Enzymatic profiling of tetanus and botulinum neurotoxins based on vesicle-associated-membrane protein derived fluorogenic substrates.

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.

TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models.

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.

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom.

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.

Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates.

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.

Biochemical and pharmacological aspects of two bradykinin-potentiating peptides obtained from tryptic hydrolysis of casein.

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.

Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification.

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).

A fluorimetric method for the determination of pepsin activity.

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.

About the neurolinguistics of the implicatures: abstract of a study

Considerando dados acerca da linguística da produçäo e compreensäo em afasias, foi elaborado protocolo que abrange os diversos tipos de implicaturas. O protocolo foi aplicado a 90 pacientes classificados segundo o tupo de lesäo cerebral evidenciada pela TC. Os resultados säo discutidos tomando em conta as manifestaçöes clínicas das lesöes cerebrais, com afasia , apraxia, agnosia e alteraçöes da inteligência e da pragmática. A discussäo permite aceitar a imrpessäo da existência de um aparelho ('device') que correlaciona (pacreador de) contextos extra-linguísticos com o 'dito' (aspectos semânticos formais)., aposto no hemisfério cerebral direito

Elementos pragmáticos de fala de afásicos de broca: estudo clínico preliminar / Pragmatic elements of the speech of patients with Broca's aphasia: preliminary clinical study

Estudam-se três afásicos de Broca, assim caracterizados com o "Boston Diagnostic Aphasia Examination" e com os critérios de A.R. Luria, quanto ao uso e compreensäo dos elementos pragmáticos da fala - deixis de tempo, pessoa, lugar, discurso e social, implicaturas de Grice, convencionais, generalizadas e particularizadas, e pressuposiçäo. Para tal, utiliza toda a produçäo da fala de três horas de entrevista e de testes específicos. Um caso perdeu o conteúdo proposicional da fala e a linguagem interna, típicos da afasia dinâmica severa. Os outros dois mostram instabilidade na compreensäo e uso da deixis de tempo e lugar apenas nos testes, o que näo ocorre em condiçöes näo artificiais. Estes achados sugerem acometimento da metalinguagem, com afetaçäo de elementos têmporo-espaciais específicos, e é possível que haja uma graduaçäo da perda dos vínculos do discurso com o contexto verbal e näo verbal, cuja mediaçäo é feita por relaçöes pragmáticas, em lesöes frontais

Plasmaferese na síndrome de Landry Guillain Barré Strohl com insuficiência ventilatória ou rapidamente ascendente / Plasma exchange in acute inflammatory polyradiculoneuropathy with ventilatory insufficiency or fastly ascendent

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

Paralisia do olhar conjugado lateral associada a oftalmoplegia internuclear unilateral em hemorragia mesencéfalo-pontina alta: relato de um caso / Combined lateral gaze palsy with unilateral internuclear ophthalmoplegia in high mesencephalo-pontine hemorrhage: report of a case

Relata-se um caso de hemorragia mesencéfalo-pontina alta direita, com oftalmoplegia extrínseca, permanecendo apenas a convergência bilateral, a abduçäo voluntária e automático-reflexa do olho esquerdo, e a aduçäo deste olho na pesquisa dos reflexos vestíbulo-oculares ("one-and-a-half syndrome") Ainda, havia dissociaçäo voluntária-automático da motricidade das pálpebras e dos movimentos conjugados verticais e, também, hemiplegia sensitivo-motora à esquerda e asterixis

As apraxias e as agnosias: uma breve revisao

O autor revisa os principais conceitos e suas concepcoes teoricas subjacentes sobre as apraxias e as agnosias, a partir de uma perspectiva historica e enfocando principalmente aspectos fisiopatologicos e semiologicos, abarcando os seguintes topicos: apraxias ideatoria, ideomotora, do vestir, buco-facial, ocular e cineticas; agnosis visual para objetos, para cores, as estereoagnosias, a autotopoagnosia, a sindrome de Gerstmann, a simultaneagnosia, as negligencias espaciais, as hemiassomatognosias, a sindrome de Balint e as desconexoes inter-hemifericas.

Inteligencia e linguagem: demencia versus afasia: a controversia Pierre Marie-Wenicke cem anos depois

Apos revisamos o problema da relacao entre a linguagem e a inteligencia no afasico e no demente, aplicamos as matrizes progressivas de Reven (MPR) e o 'token test' a 90 sujeitos separadas pela localizacao da lesao com o auxilio da tomografia computadorizada, em focal hemisferica direita (anterior e posterior), esquerda (anterior, posterior, afasicos e nao afasicos), difusa (dementes) e controles sem lesao encefalica. Encontramos que as MPR distinguem com base estatistica os controles sem lesao encefalica dos afasicos, dementes e dos lesados focais a direita, sendo que nao separam os controles dos lesados focais a esquerda mas nao afasicos. Considerando a sindrome afasica, as MPR discriminam a afasia de Wernicke e a global, mas nao a afasia de Broca. Discutimos entao amplamente as explicacoes para estes achados.

Consideracoes sobre aspectos da neurolinguistica

O autor revisa as principais definicoes e concepcoes teoricas subjacentes sobre as afasias, agrafias e alexias, a partir da perspectiva linguistica e abordamos os problemas epistemologicos da aplicacao da linguistica estrutural e gerativo transformacional a neurolinguistica. Tambem enfatiza os aspectos semiologicos e fisiopatologicos, sempre buscando a utilidade clinica.

Consideracoes sobre aspectos da neurolinguistica

O autor revisa as principais definicoes e concepcoes teoricas subjacentes sobre as afasias, agrafias e alexias, a partir da perspectiva linguistica e abordando os problemas epistemologicos da aplicacao da linguistica estrutural e gerativo transformacional a neurolinguistica. Tambem enfatiza os aspectos semiologicos e fisiopatologicos, sempre buscando a utilidade clinica.

Semiologia das afasias: problemas metodologicos e conceptuais dos modelos associacionista e linguistico

O autor relata a evoluco dos modelos de analise das sindromes afasicas, a substituicao do associacionismo pela visao linguistica com seus recentes avancos, em especial no estudo do significado, a interacao entre ambas as concepcoes, enfatizando as insuficiencias metodologicas e conceptuais. Tambem faz previsoes teoricas baseados em ROMAN JAKOBSON do comportamento dos elementos pragmaticos da fala de afasicos, com vistas a verificacao posterior.

Aspectos linguisticos da producao e compreensao na afasia

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.

Discinesia tardia residual 24 meses apos a suspensao do uso da flunarizina

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.

Apraxia cinetica de evitacao de D. Denny-Brown e apraxia cinestesica de A.R.Luria:estudo a proposito de um caso associado a defice bilateral de sensibilidade cineticopostural

O presente trabalho relata um caso classificavel como apraxia de evitacao segundo DENNY-BROWN e apraxia cinestesica aferente de LURIA, resultante de provavel disfuncao dos funiculos posteriores. Discute as alteracoes de tono muscular frequentemente encontradas e os conceitos de apraxia e ataxia aplicados a estes casos, bem como a transicao de manifestacoes clinicas da paralisia as formas superiores das apraxias.