ABSTRACT
Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.
ABSTRACT
This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.
Subject(s)
Crotalid Venoms/enzymology , Crotalus/metabolism , Serine Proteases/metabolism , Amino Acid Sequence , Animals , Arginine/chemistry , Arginine/metabolism , Binding Sites , Biocatalysis/drug effects , Circular Dichroism , Crotalid Venoms/chemistry , Crotalid Venoms/metabolism , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Sequence Data , Neurotoxins/chemistry , Neurotoxins/metabolism , Peptides/chemistry , Peptides/metabolism , Serine Proteases/chemistry , Sodium Chloride/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , TemperatureABSTRACT
Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (K(cat)/K(M) from 0.028 to 75.9 microM.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.
Subject(s)
Botulinum Toxins, Type A/metabolism , Botulinum Toxins/metabolism , Metalloendopeptidases/metabolism , Peptides/metabolism , R-SNARE Proteins/metabolism , Tetanus Toxin/metabolism , Amino Acid Sequence , Animals , Fluorescence Resonance Energy Transfer , Hydrolysis , Kinetics , Mice , Molecular Sequence Data , Peptides/chemistry , R-SNARE Proteins/chemistryABSTRACT
Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.
Subject(s)
Bordetella pertussis/enzymology , Corynebacterium diphtheriae/enzymology , Culture Media/chemistry , Peptide Hydrolases/analysis , Peptides/metabolism , Amino Acid Sequence , Bacterial Toxins/analysis , Bordetella pertussis/growth & development , Buffers , Chromatography, Gel , Corynebacterium diphtheriae/growth & development , Electrophoresis, Polyacrylamide Gel , Filtration , Fluorescent Dyes , Hydrogen-Ion Concentration , Hydrolysis , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Peptide Hydrolases/isolation & purification , Peptides/chemistry , Pertussis Toxin/analysis , Sodium Chloride/chemistry , Substrate Specificity , Tromethamine/chemistryABSTRACT
An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.
Subject(s)
Fluorometry/methods , Pepsin A/analysis , Gastric Juice/drug effects , Gastric Juice/enzymology , Humans , Kinetics , Pepstatins/pharmacology , Time FactorsABSTRACT
Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.
Subject(s)
Bradykinin/physiology , Caseins/metabolism , Oligopeptides/pharmacology , Trypsin/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Blood Pressure/physiology , Bradykinin/metabolism , Caseins/chemistry , Female , Guinea Pigs , Hydrolysis , Ileum/drug effects , Ileum/metabolism , In Vitro Techniques , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Oligopeptides/administration & dosage , Oligopeptides/chemical synthesis , Pain/physiopathology , Rats , Uterus/drug effects , Uterus/metabolismABSTRACT
Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.
Subject(s)
Animals , Guinea Pigs , BradykininABSTRACT
Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).
Subject(s)
Membrane Proteins/chemistry , Metalloendopeptidases/analysis , Metalloendopeptidases/chemistry , Spectrometry, Fluorescence/methods , Tetanus Toxin/analysis , Tetanus Toxin/chemistry , Amino Acid Sequence , Enzyme Activation , Fluorescent Dyes/chemistry , Hydrolysis , Metalloendopeptidases/classification , Molecular Sequence Data , Peptide Fragments/chemistry , R-SNARE Proteins , Sensitivity and Specificity , Substrate Specificity , Tetanus Toxin/classificationABSTRACT
O autor descreve um caso de discinesia tardia (DT) de aparecimento após a suspensão do uso da flunarizina (Fz). A paciente procurou atendimento por síndrome de Parkinson (SP) atípica, pelo parasitismo de movimentos córeo-atetósicos, refratária a L-dopa e anticolinérgicos. Usava a Fz para o tratamento de vertigens por provável insuficiência vértebro-basilar. Suspenso o uso da Fz houve melhora progressiva da SP mas na mesma medida que desaparecia a SP a DP se fez mais evidente e persiste 24 meses após a suspensão da Fz. O objetivo é alertar para esse efeito colateral grave da Fz, uma droga cuja comercialização não é liberada em vários países do mundo mas que se indica atualmente para afecções comuns como a enxaqueca como faz o Goodman e Gilman (1990) mas não aponta como efeitos colaterais nem a SP nem a DT
Subject(s)
Humans , Female , Aged , Diagnosis, Differential , Diagnostic Errors , Dyskinesia, Drug-Induced/drug therapy , Flunarizine/adverse effects , Parkinson Disease , Vertebrobasilar Insufficiency/drug therapy , Vertigo/drug therapyABSTRACT
O autor revisa os estudos recentes mais relevantes que buscam definir a utilidade do critério lingüístico para a delimitaçäo do fenômeno das afasias, abarcando a fonologia, a sintaxe, a semântica e a metalinguagem. Cabe salientar que recentemente, com Goodglass, dado o fracasso da "navalha de Occam" lingüística, valoriza-se o critério de "salientitude" lingüística, um critério, em realidade, psicológico e näo lingüístico, retomando os conceitos tradicionais da teoria da "gestalt" de Golstein
Subject(s)
Anomia/psychology , Aphasia/psychology , Semantics , Linguistics , Speech PerceptionABSTRACT
O autor revisa os estudos recentes mais relevantes que buscam definir a utilidade do criterio linguistico para a delimitacao do fenomeno das afasias, abarcando a fonologia, a sintaxe, a semantica e a metalinguagem. Cabe salientar que recentemente, com Goodglass, dado o fracasso da navalha de Ocean linguistica, valoriza-se o criterio de salientitude linguistica, um criterio, em realidade, psicologico e nao linguistico, retomando os conceitos tradicionais da teoria da gestalt de Goldstein.
Subject(s)
Linguistics , Semantics , Anomia , Aphasia , Linguistics , Semantics , Anomie , AphasiaABSTRACT
O autor descreve um caso de discinesia tardia (DT) de aparecimento apos a suspensao do uso da flunarizina (Fz). A paciente procurou atendimento por sindrome de Parkinson (SP) atipica, pelo parasitismo de movimentos coreoatetosicos, refrataria a ldopa e anticolinergicos. Usava a Fz para tratamento de vertigens por provavel insuficiencia vertebro-basilar. Suspenso o uso da Fz houve melhora progressiva da SP mas na mesma medida que desaparecia a SP a DT se fez mais evidente e persistente 24 meses apos a suspensao da Fz. O objetivo e alertar para esse efeito colateral grave da Fz, uma droga cuja comercializacao nao e liberada em varios paises do mundo mas que se indica atualmente para afeccoes comuns como a enxaqueca como faz o Goodman e Gilman (1990) mas nao aponta como efeitos colaterais nem a SP nem a DT.
Subject(s)
Tardive Dyskinesia , Flunarizine , Parkinson Disease , Tardive Dyskinesia , FlunarizineABSTRACT
O autor observa se um modelo linguistico recente - o das implicaturas de Grice - pode clivar o desempenho de varios grupos de sujeitos com lesoes cerebrais. Esta teoria tem como preocupaçao fundamental, explicar como pode o significado do falante ser diferente do significado do estritamente dito (limitado aos verbetes dos dicionarios) e para a explicaçao de sua teoria, oferece um exemplo que se tornou classico: suponha que (A) e (B) estejam conversando sobre um amigo comum (C) que esta atualmente trabalhando num banco. (A) pergunta para (B) como (C) esta se dando em seu novo emprego, e (B) retruca: "Oh! Muito bem, eu acho: ele gosta de seus colegas e ainda nao foi preso". Neste ponto (A) deve procurar o que (B) estava implicando (implicitar, implicatura - 'implicature' -- 'implicatum'), o que ele estava sugerindo, ou ate mesmo o que ele quis dizer ou dizer que (C) ainda nao tinha sido preso. Elaboramos um protocolo com vinte itens assemelhados aos do exemplo de Grice, abarcando os varios tipos de implicaturas e, justamente com um teste de inteligencia nao verbal e um teste verbal simples, aplicamo-lo a 90 sujeitos, classificados pela localizaçao da lesao conforme a tomografia computadorizada do encefalo: 30 sujeitos com lesoes focais hemisfericas esquerdas, sendo que destes, 24 eram afasicos e 6 eram nao afasicos; 20 com lesoes focais hemisfericas direitas; 20 com lesoes encefalicas difusas dementes, excluidos os com lesoes multifocais) e 20 controles sem lesao encefalica pareados aos demais pela procedencia, nivel socio-economico e anos de escolarizaçao. Pelo tratamento estatistico, observamos que o protocolo das implicaturas separou os controles dos dementese dos afasicos. No grupo de dementes, e com menor intensidade no de lesados a direita, o desmepenho no protocolo com o teste de inteligencia nao-verbal e nao com o verbal, mostrando-nos que o protocolo avalia "inteligencia na linguagem". Pela analise dos rros varificamos que os dementese, em menor intensidade, os com lesoes hemisfericas direitas, tem dificuldades em parear os contextos extra-linguisticos com o dito, manifestas por tipos de erros que quase so ocorrem nestes dois grupos, poupando os afasicos, os sujeitos com lesoes hemisfericas esquerdas mas nao afasicos e os controles. Ficam-se com a impressao de que existe um aparelho pareador de contestos (extra-linguisticos) com odito no hemisferio direito
Subject(s)
Cerebrum/injuries , Intelligence Tests , Linguistics , Natural Language Processing , Sign LanguageABSTRACT
Os autores estudam a fonologia de um afásico global buscando definir os processos fonológicos subjacentes a sua desorganizaçäo. Observam que as consoantes mais resistentes a mudança, em ordem decrescente, säo as seguintes: oclusiva surda ('p','t'), oclusiva dento-alveolar('t), oclusiva nasal('m','n'), oclusiva lábio-labial('p'), fricativa surda dento-alveolar('s'), oclusiva pálato-velar('k'), fricativa uvular('x','rr'), oclusiva sonora('b','d','g') e as líquidas('r','l'). A fricativa uvular sonora 'j' é o único fonema do português ausente de seu inventário fonológico. O paciente desdiferenciou sua fala, tornando o sonoro em surdo, o fricativo ou africado em oclusivo e deslocou a articulaçäo para a crista alveolar. As dentais 't','d' e mesmo a fricativa 's' mostraram-se mais resistentes que o 'g' e o 'b'. Também observamos a estabilidade das nasais (oclusivas sonoras) 'n' e 'm'. Os achados desse paciente säo muito regulares e correspondem ao que se vê na estrutura das línguas e também na aquisiçäo da linguagem conforme a hipótese de Jakobson. O único achado discordante é a resistência da fricativa sonora 'v', maior do que a da fricativa surda 'f'. Esse estudo é parte de uma tentativa de reeducaçäo baseada na correçäo do traço distintivo instável
Subject(s)
Middle Aged , Humans , Male , Speech Disorders/rehabilitation , Phonetics , Speech Therapy , SyndromeABSTRACT
Os autores revisam as síndromes paraneoplásicas neurológicas com lesöes nervosas periféricas, radiculares, medulares e encefálicas, enfatizando as manifestaçöes clínicas e relatam um caso associado a um carcinoma ductal indiferenciado de mama com síndrome vestibular e arreflexia patelar e diminuiçäo da palestesia é força nos membros inferiores que, no pós-operatório imediato da mastectomia, acrescentou-se de síndrome demencial e astasia (cerebelar)
Subject(s)
Adult , Humans , Male , Female , Encephalomyelitis/etiology , Paraneoplastic Syndromes/etiology , Breast Neoplasms/complications , Carcinoma, Ductal, Breast/complicationsABSTRACT
Os autores revisam as sindromes paraneoplasicas neurologicas com lesoes nervosas perifericas, radiculares, medulares e encefalicas, enfatizando as manifestacoes clinicas e relatam um caso associado a um carcioma ductal indiferenciado de mama com sindrome vestibular e arreflexia potelar e diminuicao da palestesia e forca nos membros inferiores que, no pos-operatorio imediato da mastectomia, acrescentou-se de sindrome demencial e astasia (cerebelar).
Subject(s)
Paraneoplastic Syndromes , Encephalomyelitis , Carcinoma, Ductal, Breast , Breast Neoplasms , Adult , Case Reports , Paraneoplastic Syndromes , Encephalomyelitis , Breast Neoplasms , AdultABSTRACT
Os autores estudam a fonologia de um afasico global buscando definir os processo fonologicos subjacentes a sua desorganizacao. Observam que as consoantes mais resistentes a mudanca, em ordem decrescente, sao as seguintes: oclusiva surda ('p', 't'), oclusiva dentro-al-veolar ('t'), oclusiva nasal ('m', 'n'), oclusiva labio-labial ('p'), fricativa surda dento-alveolar ('s'), oclusiva palato-velar ('k'), fricativa uvular ('x', 'rr'), oclusiva sonora ('b', 'd', 'g') e as liquidas ('r', 'l'). A fricativa uvular sonora 'j' e o unico fonema do portugues ausente de seu inventario fonologico. O paciente desdiferenciou sua fala, tornando o sonoro em surdo, o fricativo ou africado em oclusivo e deslocou a articulacao para a crista alveolar. As demais 't', 'd' e mesmo as africativa's' mostraram-se mais resistentes que o 'g' e o 'b'. Tambem observamos a estabilidade das nasais (oclusiva sonoras) 'n' e 'm'. Os achados desse paciente sao muito regulares e correspondem ao que se ve na estrutura das linguas e tambem na aquisicao da linguagem conforme a hipotese de Jakobson. O unico achado discordante e a resistencia da fricativa sonora 'v', maior do que a da fricativa surda 'f'. Esse estudo e parte de uma tentativa de reeducacao baseada na correcao do traco distivo instavel.
Subject(s)
Speech Disorders , Speech Therapy , Phonetics , Syndrome , Middle Aged , Speech Disorders , Speech Therapy , Phonetics , Syndrome , Middle AgedABSTRACT
O autor revisa as principais definiçöes e concepçöes teóricas subjacentes sobre as afasias, agrafias e alexias, a partir da perspectiva lingüística e abordamos os problemas epistemológicos da aplicaçäo da lingüística estrutural e gerativo transforcional a neurolingüística. Também enfatiza os aspectos semiológicos e fisiopatológicos, sempre buscando a utilidade clínca
Subject(s)
Humans , Agraphia , Aphasia , Language , Dominance, CerebralABSTRACT
Os autores descrevem três casos de síndrome de Landry-Guillain-Barré-Sthohl tratados com sucesso pela plasmaferese - os únicos do Rio Grande do Sul - precedidos por breve revisäo e detendo-se no procedimento, fixando critérios para sua indicaçäo
