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Electron. j. biotechnol ; 32: 63-68, Mar. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1022642


Background: Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity has a major issue for long-term treatment. One method to improve therapies and methods is the application of immunomodulatory agents from parasites such as Hypoderma lineatum. Hypodermin A (HA) is a serine esterase secreted by the larvae of Hypoderma lineatum, several studies demonstrated its immunosuppressive mechanism in vitro, and recently we discovered that HA inhibits the expression of interferon (IFN)-γ and interleukin (IL)-2 and activates IL-10 expression. Therefore, we hypothesized that it might be a potential agent used to block allograft rejections. However, most studies of the immunosuppressive mechanisms associated with HA were undertaken at the cellular level. In order to augment these studies, we evaluated the immunosuppressive effects of HA in vivo using an HA transgenic mouse model. Result: Our results revealed similar findings to those reported by in vitro studies, specifically that HA induced prostaglandin E2 expression, downregulated IFN-γ and IL-2 expression, and promoted IL-10 secretion via E-type prostanoid receptor 4. Additionally, we observed that HA overexpression inhibited lipopolysaccharide-induced TLR4 activation. These findings provide insight into a new potential agent capable of blocking graft rejection. Conclusion: Our founding suggested that HA-related treatment could be a promising option to improve the viability of grafts in human.

Animais , Camundongos , Serina Endopeptidases/imunologia , Dípteros/enzimologia , Dípteros/imunologia , Rejeição de Enxerto/imunologia , Ensaio de Imunoadsorção Enzimática , Serina Endopeptidases/metabolismo , Western Blotting , Citocinas , Imunossupressão , Interleucinas/antagonistas & inibidores , Interferons/antagonistas & inibidores , Interleucina-10/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like , Reação em Cadeia da Polimerase em Tempo Real , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/prevenção & controle
Electron. j. biotechnol ; 29: 7-12, sept. 2017. ilus, graf, tab
Artigo em Inglês | LILACS | ID: biblio-1016095


Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Serina Endopeptidases/genética , Proteínas Periplásmicas/genética , Chromohalobacter/enzimologia , Proteólise , Proteínas de Choque Térmico/genética , Proteínas Recombinantes , Serina Endopeptidases/metabolismo , Caseínas , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Proteínas Periplásmicas/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Salinidade , Chromohalobacter/genética , Proteínas de Choque Térmico/metabolismo , Peso Molecular
Acta cir. bras ; 31(5): 314-319, May 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-783800


ABSTRACT PURPOSE : To evaluate the effects of platelet rich plasma (PRP) on the healing of fascia wherein peritonitis has been created. METHODS: Twenty eight Wistar Albino rats were divided into four groups. Only a primary fascial repair following laparotomy was performed on Group 1, a primary fascial repair performed and PRP treatment applied following laparotomy on Group 2, and a fecal peritonitis created following laparotomy and a primary fascial repair carried out on Group 3. A fecal peritonitis was created following laparotomy and primary fascial repair and PRP treatment on the fascia was carried out on Group 4. RESULTS: TNF-α was found to be significantly lower in the control group (Group 1). It was detected at the highest level in the group in which fecal peritonitis was created and PRP applied (Group 4). TGF-β was determined as being significantly higher only in Group 4. Histopathologically, the differences between the groups in terms of cell infiltration and collagen deposition were not found to be significant. CONCLUSION: When platelet rich plasma was given histologically and biochemicaly as wound healing parameters cellular infiltration, collagen accumulation, and tissue hydroxyiproline levels were not increased but neovascularization, fibroblast activation and TNF Alfa levels were increased and PRP accelerated wound healing.

Animais , Peritonite/complicações , Cicatrização , Plasma Rico em Plaquetas , Fáscia/fisiologia , Peritonite/metabolismo , Serina Endopeptidases/metabolismo , Distribuição Aleatória , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Ratos Wistar , Gelatinases/metabolismo , Neovascularização Fisiológica , Modelos Animais , Fáscia/irrigação sanguínea , Hidroxiprolina/análise , Hidroxiprolina/metabolismo , Proteínas de Membrana/metabolismo
Braz. j. microbiol ; 46(3): 879-883, July-Sept. 2015. tab
Artigo em Inglês | LILACS | ID: lil-755800


Studies investigating rickettsial infections in ticks parasitizing wild animals in the Northeast region of Brazil have been confined to the detection of Rickettsia amblyommii in immature stages of Amblyomma longirostre collected from birds in the state of Bahia, and in immatures and females of Amblyomma auriculariumcollected from the striped hog-nosed skunk (Conepatus semistriatus) and armadillos (Euphractus sexcinctus) in the state of Pernambuco. The current study extends the distribution of R. amblyommii (strain Aranha), which was detected in A. longirostre collected from the thin-spined porcupine Chaetomys subspinosus and the hairy dwarf porcupine Coendou insidiosus. In addition, we report the first detection of Rickettsia bellii in adults of A. longirostre collected from C. insidiosus in the state of Bahia.


Animais , Ixodidae/microbiologia , Infecções por Rickettsia/microbiologia , Rickettsia/genética , Rickettsia/isolamento & purificação , Infestações por Carrapato/microbiologia , Animais Selvagens , Tatus , Sequência de Bases , Aves , Brasil , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Citrato (si)-Sintase/genética , DNA Bacteriano , Proteínas de Choque Térmico/genética , Mephitidae , Dados de Sequência Molecular , Tipagem Molecular , Porcos-Espinhos , Proteínas Periplásmicas/genética , Análise de Sequência de DNA , Serina Endopeptidases/genética
Rev. Assoc. Med. Bras. (1992) ; 61(3): 263-268, May-Jun/2015.
Artigo em Inglês | LILACS | ID: lil-753178


Summary In southern Brazil, since 1989, several cases of accidents produced by unwilling contact with the body of poisonous caterpillars of the moth species Lonomia obliqua Walker, 1855 (Lepidoptera: Saturniidae), were described. L. obliqua caterpillars have gregarious behavior and feed on leaves of host trees during the night, staying grouped in the trunk during the day, which favors the occurrence of accidents with the species. This caterpillar has the body covered with bristles that on contact with the skin of individuals, breaks and release their contents, inoculating the venom into the victim. The basic constitution of the venom is protein and its components produce physiological changes in the victim, which include disturbances in hemostasis. Hemorrhagic syndrome associated with consumption coagulopathy, intravascular hemolysis and acute renal failure are some of the possible clinical manifestations related to poisoning by L. obliqua. Specific laboratory tests for diagnosis of poisoning have not been described previously. The diagnosis of poisoning is made based on the patient's medical history, clinical manifestations, erythrocyte levels, and, primarily, parameters that evaluate blood coagulation. Treatment is performed with the use of supportive care and the administration of specific hyperimmune antivenom. Poisoning can be serious and even fatal.

Na região Sul do Brasil, a partir de 1989, foram descritos inúmeros casos de contato acidental com o corpo de lagartas venenosas da mariposa da espécie Lonomia obliqua Walker, 1855 (Lepidoptera: Saturniidae). As lagartas de L. obliqua possuem comportamento gregário e alimentam- -se de folhas das árvores hospedeiras durante a noite, permanecendo agrupadas no tronco durante o dia, o que favorece a ocorrência de acidentes. Essa lagarta possui o corpo recoberto por cerdas urticantes, que, ao contato com a pele dos indivíduos, se rompem e liberam seu conteúdo, inoculando o veneno na vítima. A constituição básica do veneno é proteica e seus componentes produzem modificações fisiológicas no acidentado, que incluem distúrbios na hemostasia. Síndrome hemorrágica associada a coagulopatia de consumo, hemólise intravascular e insuficiência renal aguda são algumas das manifestações clínicas possíveis relacionadas ao envenenamento por L. obliqua. Ainda não foram descritas avaliações laboratoriais específicas para diagnóstico do envenenamento, que é feito com base na anamnese do paciente, nas manifestações clínicas, nos índices hematimétricos e, principalmente, nos parâmetros que avaliam a coagulação sanguínea. A terapêutica é feita com a utilização de medidas de suporte e com a administração de um soro heterólogo hiperimune específico. Os envenenamentos podem ser graves e até mesmo fatais.

Animais , Humanos , Venenos de Artrópodes/envenenamento , Hemostasia , Lepidópteros , Venenos de Artrópodes/metabolismo , Brasil , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Intravascular Disseminada/etiologia , Hemorragia/induzido quimicamente , Mariposas , Serina Endopeptidases/metabolismo
Rev. méd. Chile ; 142(7): 880-888, jul. 2014. tab
Artigo em Espanhol | LILACS | ID: lil-726179


Statins are the preferred treatment for hypercholesterolemia and several studies have demonstrated their long-term safety and efficacy in reducing cardiovascular morbidity and mortality. However, in some cases of severe hypercholesterolemia such as homozygous and heterozygous familial hypercholesterolemia or statin intolerant patients, statins can be less efficient. In recent years, new lipid-lowering agents with novel mechanisms of action have been developed to reduce LDL-cholesterol in patients with severe hypercholesterolemia, associated or not to conventional lipid-lowering therapy. These therapies include microsomal transfer protein inhibitor (Lomitapide), antisense oligonucleotide to Apo B100 (Mipomersen) and monoclonal antibodies against Proprotein convertase subtilisin/kexin type 9 (PCSK9). Different studies have shown the great effectiveness of these new therapies. Short-term studies confirmed their adequate security profile, especially in patients with homozygous familiar hypercholesterolemia or severe hypercholesterolemia. Some of these agents have been also tested in statin-intolerant patients. However, long-term studies are needed to evaluate their safety, effectiveness and impact on cardiovascular risk reduction.

Humanos , Anticolesterolemiantes/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Benzimidazóis/uso terapêutico , Ensaios Clínicos como Assunto , Oligonucleotídeos/uso terapêutico , Pró-Proteína Convertases/uso terapêutico , Serina Endopeptidases/uso terapêutico
Trends psychiatry psychother. (Impr.) ; 36(1): 52-58, Jan-Mar/2014. tab
Artigo em Inglês | LILACS | ID: lil-707279


INTRODUCTION: Day hospitals in psychiatry are a major alternative to inpatient care today, acting as key components of community and social psychiatry. Objective: To study trends in the use of psychiatric day hospitals over the last decades of the 20th century and the first decade of the 21st century, focusing on patient age, sex, and diagnostic group, using data from Centro Hospitalar São João, Porto, Portugal. METHODS: Data corresponding to years 1970 to 2009 were collected from patient files. Patients were classified into seven diagnostic groups considering their primary diagnoses only. RESULTS: Mean age upon admission rose from 32.7±12.1 years in the second half of the 1970s to 43.5±12.2 years in 2005-2009 (p for trend < 0.001). Most patients were female (63.2%), however their proportion decreased from nearly 70% in the 1970s to 60% in the first decade of the 21st century. In males, until the late 1980s, neurotic disorders (E) were the most common diagnosis, accounting for more than one third of admissions. In the subsequent years, this proportion decreased, and the number of admissions for schizophrenia (C) exceeded 50% in 2004- 2009. In females, until the late 1980s, affective disorders (D) and neurotic disorders (E), similarly distributed, accounted for most admissions. From the 1990s on, the proportion of neurotic disorders (E) substantially decreased, and affective disorders (D) came to represent more than 50% of all admissions. CONCLUSIONS: Mean age upon admission rose with time, as did the percentage of female admissions, even though the latter tendency weakened in the last 10 years assessed. There was also an increase in the proportion of patients with schizophrenia. .

INTRODUÇÃO: Os hospitais de dia em psiquiatria representam atualmente uma das principais alternativas ao internamento, atuando como componentes chave na psiquiatria comunitária e social. OBJETIVO: Avaliar tendências na utilização de um hospital de dia no período compreendido entre as últimas décadas do século 20 e a primeira década do século 21, com foco em idade, sexo e grupo diagnóstico, usando dados do Centro Hospitalar São João, Porto, Portugal. MÉTODOS: Dados correspondentes aos anos 1970 a 2009 foram coletados dos prontuários clínicos. Os pacientes foram classificados em sete grupos diagnósticos, tendo em conta o diagnóstico principal. Resultados: A idade média na admissão aumentou de 32.7±12.1 anos na segunda metade da década de 1970 para 43.5±12.2 anos em 2005-2009 (p < 0.001). A maioria dos pacientes era do sexo feminino (63.2%), no entanto sua proporção diminuiu de cerca de 70% na década de 1970 para 60% na primeira década do século 21. Nos homens, até o final dos anos 1980, o grupo das perturbações neuróticas (E) era o diagnóstico mais comum, representando mais de um terço das admissões. Durante os anos seguintes, essa proporção diminuiu, e o número de admissões por esquizofrenia (C) alcançou mais de 50% no período de 2004-2009. Nas mulheres, até o final dos anos 1980, as perturbações afetivas (D) e as perturbações neuróticas (E), distribuídas similarmente, respondiam pela maioria das admissões. A partir dos anos 1990, a proporção das perturbações neuróticas (E) diminuiu substancialmente, e as perturbações afetivas (D) passaram a corresponder a mais de 50% do total das admissões. Conclusões: A idade média na admissão ...

Adolescente , Criança , Feminino , Humanos , Masculino , Adulto Jovem , Adenoma/genética , Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias/genética , Serina Endopeptidases/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/metabolismo , Adenoma/patologia , Predisposição Genética para Doença , Linhagem , Análise de Sequência de DNA , Serina Endopeptidases/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
Mem. Inst. Oswaldo Cruz ; 109(1): 38-50, 02/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-703647


Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Animais , Bovinos , Flavivirus/química , RNA Viral/isolamento & purificação , Rhipicephalus/virologia , Proteínas não Estruturais Virais/química , Brasil , Sequência Conservada/genética , Flavivirus/classificação , Flavivirus/isolamento & purificação , Biblioteca Gênica , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Reação em Cadeia da Polimerase , RNA Helicases/química , Alinhamento de Sequência/estatística & dados numéricos , Análise de Sequência de Proteína/métodos , Serina Endopeptidases/química , Extratos de Tecidos/análise , Transcriptoma/genética
Braz. j. med. biol. res ; 46(6): 546-554, 02/jul. 2013. tab, graf
Artigo em Inglês | LILACS | ID: lil-679208


Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Laríngeas/genética , MicroRNAs/isolamento & purificação , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , RNA Mensageiro/isolamento & purificação , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Citometria de Fluxo , Fluoruracila/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Genes MDR , Neoplasias Laríngeas/tratamento farmacológico , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas RGS/genética , /farmacocinética , Serina Endopeptidases/genética , Análise Serial de Tecidos , Vincristina/farmacologia
Rio de Janeiro; s.n; 2013. xiv,140 p. ilus, tab, graf, mapas.
Tese em Português | LILACS | ID: lil-746873


Os mosquitos Aedes albopictus (Skuse, 1894) e Aedes aegypti (Linnaeus, 1762) apresentam significativa importância epidemiológica dentro da família Culicidae por serem os principais vetores da febre amarela e dos quatro sorotipos do vírus da dengue. As peptidases ou proteases são enzimas que clivam ligações peptídicas. No sistema digestivo de invertebrados as peptidases, especificamente aquelas da classe das serino-peptidases, são abundantes e participam de vários processos fisiológicos, desempenhando um papel principal na hidrólise de proteínas destinadas à nutrição. Estas enzimas podem apresentar características espécie-específicas e essas diferenças são respostas adaptativas a diferentes estilos de vida, ambientes e habilidade de sobrevivência entre as espécies. Neste trabalho descreve-se a caracterização das atividades proteolíticas detectadas em extratos totais das formas pré-imaginais de A. aegypti e A. albopictus por enzimografia em uma dimensão. A estabilidade da expressão de peptidases nessas espécies foi avaliada pela comparação do perfil proteolítico de formas larvais obtidas de ovos de insetos recém coletados no ambiente com ovos de insetos mantidos em colônia por longo período. Foram também comparados os perfis proteolíticos das pupas com seus respectivos imagos, bem como a estabilidade térmica das peptidases detectadas. Observaram-se complexos perfis de serino-peptidases do tipo tripsina em ambas as espécies vetoras.

Contudo esses perfis apresentam diferenças espécie-específicas e também diferenças entre os distintos estágios evolutivos dentro de uma mesma espécie. Também, neste trabalho, descreve-se a caracterização das atividades proteolíticas detectadas no intestino médio de fêmeas de A. albopictus alimentadas com açúcar bem como o primeiro mapa proteô mico e a identificação de peptidases por eletroforese bi-dimensional e espectrometria de massas neste órgão. São discutidas aqui as estratégias usadas para analisar as peptidases expressas nesse tecido. As proteínas expressas no intestino médio de A. albopictus foram identificadas por similaridade com as sequencias de genoma de A. aegypti e distintas ferramentas bioinformáticas foram usadas para obter informação funcional de muitas dessas sequencias que estão pobremente anotadas. Foram identificadas 59 proteínas entre v Resumo as quais três serino-peptidases, e dessas, duas do tipo tripsina e uma quimiotripsina. Os resultados obtidos nos permitiram atribuir de maneira confiável um local de expressão para os genes de tripsina, tripsina alfa e quimotripsina. Em outras palavras, podemos afirmar que os genes acima mencionados se expressam no intestino médio de fêmeas adultas de A. albopictus alimentadas com açúcar. Este achado representa um pequeno, mas importante passo, para a atribuição funcional, ao nível de proteína, de genes codificantes para serino-peptidases do tipo tripsina e quimiotripsina no gênero Aedes.

The mosquitoes Aedes albopictus (Skuse, 1894) and Aedes aegypti (Linnaeus, 1762) havesignificant epidemiological importance within the family Culicidae because they are themain vectors of the yellow fever virus and the four serotypes of Dengue virus. Peptidasesor proteases are enzymes that hydrolyze peptide bonds. In the digestive system ofinvertebrates, the peptidases, specifically those from the class of serine peptidases, areabundant and participate in various physiological processes, playing a major role in thehydrolysis of proteins destined to nutrition. These enzymes may have species-specificcharacteristics, and such differences reflect adaptive responses to different lifestyles andenvironments. In this work we describe the characterization, using zymography, of theproteolytic activities detected in total extracts of larval forms of A. aegypti and A.albopictus. The stability of expression of peptidases in these species was evaluated bycomparing the proteolytic profile of larval forms obtained from eggs of newly collectedinsects to that from eggs of insect kept in colony for a long period. We also compared theproteolytic profiles of pupae as well as the thermal stability of the peptidases detected.Complex profiles of trypsin-like serine peptidases were observed in both species.

However, these profiles differ between species and also differences between the differentevolutionary stages were detected within the same species. Also, in this work, we describethe characterization of the proteolytic activities detected in the midgut of A. albopictusfemales fed on sugar as well as the first proteome map and proteomic identification ofpeptidases in this organ, using two-dimensional electrophoresis and mass spectrometry.The strategies used to analyze the proteases expressed in this tissue are discussed here. Theproteins expressed in the midgut of A. albopictus were identified by similarity with thegenome sequences of A. aegypti and various bioinformatic tools were used to obtainfunctional information for many of these sequences that are poorly annotated. Weidentified 59 proteins including three serine peptidases, and among these, two types oftrypsin-like and one chymotrypsin. The results obtained here allowed us to reliably assignviiiAbstracta localization for the expression of trypsin, trypsin-alpha and chymotrypsin genes. In otherwords, we can say that the above mentioned genes are expressed in the midgut of adultfemales of A. albopictus fed with sugar. This finding represents a small but important stepfor the functional assignment, at the protein level, of the genes coding for trypsin-like andchymotrypsin serine peptidases of the Aedes genus.

Aedes/classificação , Peptídeo Hidrolases , Proteômica , Serina Endopeptidases , Dengue , Febre Amarela
Einstein (Säo Paulo) ; 10(4): 526-527, Oct.-Dec. 2012. tab
Artigo em Inglês | LILACS | ID: lil-662482


This is a remarkable progress; since the finding of statins, there was no new way of reducing, significantly, cholesterol and LDL fraction. It is also clear that this decrease, by statins, is related to future cardiovascular lesions, being useful in its primary and secondary prophylaxis. The authors presented studies on research to promote the falling of blood cholesterol by means of antibodies, which inhibit the pro-protein PCSK9, as well as agents that act performing the RNA interference. We had two advantages immediately: for patients with myopathy associated with statins, and the fact of being injected every 15 days, that may contribute to better treatment adherence.

Este é um progresso sensível; desde a descoberta das estatinas, não havia novas maneiras de diminuir, de maneira significativa, o colesterol e a fração LDL. Também está claro que essa redução, pelas estatinas, tem relação com futuras lesões cardiovasculares, sendo útil na profilaxia primária e secundária destas. Os autores apresentaram estudos sobre pesquisas para promover a queda do colesterol sanguíneo por meio de anticorpos que inibem a pró-proteína PCSK9, bem como agentes que atuam realizando a interferência no RNA. Duas vantagens se afiguram imediatamente: para pacientes que têm a miopatia relacionada às estatinas e por ser droga injetável a cada 15 dias, o que pode colaborar para maior adesão ao tratamento.

Humanos , Dislipidemias/terapia , Pró-Proteína Convertases/imunologia , Serina Endopeptidases/imunologia , Anticorpos Monoclonais/administração & dosagem , Ensaios Clínicos como Assunto , LDL-Colesterol/sangue , Interferência de RNA
Braz. j. med. biol. res ; 45(10): 929-934, Oct. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-647751


Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Animais , Masculino , Coelhos , Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemorragia/tratamento farmacológico , Antagonistas de Heparina/farmacologia , Heparina de Baixo Peso Molecular/antagonistas & inibidores , Protrombina/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Hemorragia/induzido quimicamente
Botucatu; s.n; 2012. 89 p. ilus, tab.
Tese em Português | LILACS | ID: lil-691817


Entre os alvos mais promissores para o desenvolvimento de novos agentes antiparasitários, destacam-se as proteases que nos protozoários participam de processos metabólicos e fisiológicos, atuando como importantes fatores de virulência. Como a atividade dessas moléculas pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial terapêutico em diferentes infecções parasitárias, inclusive por Giardia. O presente estudo foi desenvolvido com o objetivo de avaliar o efeito in vitro dos inibidores de cisteína (IAA e E-64) e serina-proteases (antipaina, leupeptina e TLCK) sobre o crescimento, aderência, viabilidade e ultraestrutura de trofozoítos de cepa de Giardia isolada e axenizada em Botucatu. Para isso, trofozoítos foram incubados em meio contendo os inibidores a diferentes concentrações durante 24, 48 e 72 horas. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado a partir de contagens em hemocitômetro, enquanto que a viabilidade celular e as alterações ultraestruturais foram avaliadas, respectivamente, pelo método de redução do MTT e por microscopia eletrônica de transmissão. De acordo com as observações feitas no presente estudo, todos os inibidores de proteases apresentaram efeito sobre o crescimento, aderência e viabilidade dos trofozoítos. Entretanto, melhor desempenho quanto à capacidade de reduzir os parâmetros avaliados foi demonstrado nos ensaios com os inibidores de cisteína-proteases, especialmente a IAA. As maiores porcentagens de inibição do crescimento e aderência e as menores taxas de viabilidade foram observadas após o tratamento com IAA...

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In light of this, the proteolytic enzymes or proteases have excited the researcher’s interest, once they have been identified as important virulence factors as well as potential chemotherapeutic targets in parasites. Considering that proteases are naturally regulated by specific inhibitors, these substances have been evaluated for their therapeutic potential in parasitic infections including Giardia. In this way, we proposed to evaluate the in vitro effect of inhibitors of cysteine (IAA and E-64) and serine proteases (antipain, leupeptin and TLCK) on growth, adherence, viability and ultrastructure of Giardia trophozoites of a strain isolated and axenized in Botucatu. For this, trophozoites were incubated in medium containing the inhibitors at various concentrations for 24, 48 and 72 hours. In growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability and ultrastructural changes were evaluated, respectively, by the method of MTT and transmission electron microscopy. In this study, all protease inhibitors showed effect on growth, adherence and viability of trophozoites. However, better performance in their ability to reduce the parameters assessed was demonstrated in experiments with cysteine proteases inhibitors, especially IAA...

Humanos , Antipaína/antagonistas & inibidores , Cisteína/antagonistas & inibidores , Giardia lamblia/isolamento & purificação , Técnicas In Vitro , Leupeptinas/antagonistas & inibidores , Inibidores de Proteases , Serina Endopeptidases , Tosilina Clorometil Cetona/antagonistas & inibidores , Trofozoítos
Braz. j. med. biol. res ; 44(9): 914-919, Sept. 2011. ilus
Artigo em Inglês | LILACS | ID: lil-599662


The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.

Animais , Humanos , Camundongos , Ratos , Angiotensina II/biossíntese , Sistema Cardiovascular/metabolismo , Sistema Renina-Angiotensina/fisiologia , Serina Endopeptidases/fisiologia , Angiotensina I/biossíntese , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Quimases/metabolismo , Serina Endopeptidases/farmacologia
Mem. Inst. Oswaldo Cruz ; 104(8): 1132-1138, Dec. 2009. tab, ilus
Artigo em Inglês | LILACS | ID: lil-538173


Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Humanos , Mycobacterium leprae/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Mycobacterium leprae/genética , Espectroscopia de Infravermelho com Transformada de Fourier
Rev. bras. parasitol. vet ; 18(1): 1-7, Mar. 2009.
Artigo em Inglês | LILACS | ID: lil-606757


The tick Rhipicephalus (Boophilus) microplus (formerly Boophilus microplus) is the major ectoparasite affecting livestock in America, Asia, Africa, and Oceania. Conventional tick control is based on the use of acaricides but immunization of bovines with tick gut proteins induces only a partial protective immune response. Based on this information, distinct research groups have explored the possibility of protecting the animals by inducing an immune response against other tick proteins. However, the antigens so far described do not induce the necessary protection for suppressing the use of acaricides. Currently, several groups are engaged in identifying new tick proteins to be used as targets for the development of new vaccines. This approach focuses on the enhancement of the immunogenicity of antigens already tested by incorporating new adjuvants or formulations and by searching for new antigens. This paper reviews the work done by Brazilian researchers to develop a vaccine against this tick.

O carrapato Rhipicephalus (Boophilus) microplus (anteriormente Boophilus microplus) é o principal ectoparasita que afeta bovinos na América, Ásia, África e Oceania e o seu controle é tradicionalmente realizado através do uso de acaricidas. Experimentos de imunização com proteínas do carrapato mostram que a resposta imune desenvolvida pelos bovinos vacinados protege, em parte, os animais do parasitismo. Baseado nessas observações, vários grupos de pesquisa exploram a possibilidade de proteger os animais pela indução de uma resposta imune contra proteínas do carrapato. Entretanto, os antígenos já caracterizados não asseguram o grau de proteção necessário para suprimir o uso de acaricidas. Portanto, esses grupos de pesquisa estão engajados na tentativa de identificar novas proteínas que possam ser utilizadas para o desenvolvimento de novas vacinas, as quais possam induzir maior imunogenicidade de que os antígenos já testados, através do uso de novas formulações e/ou pela incorporação de adjuvantes. O presente artigo apresenta uma revisão da literatura sobre os resultados obtidos por pesquisadores brasileiros no desenvolvimento de vacinas contra o carrapato.

Animais , Rhipicephalus , Infestações por Carrapato/prevenção & controle , Vacinas , Ácido Aspártico Endopeptidases , Precursores Enzimáticos , Serina Endopeptidases
Clinics ; 64(7): 641-644, 2009.
Artigo em Inglês | LILACS | ID: lil-520795


INTRODUCTION: Ovarian cancer is generally diagnosed at advanced stages of the disease; therefore, poor prognoses are typical. The development of tumor markers is thus of utmost importance. Prostasin is a protease that in normal tissues is highly expressed only in the prostate gland and seminal fluid. A previous study showed that prostasin is highly overexpressed in ovarian cancer cell lines. This study sought to evaluate the expression of prostasin in ovarian cancer. METHODS: Fresh tumor samples of ovarian epithelial cancer (n: 12) were analyzed for expression of prostasin mRNA (messenger ribonucleic acid) by conventional and real time quantitative PCR (polymerase chain reaction). As a standard control, a normal prostate sample was analyzed. RESULTS: Using conventional PCR, prostasin was detected in all but one sample. Using quantitative PCR, prostasin was over-expressed in all but one of the samples as compared to the control (prostate). CONCLUSIONS: These findings indicate that prostasin is overexpressed in many epithelial ovarian cancers. Further studies of prostasin as a potential biomarker for this disease are warranted.

Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica , Programas de Rastreamento/métodos , Proteínas de Neoplasias/sangue , Neoplasias Ovarianas/enzimologia , Serina Endopeptidases/sangue , Biomarcadores Tumorais/sangue , Neoplasias Ovarianas/diagnóstico , Projetos Piloto , Reação em Cadeia da Polimerase Via Transcriptase Reversa
Mem. Inst. Oswaldo Cruz ; 103(5): 504-506, Aug. 2008. ilus
Artigo em Inglês | LILACS | ID: lil-491976


We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Animais , Dípteros/enzimologia , Serina Endopeptidases/metabolismo , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Larva/enzimologia , Serina Endopeptidases/isolamento & purificação
Braz. j. med. biol. res ; 41(1): 12-17, Jan. 2008. graf
Artigo em Inglês | LILACS | ID: lil-469981


A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Animais , Camundongos , Coagulação Sanguínea , Bothrops , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia em Agarose , Cromatografia por Troca Iônica , Costa Rica , Coagulantes/administração & dosagem , Coagulantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Fibrinogênio/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Mordeduras de Serpentes/fisiopatologia , Trombina/química