RESUMEN
Sjögren's Syndrome (SS) is an autoimmune exocrinopathy characterized by the progressive damage of salivary and lacrimal glands associated with lymphocytic infiltration. Identifying new non-invasive biomarkers for SS diagnosis remains a challenge, and alterations in saliva composition reported in patients turn this fluid into a source of potential biomarkers. Among these, proteases are promising candidates since they are involved in several key physio-pathological processes. This study evaluated differentially expressed proteases in SS individuals' saliva using synthetic fluorogenic substrates, zymography, ELISA, and proteomic approaches. Here we reported, for the first time, increased activity of the serine protease dipeptidyl peptidase-4/CD26 (DPP4/CD26) in pSS saliva, the expression level of which was corroborated by ELISA assay. Gelatin zymograms showed that metalloproteinase proteolytic band profiles differed significantly in intensity between control and SS groups. Focusing on matrix metalloproteinase-9 (MMP9) expression, an increased tendency in pSS saliva (p = 0.0527) was observed compared to the control group. Samples of control, pSS, and sSS were analyzed by mass spectrometry to reveal a general panorama of proteases in saliva. Forty-eight protein groups of proteases were identified, among which were the serine proteases cathepsin G (CTSG), neutrophil elastase (ELANE), myeloblastin (PRTN3), MMP9 and several protease inhibitors. This work paves the way for proteases to be explored in the future as biomarkers, emphasizing DPP4 by its association in several autoimmune and inflammatory diseases. Besides its proteolytic role, DPP4/CD26 acts as a cell surface receptor, signal transduction mediator, adhesion and costimulatory protein involved in T lymphocytes activation.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Péptido Hidrolasas/análisis , Proteómica/métodos , Saliva/metabolismo , Síndrome de Sjögren/metabolismo , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Catepsina G , Femenino , Humanos , Elastasa de Leucocito , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Serina Endopeptidasas , Transducción de Señal , Síndrome de Sjögren/diagnósticoRESUMEN
Keratinases present promising biotechnological applications, due to their ability to degrade keratin. Streptomyces appears as one of the main sources of these enzymes, but complete genome sequences of keratinolytic bacteria are still limited. This article reports the complete genomes of three marine-derived streptomycetes that show different levels of feather keratin degradation, with high (strain G11C), low (strain CHD11), and no (strain Vc74B-19) keratinolytic activity. A multi-step bioinformatics approach is described to explore genes encoding putative keratinases in these genomes. Despite their differential keratinolytic activity, multiplatform annotation reveals similar quantities of ORFs encoding putative proteases in strains G11C, CHD11, and Vc74B-19. Comparative genomics classified these putative proteases into 140 orthologous groups and 17 unassigned orthogroup peptidases belonging to strain G11C. Similarity network analysis revealed three network communities of putative peptidases related to known keratinases of the peptidase families S01, S08, and M04. When combined with the prediction of cellular localization and phylogenetic reconstruction, seven putative keratinases from the highly keratinolytic strain Streptomyces sp. G11C are identified. To our knowledge, this is the first multi-step bioinformatics analysis that complements comparative genomics with phylogeny and cellular localization prediction, for the prediction of genes encoding putative keratinases in streptomycetes.
Asunto(s)
Organismos Acuáticos/química , Organismos Acuáticos/genética , Biología Computacional/métodos , Péptido Hidrolasas/análisis , Péptido Hidrolasas/genética , Streptomyces/química , Streptomyces/genética , Organismos Acuáticos/microbiología , Genómica , Filogenia , Streptomyces/aislamiento & purificación , Streptomyces/metabolismoRESUMEN
With the aim to widen the current knowledge of toxinological implications of bites from rear-fanged snakes and biological roles of their venoms, this study focuses on the biochemical composition and toxic effects of the venom of Leptodeira annulata pulchriceps from Argentina. We analyzed the protein composition by electrophoresis and mass spectrometry, and enzymatic properties by quantitative assays on different substrates. Additionally, we evaluated local and systemic toxicity in mice, and tested its cross-reactivity with elapid and viperid antivenoms used in Argentina. This venom showed features reminiscent of venoms from snakes of Bothrops genus, containing components ranging from ~17 to 75â¯kDa, which are mainly tissue-damaging toxins such as proteinases. Although showing low lethality to mice (LD50â¯=â¯20⯵g/g body weight), prominent hemorrhage developed locally in mice intramuscularly and intradermally injected with the venom, and the minimum hemorrhagic dose was found to be 12.7⯵g/mouse. This study is the first comprehensive investigation of the venom of L. a. pulchriceps, and sheds new light on differences between this and those of the other two subspecies of L. annulata. Additionally, the study provides new insights into the venom components of "colubrid" snakes, advocating for considering bites from this rich diversity of snakes as a public health problem that needs to be addressed worldwide.
Asunto(s)
Colubridae/metabolismo , Venenos de Serpiente , Animales , Argentina , Masculino , Ratones , Péptido Hidrolasas/análisis , Venenos de Serpiente/química , Venenos de Serpiente/toxicidadRESUMEN
Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.(AU)
Asunto(s)
Pleurotus/química , Agentes de Coagulación , Péptido Hidrolasas/análisis , Queso/análisisRESUMEN
Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.
Asunto(s)
Agentes de Coagulación , Péptido Hidrolasas/análisis , Pleurotus/química , Queso/análisisRESUMEN
Abstract Bacteriocin has been identified as an excellent alternative to chemical preservatives due to its astonishing antimicrobial activity against food spoiling and food-borne pathogens. So there is a need to identify the newer and potent sources of bacteriocin producers. This study aims the isolation of potent bacteriocin producing microorganism from fresh fruits and vegetables, its production, purification, and characterization. Firstly, 43 isolates were analysed for its antimicrobial potential, out of which7 were found to inhibit the growth of various pathogens. Considering the results of antimicrobial activity; the microorganism isolated from mango was regarded as the most potent one; which was identified as Bacillus subtilis VS.70% ammonium sulphate precipitated and dialysed bacteriocin was purified using DEAE cellulose and sephadex G75 chromatography. Bacteriocin was purified by 24.64 fold with 8.65% recovery and its molecular weight was found to be 31.2kDa. The Purified bacteriocin was found to be stable at broad pH and temperature. It was found to be degraded by various proteases studied confirming its proteinaceous nature. Considering all these attributes; the purified bacteriocin isolated from Bacillus subtilis VS can be exploited by various food industries.
Asunto(s)
Péptido Hidrolasas/análisis , Bacteriocinas/análisis , Antiinfecciosos/análisis , Bacillus subtilis , CromatografíaRESUMEN
Cocoa contains bioactive components, which vary according to genetic and environmental factors. The present study aimed to ascertain the anti-allergic properties of native Peruvian cocoa populations ("Blanco de Piura" or BPC, "Amazonas Peru" or APC, "Criollo de Montaña" or CMC, "Chuncho" or CCC, and an ordinary cocoa or OC). To do so, after an initial in vitro approach, an in vivo study focused on the induction of an anaphylactic response associated with allergic asthma in Brown Norway rats was carried out. Based on their polyphenol content, antioxidant activity and in vitro effects, the APC and CMC were selected to be included in the in vivo study. Cocoa diets were tested in a model of allergic asthma in which anaphylactic response was assessed by changes in body temperature, motor activity and body weight. The concentration of specific immunoglobulin E (IgE), mast cell protease and leukotrienes was also quantified in serum and/or bronchoalveolar lavage fluid. CMC and OC populations exhibited a protective effect on the allergic asthma rat model as evidenced by means of a partial protection against anaphylactic response and, above all, in the synthesis of IgE and the release of mast cell protease.
Asunto(s)
Antioxidantes/farmacología , Asma/prevención & control , Cacao/química , Polifenoles/farmacología , Sustancias Protectoras/farmacología , Anafilaxia/inducido químicamente , Anafilaxia/prevención & control , Animales , Asma/inducido químicamente , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/química , Dieta/métodos , Modelos Animales de Enfermedad , Hipersensibilidad/prevención & control , Inmunoglobulina E/análisis , Leucotrienos/análisis , Actividad Motora/efectos de los fármacos , Péptido Hidrolasas/análisis , Perú , RatasRESUMEN
This study aimed to determine the frequency of Pseudomonas fluorescens, P. putida, and P. aeruginosa in refrigerated raw milk; their proteolytic potential; and your association with the aprX gene. Of the 173 isolates confirmed as belonging to Pseudomonas spp., 37% were P. fluorescens, 25.4% P. putida, and none belongs to P. aeruginosa. The aprX gene was distributed proportionally between P. putida (68%) and P. fluorescens (75%), but it was not associated with low or high proteolytic potential in both species. P. putida (16) and P. fluorescens (14) isolates with no aprX gene identified also had proteolytic potential. Considering the synthesis of proteases other than AprX by the isolates under study, we concluded that P. fluorescens and P. putida represented 62.4% of the Pseudomonas genus, with high probability of having the aprX gene and proteolytic potential. However, there was no association between the deteriorating potential with the presence of the aprX gene.(AU)
O objetivo do estudo foi verificar a frequência, em leite cru refrigerado de Pseudomonas fluorescens, P. putida e P. aeruginosa, o potencial proteolítico dos isolados e sua associação com a presença do gene aprX. Dos 173 isolados confirmados como pertencente ao gênero Pseudomonas spp, 37% foram P. fluorescens, 25,4% P. putida e nenhum pertencia à espécie P. aeruginosa. A prevalência do gene aprX se distribuiu proporcionalmente entre P.putida (68%) e P.fluorescens (75%) porém não foi associada ao baixo ou alto potencial proteolítico nas duas espécies avaliadas. P. putida (16) e P. fluorescens (14) que não tiveram o gene aprX identificado também expressaram potencial proteolítico, considerando-se a síntese de outras proteases além da AprX pelos isolados estudados. Concluiu-se que Pseudomonas fluorescens e P. putida representaram 62,4 % do gênero Pseudomonas ssp., com alta probabilidade da presença do gene aprX e potencial proteolítico, porém sem associação da expressão do potencial deteriorante com a presença do gene aprX.(AU)
Asunto(s)
Animales , Femenino , Péptido Hidrolasas/análisis , Pseudomonas/aislamiento & purificación , Leche/microbiología , Ascorbato Peroxidasas/genéticaRESUMEN
The Central American locust, Schistocerca piceifrons piceifrons (Walker) is a major agricultural pest in Mexico and Central America. Control measures against this pest have generated much environmental damage and substantial financial costs because chemical insecticides are used. Yet various Orthoptera species also appear to be a potential source of nutrients and a source of bioactive metabolites. Here, we studied the presence of secondary metabolites in the adult stage of S. p. piceifrons by applying different colorimetric techniques. Adults were collected from the southern region of Tamaulipas, Mexico, during September-December 2017. These samples were subjected to sequential processes of eviscerating, drying, pulverizing, extracting, and detecting of metabolites. Extractions were carried out in water, 50% ethanol, and absolute ethanol. The presence of phenolic compounds, alkaloids, tannins, saponins, flavonoids, and quantity of antioxidants against the DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2'-azino-bis, 3-ethylbenzothiazoline-6-sulfonic acid) radicals were determined and reported. Proximate analysis showed that S. p. piceifrons has a high protein content (80.26%), low fat content (6.21%), and fiber content (12.56%) similar to other Orthoptera species. Chitin and chitosan contents of S. p. piceifrons were 11.88 and 9.11%, respectively; the recovery percentage of chitosan from chitin was 76.71%. Among the Orthoptera, the protein content of this pest is among the highest while its contents of chitin and chitosan are similar to those of other insect species (e.g., Bombix mori Linnaeus [Lepidoptera: Bombycidae]). Our results suggest this pest species is a potential source of bioactive compounds of biotechnological interest for use by pharmaceutical and food industries.
Asunto(s)
Saltamontes/química , Animales , Antioxidantes/análisis , Quitina/análisis , Quitosano/análisis , Proteínas de Insectos/análisis , México , Péptido Hidrolasas/análisis , Fenoles/análisisRESUMEN
Plant cell walls mostly comprise polysaccharides and proteins. The composition of monocots' primary cell walls differs from that of dicots walls with respect to the type of hemicelluloses, the reduction of pectin abundance and the presence of aromatic molecules. Cell wall proteins (CWPs) differ among plant species, and their distribution within functional classes varies according to cell types, organs, developmental stages and/or environmental conditions. In this review, we go deeper into the findings of cell wall proteomics in monocot species and make a comparative analysis of the CWPs identified, considering their predicted functions, the organs analyzed, the plant developmental stage and their possible use as targets for biofuel production. Arabidopsis thaliana CWPs were considered as a reference to allow comparisons among different monocots, i.e., Brachypodium distachyon, Saccharum spp. and Oryza sativa. Altogether, 1159 CWPs have been acknowledged, and specificities and similarities are discussed. In particular, a search for A. thaliana homologs of CWPs identified so far in monocots allows the definition of monocot CWPs characteristics. Finally, the analysis of monocot CWPs appears to be a powerful tool for identifying candidate proteins of interest for tailoring cell walls to increase biomass yield of transformation for second-generation biofuels production.
Asunto(s)
Brachypodium/metabolismo , Pared Celular/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Saccharum/metabolismo , Brachypodium/química , Metabolismo de los Hidratos de Carbono , Pared Celular/química , Metabolismo de los Lípidos , Oryza/química , Oxidorreductasas/análisis , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Péptido Hidrolasas/análisis , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Proteómica , Saccharum/química , Transducción de SeñalRESUMEN
Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.
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Antifúngicos/farmacología , Biopelículas/efectos de los fármacos , Farnesol/farmacología , Trichosporon/efectos de los fármacos , Trichosporon/crecimiento & desarrollo , Adhesión Celular/efectos de los fármacos , Humanos , Metabolismo/efectos de los fármacos , Péptido Hidrolasas/análisis , Trichosporon/aislamiento & purificación , Trichosporon/metabolismo , Tricosporonosis/microbiologíaRESUMEN
This study aimed to evaluate the effect of different reductions in digestible amino acids content (lysine, methionine, and threonine), according to two nutritional requirements in corn, soybean meal, and meat and bone meal based diets, with protease supplementation, on performance parameters and carcass characteristics. A total of 1080 day-old chicks, male, Cobb 500, were allotted to a completely randomized design, in a factorial arrangement 3 x 2, three reductions in digestible amino acids content (lysine, methionine, and threonine) and two nutritional requirements (Rostagno et al. 2005 and Cobb-Vantress Guidelines 2008), and all diets were supplemented with protease (200 ppm) with 6 replicates of 30 birds per pen. There was no significant interaction (p 0.05) between digestible amino acid reductions and both nutritional requirements for the performance variables and carcass yield and cuts. There was an effect of amino acid reduction and protease supplementation only on slaughter weight (p 0.05). Broilers fed according to the nutritional requirements of Rostagno et al. (2005) showed better (p 0.05) performance when compared to broilers fed as specified by the nutritional requirements of Cobb-Vantress (2008) with no significant differences in carcass characteristics. Protease supplementation of corn, soybean meal, and meat and bone meal based diets allows a reduction in the inclusion of crystalline amino acids (lysine, methionine, and threonine).(AU)
Asunto(s)
Animales , Péptido Hidrolasas/análisis , Pollos/fisiología , Alimentación Animal/análisis , Alimentación AnimalRESUMEN
This study aimed to evaluate the effect of different reductions in digestible amino acids content (lysine, methionine, and threonine), according to two nutritional requirements in corn, soybean meal, and meat and bone meal based diets, with protease supplementation, on performance parameters and carcass characteristics. A total of 1080 day-old chicks, male, Cobb 500, were allotted to a completely randomized design, in a factorial arrangement 3 x 2, three reductions in digestible amino acids content (lysine, methionine, and threonine) and two nutritional requirements (Rostagno et al. 2005 and Cobb-Vantress Guidelines 2008), and all diets were supplemented with protease (200 ppm) with 6 replicates of 30 birds per pen. There was no significant interaction (p 0.05) between digestible amino acid reductions and both nutritional requirements for the performance variables and carcass yield and cuts. There was an effect of amino acid reduction and protease supplementation only on slaughter weight (p 0.05). Broilers fed according to the nutritional requirements of Rostagno et al. (2005) showed better (p 0.05) performance when compared to broilers fed as specified by the nutritional requirements of Cobb-Vantress (2008) with no significant differences in carcass characteristics. Protease supplementation of corn, soybean meal, and meat and bone meal based diets allows a reduction in the inclusion of crystalline amino acids (lysine, methionine, and threonine).
Asunto(s)
Animales , Pollos/fisiología , Péptido Hidrolasas/análisis , Alimentación Animal , Alimentación Animal/análisisRESUMEN
Abstract Introduction Candida albicans is the yeast most commonly affecting the oral cavity, sometimes causing infection. However, several factors may be associated with the onset of candidiasis, which may be related not only to the hygiene and health of individuals, but also to the pathogenicity of these microorganisms. Objective To evaluate the virulence factors of Candida yeasts isolated from the oral mucosa of elderly people living in the "Comunidade Lago do Limão", municipality of Iranduba, Amazonas state, Brazil. Material and method Testes were performed to assess the production of urease, proteinase, phospholipase and hemolysin. Statistical analysis used the Fisher's exact test and the Chi-squared test. Result Prevalence of non-albicans species was observed. As for virulence factors, all isolates were negative ureases, and there was prevalence of very strong proteinase production, whereas most isolates did not produce this enzyme in the phospholipase test. All yeasts analyzed presented hemolysin production, with grade IV hemolysis as the most prevalent. There was no statistically significant difference between the virulence of isolates from the oral cavity and the prostheses of the elderly analyzed. Conclusion Several virulence factors may present with high intensity in the presence of oral microbiota changes. In addition, non-albicans species present number of virulence factors similar to that of C. albicans, with high pathogenicity. This study allows a better analysis of candidiasis prevention strategies aiming to promote improvement in the health and quality of life for the elderly.
Resumo Introdução A Candida albicans é a levedura que mais acomete a cavidade oral, podendo causar infecção. Porém diversos fatores podem estar associados ao aparecimento da candidíase, que podem estar relacionados com a higiene e saúde dos indivíduos, mas também com a patogenicidade destes microrganismos. Objetivo Avaliar os fatores de virulência de leveduras do gênero Candida isoladas da mucosa oral dos idosos residentes na Comunidade Lago do Limão - Iranduba - Amazonas - Brasil. Material e método Foram realizados os testes de urease, proteinase, fosfolipase, e avaliação da produção de hemólise. Na análise estatística utilizou-se teste Exato de Fisher e Quiquadrado. Resultado Obteve-se a prevalência de espécies não-albicans, quanto aos fatores de virulência, todos os isolados foram ureases negativos, houve prevalência de produção muito forte de proteinase, enquanto que no teste da fosfolipase, a maioria dos isolados não apresentou produção desta enzima; todas as leveduras analisadas apresentaram produção de hemolisina, sendo mais prevalente a hemólise grau IV. Não houve diferença estatisticamente significativa entre a virulência dos isolados oriundos da cavidade oral e da prótese dos idosos analisados. Conclusão Diversos fatores de virulência podem apresentar-se com alta intensidade na presença de alterações da microbiota oral. Além disso, as espécies não-albicans apresentam fatores de virulência tanto quanto a C. albicans, com graus de patogenicidade elevados. Este estudo permite a análise de estratégias de prevenção da candidíase, com intuito de promover melhor saúde e qualidade de vida para os idosos.
Asunto(s)
Humanos , Anciano , Candida , Infecciones Relacionadas con Prótesis , Factores de Virulencia , Mucosa Bucal/fisiopatología , Péptido Hidrolasas/análisis , Brasil , Candida albicans , Grupos de PoblaciónRESUMEN
BACKGROUND: Angiostrongylus costaricensis is a relatively uncharacterized nematode that causes abdominal angiostrongyliasis in Latin America, a human parasitic disease. Currently, no effective pharmacological treatment for angiostrongyliasis exists. Peptidases are known to be druggable targets for a variety of diseases and are essential for several biological processes in parasites. Therefore, this study aimed to systematically characterize the peptidase activity of A. costaricensis in different developmental stages of this parasitic nematode. METHODOLOGY/PRINCIPAL FINDINGS: A library of diverse tetradecapeptides was incubated with cellular lysates from adult worms and from first-stage larvae (L1) and cleaved peptide products were identified by mass spectrometry. Lysates were also treated with class specific peptidase inhibitors to determine which enzyme class was responsible for the proteolytic activity. Peptidase activity from the four major mechanistic classes (aspartic, metallo, serine and cysteine) were detected in adult worm lysate, whereas aspartic, metallo and serine-peptidases were found in the larval lysates. In addition, the substrate specificity profile was found to vary at different pH values. CONCLUSIONS/SIGNIFICANCE: The proteolytic activities in adult worm and L1 lysates were characterized using a highly diversified library of peptide substrates and the activity was validated using a selection of fluorescent substrates. Taken together, peptidase signatures for different developmental stages of this parasite has improved our understanding of the disease pathogenesis and may be useful as potential drug targets or vaccine candidates.
Asunto(s)
Angiostrongylus/enzimología , Péptido Hidrolasas/análisis , Animales , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Larva/enzimología , Péptido Hidrolasas/química , Proteolisis , Especificidad por SustratoRESUMEN
To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.
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Proteínas de Artrópodos/análisis , Venenos de Escorpión/química , Animales , Bloqueadores de los Canales de Calcio/análisis , Bloqueadores de los Canales de Calcio/química , Femenino , Perfilación de la Expresión Génica , Hialuronoglucosaminidasa/análisis , Hialuronoglucosaminidasa/química , Masculino , Péptido Hidrolasas/análisis , Péptido Hidrolasas/química , Péptidos/análisis , Péptidos/química , Fosfolipasas A2/análisis , Fosfolipasas A2/química , Bloqueadores de los Canales de Potasio/análisis , Bloqueadores de los Canales de Potasio/química , Proteoma , Proteómica , Escorpiones , Bloqueadores de los Canales de Sodio/análisis , Bloqueadores de los Canales de Sodio/químicaRESUMEN
Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.
Asunto(s)
Interacciones Microbiota-Huesped , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma/metabolismo , Neumonía Porcina por Mycoplasma/microbiología , Proteoma/metabolismo , Adhesinas Bacterianas/análisis , Animales , Proteínas Bacterianas/análisis , Espectrometría de Masas , Mycoplasma hyopneumoniae/patogenicidad , Péptido Hidrolasas/análisis , Especificidad de la Especie , Porcinos , Factores de VirulenciaRESUMEN
The effects of increasing doses of three exogenous enzymes preparations with fibrolytic activity (FIB- 0, 0.6, 1.2, 1.8, and 2.4 mg mL-¹liquid volume incubated), amylolytic activity (AMZ - 0, 0.05, 0.10,0.15, and 0.20 mg mL-¹liquid volume incubated), and proteolytic activity (PRO - 0, 0.05, 0.10, 0.15, and 0.20 mg mL-¹ liquid volume incubated ) on gas production (GP), kinetic parameters, and fermentation profile of Brachiaria brizantha cv. Marandu were evaluated using the in vitro gas production technique. Ruminal liquid was obtained from two rumen-cannulated Santa Inês sheep maintained on pasture. Accumulated gas production was measured during 96 hours of incubation, measured at 18 different time points. The determined parameters were pH, asymptotic gas production (mL g-¹), rate of gas production (h-¹), lag time (h), organic matter digestibility (OMD, g g-¹ DM), metabolizable energy (ME, MJ kg-¹DM), and neutral detergent fiber digestibility (NDFD, mg g-¹ DM). Increasing the FIB dose linearly increased (P < 0.05) the asymptotic gas production. However, the rate of gas production and the lagtime showed linear decreases (P < 0.05). Addition of FIB also linearly increased (P < 0.05) the GP at all incubation times, as well as the OMD, NDFD and ME. Addition of AMZ linearly increased (P <0.05) the asymptotic gas production, but GP linear increased (P < 0.05) only at the 6-hour and 12-hour time points. The rate of gas production and the lag time decreased linearly (P < 0.05) in response to increasing AMZ addition. Inclusion of PRO did not affect (P > 0.05) asymptotic gas production, but there was quadratic effect (P < 0.05) on the rate of gas production, the lag time, and the GP at the 6-hour and 12-hour time points. The OMD, NDFD and ME were not affected by PRO addition. Thus, fibrolytic, amylolytic and proteolytic enzymes are effective in reducing the lag time and increasing thein vitro gas production from Brachiaria Brizantha cv.[...](AU)
Avaliou-se os efeitos de doses crescentes de três enzimas exógenas: fibrolíticas (FIB), 0; 0,6; 1,2; 1,8 e 2,4 mg mL-¹ volume de líquido incubado; amilolíticas (AMZ), 0; 0,05; 0,10; 0,15 e 0,20 mg mL-¹ volume de líquido incubado, proteolíticas (PRO), 0; 0,05; 0,10; 0,15 e 0,20 mg mL-¹ volume de líquido incubado, sobre a produção de gases (PG), parâmetros cinéticos e perfil da fermentação da Brachiaria brizantha cv. Marandu usando a técnica in vitro de produção de gases. O líquido ruminal foi obtido de dois ovinos Santa Inês canulados no rúmen, mantidos em regime de pastejo. A produção de gases acumulada foi obtida durante 96h00 de incubação, mensurada em 18 tempos. Ao final da incubação foram determinados o pH, produção de gás assintótica (mL g-¹), taxa de degradação fracional (h-¹), lag time (h), digestibilidade da matéria orgânica (DMO, g/g MS incubada por 24h00), energia metabolizável (EM, MJ kg-¹ MS) e a digestibilidade da fibra insolúvel em detergente neutro (DFDN, mg g-¹ MS). As doses de FIB aumentaram linearmente (P < 0,05) a produção de gases assintótica. Entretanto, a taxa de degradação fracional e lag time reduziram linearmente (P < 0,05). A adição de FIB também aumentou linearmente (P < 0,05) a PG em todos os tempos de incubação, DMO, DFDN e EM. A inclusão de AMZ aumentou linearmente (P < 0,05) a produção de gases assintótica, e a PG somente nos tempos 6 e 12 horas após a incubação. A taxa de degradação fracional e lag time reduziu linearmente (P < 0,05) com a inclusão de AMZ. A inclusão de PRO não modificou (P > 0,05) a produção de gás assintótica, mas promoveu efeito quadrático (P < 0,05) na taxa de degradação fracional, lag time, e PG nos tempos 6 e 12. No entanto, PRO não afetou DMO, DFDN e EM. As enzimas fibrolíticas, amilolíticas e proteolíticas são efetivas em reduzir o lag time e aumentar a produção de gases in vitro da Brachiaria Brizantha cv.[...](AU)
Asunto(s)
Animales , Ovinos/fisiología , Péptido Hidrolasas/análisis , Fermentación , Pruebas de Enzimas/veterinaria , RumiantesRESUMEN
The aim was to evaluate the microbiological, chemical- physical, and shelf-life quality of milk samples after pasteurization (HTST) for 10 days or ultra-high temperature (UHT) treatment for 120 days. Raw milk counts of mesophilic aerobic microorganisms, Staphylococcus spp. and thermotolerant coliforms before HTST and UHT processing were 6.73 and 7.77; 2.84 and 4.30, and 4.68 and 4.37log10, respectively. Pseudomonas spp. were found in raw milk samples. No presence of any other microorganisms studied was detected and no microbial inhibitor was found. Processed samples met microbiological legal requirements. However, aerobic mesophilic counts for HTST pasteurized milk samples stored for 5 and 10 days increased to values comparable to those in raw milk. Composition chemical- physical of all samples were within legal limits. These results demonstrate that, although HTST and UHT processed milk comply with the microbiological standards required by Brazilian law, high microbial counts in raw milk are an issue, possibly due to failures in the early stages of the production chain. Increase in casein macropeptide (CMP), probably because of proteases psychrotrophic bacteria. It is concluded that the quality of raw milk directly influences the progressive increase of the CMP values.(AU)
O objetivo da presente pesquisa foi avaliar a qualidade microbiológica, fisco-química e a vida de prateleira de amostras de leite, após o processo de pasteurização rápida (HTST) ou de ultra-alta temperatura (UHT) durante 10 dias, ou de ultra-alta temperatura (UHT) por 120 dias. As contagens de micro-organismos aeróbios mesófilos, Staphylococcus spp. e de coliformes termotolerantes do leite cru utilizado para tratamentos HTST e UHT foram, respectivamente (log10): 6,73 e 7,77; 2,84 e 4,30 e 4,68 e 4,37. Foi constatada a presença de Pseudomonas spp. no leite cru. Não foi detectada a presença de nenhum outro micro-organismo estudado, e as amostras estavam isentas de inibidores microbianos. Após a pasteurização, todas as amostras apresentaram contagens microbianas compatíveis com os limites legais. No entanto, as amostras de leite pasteurizado apresentaram contagens de aeróbios mesófilos semelhantes ao leite cru após cinco e 10 dias de armazenamento. A composição físico-química de todas as amostras estava de acordo com os limites legais. Observou-se acréscimo dos níveis de caseinomacropeptídeo (CMP) no leite UHT, provavelmente em função das proteases de bactérias psicrotróficas. Conclui-se que a qualidade do leite cru influencia diretamente os valores de CMP.(AU)
Asunto(s)
Leche/química , Leche/microbiología , Péptido Hidrolasas/análisis , Caseína Quinasas/análisisRESUMEN
The aim was to evaluate the microbiological, chemical- physical, and shelf-life quality of milk samples after pasteurization (HTST) for 10 days or ultra-high temperature (UHT) treatment for 120 days. Raw milk counts of mesophilic aerobic microorganisms, Staphylococcus spp. and thermotolerant coliforms before HTST and UHT processing were 6.73 and 7.77; 2.84 and 4.30, and 4.68 and 4.37log10, respectively. Pseudomonas spp. were found in raw milk samples. No presence of any other microorganisms studied was detected and no microbial inhibitor was found. Processed samples met microbiological legal requirements. However, aerobic mesophilic counts for HTST pasteurized milk samples stored for 5 and 10 days increased to values comparable to those in raw milk. Composition chemical- physical of all samples were within legal limits. These results demonstrate that, although HTST and UHT processed milk comply with the microbiological standards required by Brazilian law, high microbial counts in raw milk are an issue, possibly due to failures in the early stages of the production chain. Increase in casein macropeptide (CMP), probably because of proteases psychrotrophic bacteria. It is concluded that the quality of raw milk directly influences the progressive increase of the CMP values.(AU)
O objetivo da presente pesquisa foi avaliar a qualidade microbiológica, fisco-química e a vida de prateleira de amostras de leite, após o processo de pasteurização rápida (HTST) ou de ultra-alta temperatura (UHT) durante 10 dias, ou de ultra-alta temperatura (UHT) por 120 dias. As contagens de micro-organismos aeróbios mesófilos, Staphylococcus spp. e de coliformes termotolerantes do leite cru utilizado para tratamentos HTST e UHT foram, respectivamente (log10): 6,73 e 7,77; 2,84 e 4,30 e 4,68 e 4,37. Foi constatada a presença de Pseudomonas spp. no leite cru. Não foi detectada a presença de nenhum outro micro-organismo estudado, e as amostras estavam isentas de inibidores microbianos. Após a pasteurização, todas as amostras apresentaram contagens microbianas compatíveis com os limites legais. No entanto, as amostras de leite pasteurizado apresentaram contagens de aeróbios mesófilos semelhantes ao leite cru após cinco e 10 dias de armazenamento. A composição físico-química de todas as amostras estava de acordo com os limites legais. Observou-se acréscimo dos níveis de caseinomacropeptídeo (CMP) no leite UHT, provavelmente em função das proteases de bactérias psicrotróficas. Conclui-se que a qualidade do leite cru influencia diretamente os valores de CMP.(AU)