise , Eletricidade Estática , Propriedades de Superfície , Tripsina/química
11.
J Insect Sci ; 11: 72, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21867440

RESUMO

In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps.


Assuntos
Heterópteros/efeitos dos fármacos , Proteínas de Insetos/antagonistas & inibidores , Inibidores de Serino Proteinase/farmacologia , Animais , Benzoilarginina Nitroanilida , Peso Corporal , Caseínas , Eletroforese em Gel de Poliacrilamida , Trato Gastrointestinal/enzimologia , Heterópteros/enzimologia , Heterópteros/crescimento & desenvolvimento , Oligopeptídeos
12.
BMC Biochem ; 12: 43, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21827688

RESUMO

BACKGROUND: The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. RESULTS: We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k(cat)/K(M)) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin. CONCLUSIONS: These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.


Assuntos
Aedes/enzimologia , Insetos Vetores/enzimologia , Serina Proteases/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Dengue/transmissão , Sistema Digestório/enzimologia , Enteropeptidase/metabolismo , Ativação Enzimática , Hemoglobinas/metabolismo , Cinética , Desnaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/genética , Albumina Sérica/metabolismo , Homologia Estrutural de Proteína , Tripsina/metabolismo
13.
FEBS Lett ; 585(6): 829-33, 2011 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-21320498

RESUMO

Acetaldehyde and malondialdehyde react covalently with cellular proteins forming protein-malondialdehyde-acetaldehyde adducts thus modulating their biochemical functions. Alpha-2 macroglobulin, an acute phase protein produced by liver binds to cytokines, growth factors and neutralizes proteinases. In this study we examined the formation of MAA adducts of N-terminal and bait region of mouse A2M and their effect on modulating its proteinase and TGF-ß1 binding activities. Adduct formation abrogated the binding of bait region with TGF-ß1, trypsin, and elastase. TGF-ß1 induced NO production was also suppressed. Acetaldehyde and MDA adduction of A2M may have physiological consequences in alcoholic patients.


Assuntos
Acetaldeído/metabolismo , Malondialdeído/metabolismo , Peptídeo Hidrolases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , alfa-Macroglobulinas/metabolismo , Acetaldeído/química , Animais , Benzoilarginina Nitroanilida , Sítios de Ligação , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Epitopos/química , Epitopos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Malondialdeído/química , Camundongos , Óxido Nítrico/metabolismo , Elastase Pancreática/metabolismo , Ligação Proteica , Tripsina/metabolismo , alfa-Macroglobulinas/química , alfa-Macroglobulinas/genética
14.
Clin Oral Investig ; 15(6): 967-73, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20820824

RESUMO

Previous reports have demonstrated increased tryptase-like proteolytic activity in the crevicular fluid of patients with periodontal disease. In the present study, we have investigated the effect of tryptase inhibition with nafamostat mesilate (NM, 6-amino-2-naphtlyl p-guanidinobenzoate dimethansulfonate) on the development of experimental periodontitis in rats. Eighty (80) male Wistar rats were randomly separated into four groups: Control group, NM group (daily 0.1 mg/kg body weight of NM, i.p.), Ligature group (ligature placed at lower right first molars), and NM+Ligature group. The amount of alveolar bone loss (ABL) around the mesial root surface of the first mandibulary molar, as well as the myeloperoxidase (MPO) activity, and total proteolytic activity [N-benzoyl-L: -arginine-p-nitroanilide (BApNA) substrate] were determined at 7 and 14 days. NM led to significantly (p < 0.05) decreased ABL in animals subjected to ligature-induced periodontitis. Tryptase inhibition prevented the onset of significant ABL at 7 days of experiment (0.44 ± 0.16 and 0.60 ± 0.22, p > 0.05, NM+Ligature and Control, respectively) and significantly decreased the ABL at 14 days (0.97 ± 0.17 versus 1.82 ± 0.26, p < 0.001, NM+Ligature versus Ligature, respectively). In addition, NM significantly decreased MPO and total proteolytic activity at 14 days (p < 0.05). These data provided evidence that tryptase inhibition with NM attenuates gingival granulocyte infiltration and ABL in an experimental model of periodontitis in rats.


Assuntos
Guanidinas/uso terapêutico , Periodontite/prevenção & controle , Inibidores da Tripsina/uso terapêutico , Triptases/antagonistas & inibidores , Perda do Osso Alveolar/prevenção & controle , Animais , Benzamidinas , Benzoilarginina Nitroanilida , Movimento Celular/efeitos dos fármacos , Compostos Cromogênicos , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengivite/prevenção & controle , Granulócitos/efeitos dos fármacos , Masculino , Doenças Mandibulares/prevenção & controle , Dente Molar/efeitos dos fármacos , Peptídeo Hidrolases/análise , Peroxidase/análise , Distribuição Aleatória , Ratos , Ratos Wistar , Receptor PAR-2/análise , Fatores de Tempo
15.
J. appl. oral sci ; 18(6): 599-606, Nov.-Dec. 2010. ilus, tab
Artigo em Inglês | LILACS | ID: lil-573730

RESUMO

OBJECTIVE: The aim of this controlled clinical trial was to evaluate the effect of topically applied povidone-iodine (PVP-I) used as an adjunct to non-surgical treatment of interproximal class II furcation involvements. MATERIAL AND METHODS: Thirty-two patients presenting at least one interproximal class II furcation involvement that bled on probing with probing pocket depth (PPD) >5 mm were recruited. Patients were randomly chosen to receive either subgingival instrumentation with an ultrasonic device using PVP-I (10 percent) as the cooling liquid (test group) or identical treatment using distilled water as the cooling liquid (control group). The following clinical outcomes were evaluated: visible plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PPD and relative horizontal attachment level (RHAL). BAPNA (N-benzoyl-L-arginine-p-nitroanilide) testing was used to analyze trypsin-like activity in dental biofilm. All parameters were evaluated at baseline and 1, 3 and 6 months after non-surgical subgingival instrumentation. RESULTS: Six months after treatment, both groups had similar means of PPD reduction, RAL and RHAL gain (p>0.05). These variables were, respectively, 2.20±1.10 mm, 1.27±1.02 mm and 1.33±0.85 mm in the control group and 2.67±1.21 mm, 1.50±1.09 mm and 1.56±0.93 mm in the test group. No difference was observed between groups at none of the posttreatment periods, regarding the number of sites showing clinical attachment gain >2 mm. However, at 6 months posttreatment, the test group presented fewer sites with PPD >5 mm than the control group. Also at 6 months the test group had lower BAPNA values than control group. CONCLUSION: The use of PVP-I as an adjunct in the non-surgical treatment of interproximal class II furcation involvements provided limited additional clinical benefits.


Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anti-Infecciosos Locais/uso terapêutico , Defeitos da Furca/tratamento farmacológico , Povidona-Iodo/uso terapêutico , Administração Tópica , Análise de Variância , Anti-Infecciosos Locais/administração & dosagem , Benzoilarginina Nitroanilida , Índice de Placa Dentária , Seguimentos , Índice Periodontal , Perda da Inserção Periodontal/tratamento farmacológico , Bolsa Periodontal/tratamento farmacológico , Povidona-Iodo/administração & dosagem , Fatores de Tempo , Resultado do Tratamento , Terapia por Ultrassom
16.
Fish Physiol Biochem ; 36(4): 893-902, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19898990

RESUMO

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS-PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants Km and kcat on BAPNA were 0.13 mM and 1.56 s(-1), respectively, while the catalytic efficiency kcat/Km was 12 s(-1) mM(-1). Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.


Assuntos
Perciformes/metabolismo , Tripsina/isolamento & purificação , Tripsina/metabolismo , Vísceras/enzimologia , Amidoidrolases/metabolismo , Animais , Aquicultura/métodos , Arginina/análogos & derivados , Benzoilarginina Nitroanilida , Cromatografia em Gel , Cromatografia por Troca Iônica , Dextranos , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Tripsina/química
17.
J Appl Oral Sci ; 18(6): 599-606, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21308291

RESUMO

OBJECTIVE: The aim of this controlled clinical trial was to evaluate the effect of topically applied povidone-iodine (PVP-I) used as an adjunct to non-surgical treatment of interproximal class II furcation involvements. MATERIAL AND METHODS: Thirty-two patients presenting at least one interproximal class II furcation involvement that bled on probing with probing pocket depth (PPD) ≥ 5 mm were recruited. Patients were randomly chosen to receive either subgingival instrumentation with an ultrasonic device using PVP-I (10%) as the cooling liquid (test group) or identical treatment using distilled water as the cooling liquid (control group). The following clinical outcomes were evaluated: visible plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PPD and relative horizontal attachment level (RHAL). BAPNA (N-benzoyl-L-arginine-p-nitroanilide) testing was used to analyze trypsin-like activity in dental biofilm. All parameters were evaluated at baseline and 1, 3 and 6 months after non-surgical subgingival instrumentation. RESULTS: Six months after treatment, both groups had similar means of PPD reduction, RAL and RHAL gain (p>0.05). These variables were, respectively, 2.20 ± 1.10 mm, 1.27 ± 1.02 mm and 1.33 ± 0.85 mm in the control group and 2.67 ± 1.21 mm, 1.50 ± 1.09 mm and 1.56 ± 0.93 mm in the test group. No difference was observed between groups at none of the posttreatment periods, regarding the number of sites showing clinical attachment gain ≥ 2 mm. However, at 6 months posttreatment, the test group presented fewer sites with PPD ≥ 5 mm than the control group. Also at 6 months the test group had lower BAPNA values than control group. CONCLUSION: The use of PVP-I as an adjunct in the non-surgical treatment of interproximal class II furcation involvements provided limited additional clinical benefits.


Assuntos
Anti-Infecciosos Locais/uso terapêutico , Defeitos da Furca/tratamento farmacológico , Povidona-Iodo/uso terapêutico , Administração Tópica , Adulto , Análise de Variância , Anti-Infecciosos Locais/administração & dosagem , Benzoilarginina Nitroanilida , Índice de Placa Dentária , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Perda da Inserção Periodontal/tratamento farmacológico , Índice Periodontal , Bolsa Periodontal/tratamento farmacológico , Povidona-Iodo/administração & dosagem , Fatores de Tempo , Resultado do Tratamento , Terapia por Ultrassom
18.
Artigo em Inglês | MEDLINE | ID: mdl-19835976

RESUMO

Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides--one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin-agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 microM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V(max)-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-alpha-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.


Assuntos
Besouros/enzimologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/toxicidade , Serina Proteases/metabolismo , Zea mays/parasitologia , Animais , Benzoilarginina Nitroanilida/metabolismo , Catálise/efeitos dos fármacos , Besouros/classificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Proteínas de Insetos/isolamento & purificação , Cinética , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Serina Proteases/isolamento & purificação , Especificidade da Espécie , Especificidade por Substrato , Temperatura , Tripsina/isolamento & purificação , Tripsina/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-18540057

RESUMO

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.


Assuntos
Carica/enzimologia , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/isolamento & purificação , Látex/química , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Benzoilarginina Nitroanilida/farmacologia , Compostos Cromogênicos/farmacologia , Cristalografia por Raios X , Peso Molecular , Difração de Raios X
20.
Parasitol Res ; 103(3): 507-16, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18496715

RESUMO

Two deltamethrin resistance-associated serine protease genes (NYD-tr and NYD-ch) were isolated from Culex pipiens pallens in our previous study. To study the function of NYD-Tr and NYD-Ch in the metabolism of deltamethrin, we constructed the recombinant plasmid pET32a(+)/NYD-tr and pET32a(+)/NYD-ch with a 6x histidine tag. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses of the recombinant proteins revealed that the molecular weights of NYD-Tr and NYD-Ch are 42 and 50 kDa. Enzyme activity assay indicated that the recombinant NYD-Tr and NYD-Ch had the corresponding features of trypsin and chymotrypsin. Using BApNA as the substrate, NYD-Tr gave optimal activity between pH 9.0 and 10.5, while NYD-Ch was optimally active over the range of pH 8.0-11.0 using the S(Ala)(2)ProPhe-pNA as the substrate. Then, we investigated the metabolism of deltamethrin by NYD-Tr and NYD-Ch. Our results showed that NYD-Tr and NYD-Ch could hydrolyze deltamethrin. The acute oral toxicity of the metabolite to Wistar rats was much lower than deltamethrin.


Assuntos
Culex/enzimologia , Inseticidas/metabolismo , Nitrilas/metabolismo , Piretrinas/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Western Blotting , Clonagem Molecular , Culex/genética , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Concentração de Íons de Hidrogênio , Inseticidas/toxicidade , Cinética , Peso Molecular , Nitrilas/toxicidade , Piretrinas/toxicidade , Ratos , Ratos Wistar , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química
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