RESUMO
The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.
The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.
Assuntos
Ácido Aspártico Proteases , Candida parapsilosis , Candida parapsilosis/enzimologia , Candida parapsilosis/genética , Humanos , Ácido Aspártico Proteases/metabolismo , Ácido Aspártico Proteases/genética , Fatores de Virulência/metabolismo , Soroalbumina Bovina , Proteólise , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Candidíase/microbiologia , Meios de Cultura/química , Catepsina D/metabolismo , Proteases Aspárticas SecretedasRESUMO
Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.
Assuntos
Halobacillus/enzimologia , Serina Proteases/análise , Meios de Cultura/química , Estabilidade Enzimática , Inibidores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Halobacillus/crescimento & desenvolvimento , Peso Molecular , Proteólise , Fluoreto de Fenilmetilsulfonil/metabolismo , Serina Proteases/química , Cloreto de Sódio/metabolismoRESUMO
O propósito das ressecções pulmonares em cães e gatos, quer sejam por lobectomia ou pneumonectomia, é a cura ou paliação de processos broncopulmonares sempre que os meios conservadores de tratamento clínico sejam considerados ineficientes. Tendo em vista as significativas alterações resultantes da pneumonectomia, novos estudos experimentais devem ser feitos para avaliar as vantagens dessa intervenção cirúrgica e determinar a maneira como aplicá-la com segurança. O presente estudo tem como objetivo avaliar as alterações eletrocardiográficas em dez cães adultos de ambos os sexos, sem raça definida, com 10-30 kg, submetidos à pneumonectomia direita. Foram avaliados diariamente os parâmetros clínicos de cada cão e as alterações em todas as derivações do eletrocardiograma. Todos os cães apresentaram um bom desenlace pós-operatório. Apenas um cão apresentou alteração de relevância clínica, um caso de complexos ventriculares prematuros, possivelmente decorrente da parada cardiorrespiratória, que foi revertido com sucesso. Houve diminuição da amplitude dos complexos QRS nos primeiros 14 dias, retornado ao normal após 60 dias de pós-operatório.
The purpose for using lobectomy or pneumonectomy in dogs and cats, is to cure or palliate of bronchopulmonary diseases whenever conservative clinical treatment proves ineffective. Considering the marked changes caused by pneumonectomy, new experimental studies have to be done to assess the advantages of this surgical intervention allow its performance without any risk. The aim of the current study was to evaluate the electrocardiographic alterations in ten adult mongrel dogs of both sexes with between 10 and 30 Kg that were submitted to right pneumonectomy. The clinical parameters of the dogs were evaluated in a daily basis and the alterations in every electrocardiogram derivation were recorded. All dogs presented a good post-operative outcome. In most cases there were no electrocardiographic alterations; when these alterations were observed they were of no clinical significance and included premature ventricular complexes in one dog, most likely resulting from a cardiorespiratory arrest that was reverted to successfully, and a decreased width in the QRS complex amplitude on the first 0-14 days post surgery which returned to normal after sixty days post surgery.
Assuntos
Animais , Cães , Eletrocardiografia/métodos , Parada Cardíaca , Pneumonectomia/métodosRESUMO
Moléstias de natureza infecciosa, traumática ou neoplásica acometem o pulmão dos cães, constituindo-se a pneumonectomia como possibilidade de tratamento para algumas dessas afecções. Assim, diante da escassez de dados encontrados na literatura, objetivou-se avaliar e comparar parâmetros hemogasométricos, pressão parcial de oxigênio (PaO2), de dióxido de carbono (PaCO2), concentração do íon hidrogênio (pH) e do íon bicarbonato [HCO3-] no sangue arterial de cães adultos, antes e após pneumonectomia esquerda. Foram estudados 18 cães, machos e fêmeas de idades variadas, pesando entre 15 e 20 kg, distribuídos aleatoriamente em dois grupos de 9 animais cada. No Grupo A, o coto brôn-quico esquerdo dos cães foi suturado manualmente com fio de polipropileno 5-0, e no Grupo B, o coto brônquico esquerdo foi suturado mecanicamente com grampeador cirúrgico. Os dados foram colhidos em 6 momentos: antes da administração da medicação pré-anestésica (T0), 1 hora após a extubação do animal (T1EXT), 48 horas após a intervenção cirúrgica (T48h), 17 (T7d), 15 (T15d) e 36 dias (T36d) após intervenção cirúrgica. Em seguida, foi realizada a análise estatística (teste de normalidade de Anderson-Darling, Wilcox e teste U de Man-Whitney). Os valores de PaO2 do Grupo A no T1EXT (67,00±11,31) mostraram-se significativamente inferiores em relação ao T0 (99,44±18,34), fato este que não ocorreu no Grupo B: T1EXT (87,00±8,35) em relação ao T0 (87,00±7,55). Não houve diferença do pH entre os momentos nos cães do grupo A, porém no grupo B observou-se uma diminuição em T1EXT (7,3644±0,0353) em relação ao T0 (7,4189±0,0136), sem que os animais tenham desenvolvido acidose. Apesar dessas alterações, concluiu-se que os cães submetidos à pneumonectomia esquerda (sutura manual ou mecânica do coto brônquico esquerdo) não apresentaram tendência a desenvolver desequilíbrio ácido-básico no período em que foram avaliados.
It is well known that different diseases of infectious, traumatic or neoplasic origin can occur in the lungs of dogs, and pneumonectomy technique may be an option for the treatment of some of these diseases. The objective was to evaluate hemogasometric parameters, oxygen partial pressure (PaO2), carbon dioxide partial pressure (PaCO2), hydrogen ion concentration (pH) and bicarbonate ion concentration on the pre- and post-operative moments of the left pneumonectomy. Eighteen adult mongrel dogs, males and females, were randomly distributed into two groups with 9 dogs each. In Group A, the left bronchial stump of the dogs was sutured manually with polypropylene 5-0, and, in Group B, the left bronchial stump of the dogs was sutured mechanically with a surgical stapler. The data were collected at 6 moments: Before the pre-anesthetic administration (T0), one hour after the extubation (T1EXT), 48 hours after the surgery (T48h), 7 days after the surgery (T7d), 15 days after the surgery (T15d), and 36 days after the surgery (T36d). The results were statistically analyzed. PaO2 values of Group A on T1EXT (67.00±11.31) were significantly lower in relation to T0 (99.4±18.34), a fact that did not happen in Group B: T1EXT (87.00±8.35) in regard to T0 (87.00±7.55). There was no difference on pH values in dogs of Group A, but in Group B was observed a decrease on T1EXT (7.3644±0.0353) in relation to T0 (7.4189±0.0136), although the animals did not develop acidosis. It was concluded that dogs submitted to left pneumonectomy (sutured manually or sutured mechanically of left bronchial) did not show immediate and mediate acid-basic instability during the study.
Assuntos
Animais , Masculino , Feminino , Cães , Pneumonectomia/métodos , Pneumonectomia/veterinária , Pulmão/patologiaRESUMO
Plant and insect trypanosomatids constitute the "lower trypanosomatids", which have been used routinely as laboratory models for biochemical and molecular studies because they are easily cultured under axenic conditions, and they contain homologues of virulence factors from the classic human trypanosomatid pathogens. Among the molecular factors that contribute to Leishmania spp. virulence and pathogenesis, the major surface protease, alternatively called MSP, PSP, leishmanolysin, EC 3.4.24.36 and gp63, is the most abundant surface protein of Leishmania promastigotes. A myriad of functions have been described for the gp63 from Leishmania spp. when the metacyclic promastigote is inside the mammalian host. However, less is known about the functions performed by this molecule in the invertebrate vector. Intriguingly, gp63 is predominantly expressed in the insect stage of Leishmania, and in all insect and plant trypanosomatids examined so far. The gp63 homologues found in lower trypanosomatids seem to play essential roles in the nutrition as well as in the interaction with the insect epithelial cells. Since excellent reviews were produced in the last decade regarding the roles played by proteases in the vertebrate hosts, we focused in the recent developments in our understanding of the biochemistry and cell biology of gp63-like proteins in lower trypanosomatids.
Tripanossomatídeos de insetos e de plantas são informalmente denominados de "tripanossomatídeos inferiores". Estes microrganismos são utilizados rotineiramente como modelos para estudos de bioquímica e de biologia molecular porque são facilmente cultivados sob condições axênicas e porque possuem homólogos aos fatores de virulência encontrados nos tripanossomatídeos que são patógenos humanos clássicos. Dentre os fatores moleculares que contribuem para a virulência e patogênese de Leishmania spp. destaca-se a principal protease de superfície, também conhecida como MSP, PSP, leishmanolisina, EC 3.4.24.36 e gp63, que é a proteína de superfície mais abundante encontrada nas formas promastigotas de Leishmania. Diversas funções foram descritas para a gp63 de Leishmania no hospedeiro vertebrado. Entretanto, pouco é conhecido sobre as funções desempenhadas por essa molécula no inseto vetor. Curiosamente, a gp63 é predominantemente expressa na forma evolutiva de Leishmania encontrada no inseto, e em todos os tripanossomatídeos de insetos e plantas analisados até o presente momento. Os homólogos da gp63 presentes nos tripanossomatídeos inferiores desempenham um papel essencial na nutrição assim como na interação com as células epiteliais do inseto. Uma vez que revisões de excelente qualidade foram produzidas na última década sobre a função de proteases nos hospedeiros vertebrados, nesta revisão nós abordamos os recentes progressos sobre os aspectos bioquímicos e as prováveis funções biológicas desempenhadas pelas proteínas homólogas à gp63 nos tripanossomatídeos inferiores.