RESUMO
Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.
Assuntos
Peptídeo Hidrolases , Reishi , Fermentação , Inibidores de Proteases/farmacologia , Tripsina , Papaína , Quimotripsina , Peptonas , BiomassaRESUMO
Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.
Ganoderma lucidum é um cogumelo medicinal amplamente reconhecido como fonte de biomoléculas com propriedades farmacológicas, entretanto, pouco se conhece acerca dos fatores que influenciam a síntese de proteínas bioativas por esse fungo, quando cultivado sob fermentação submersa. O objetivo deste trabalho foi avaliar a produção de biomassa micelial e de proteases e inibidores de proteases intracelulares por G. lucidum cultivado em diferentes condições de fermentação submersa. O cultivo foi realizado em meio contendo glicose (10 ou 20 g.L-1), peptona de soja (2,5 ou 5 g.L-1) e extrato de levedura (5 g.L-1), com incubação sob agitação (120 rpm) ou não-agitado, totalizando 8 condições experimentais. A produção de biomassa foi determinada a partir do peso seco e o consumo de glicose a partir da quantificação de açúcares redutores. As proteínas foram extraídas em NaCl (0,15 M) e os extratos proteicos foram submetidos à quantificação de proteínas pelo método de Bradford, atividade proteolítica total usando azocaseína, atividade caseinolítica e fibrinolítica em placa de Petri, atividade de inibidores de serino-proteases (tripsina e quimotripsina) e cisteíno-protease (papaína). O cultivo em condição agitada apresentou maior produção de biomassa com valor máximo de 7g.L-1, além de maiores atividades de inibidores de tripsina, quimotripsina e papaína, com 154 UI.mg-1, 153 UI.mg-1 e 343 UI.mg-1 de proteína, respectivamente. A condição não-agitada demonstrou maior potencial para a obtenção de proteínas, proteases totais, enzimas caseinolíticas e fibrinolíticas, com valores máximos de 433 mg.g-1 de extrato, 71 U.mL-1 de extrato, 63,62 mm2 e 50,27 mm2, respectivamente. Assim, recomenda-se o meio composto de peptona de soja, extrato de levedura e glicose na proporção 1:2:4, em condição agitada para a produção de inibidores de proteases, e a condição não-agitada para a síntese de proteases, principalmente enzimas caseinolíticas e fibrinolíticas.
Assuntos
Peptídeo Hidrolases , Plantas Medicinais , Reishi , FermentaçãoRESUMO
Salmonella Enteritidis and Salmonella Typhimurium are responsible for causing huge economic loses in aviculture, as they lead young broiler chicks to develop clinical disease and thus increase mortality. Salmonella's pathogenicity is considered complex and multifactorial, demanding more studies that could elucidate the interaction between host and pathogen. The present study aims to evaluate the virulence of 130S. Enteritidis isolates and 70S. Typhimurium inoculated in one-day-old chicks through the establishment of a pathogenicity index. For each strain, 10 commercial chicks from the Cobb lineage were used. Then, 200µL of a solution containing 2x108 CFU of S. Enteritidis or S. Typhimurium were inoculated in the birds by intraperitoneal via. Mortality and presence of lesions such as aerosaculitis (A), perihepatitis (Ph), pericarditis (Pc), peritonitis (Pt), onfalitis (O) and cellulitis (C) were registered daily for seven days. From the second to the seventh day there was a proportional decrease in the punctuation of the time of death (TD) for each day that the bird had survived. The pathogenicity index was calculated using the following formula: PI = (TD x 5) + A + Ph + Pc + Pt + O + C. The obtainment of the PI of each bacterial sample was achieved by calculating the rate of the ten inoculated birds. Based on the obtained results, it was possible to attribute the pathogenicity value for each strain, which enabled us to classify them in groups of low (27/200), intermediate (95/200) and high (78/200) pathogenicity. The utilization of standards like time of death and presence of septicemic lesions made it possible to determine the pathogenicity rate for each strain. Besides that, the proposed model has presented dramatic differences between the high, intermediate and low pathogenicity groups, which makes this mechanism useful for further classification of strains isolated in poultry farms.
Salmonella Enteritidis e Salmonella Typhimurium são responsáveis por imensos prejuízos econômicos ao setor avícola, podendo levar ao desenvolvimento de doença clínica e ao aumento da mortalidade em aves jovens. A patogenicidade de Salmonella é considerada complexa e multifatorial, necessitando de estudos que possam esclarecer a interação entre patógeno e hospedeiro. O presente trabalho teve por objetivo avaliar a virulência de 130 isolados de S. Enteritidis e 70 de S.Typhimurium, inoculadas em pintos de um dia de idade, por meio do estabelecimento de um índice de patogenicidade. Para cada cepa, foram utilizados 10 pintos comerciais da linhagem Cobb. As aves foram inoculadas com 200µL de uma solução contendo 2x108 UFC de S. Enteritidis ou S. Typhimurium, por via intraperitoneal. A mortalidade e a presença de lesões como aerossaculite (A), peri-hepatite (Ph), pericardite (Pc), peritonite (Pt), onfalite (O) e celulite (C) foram registradas diariamente durante sete dias. Do segundo ao sétimo dia, houve uma diminuição proporcional da pontuação no tempo de morte (TM) a cada dia em que o animal sobrevivia. O cálculo do índice de patogenicidade de cada pintinho inoculado (IP) obedeceu à seguinte fórmula: IP = (TMx5) + A + Ph + Pc + Pt + O + C. Para obtenção do IP de cada amostra, foi realizada a média do IP obtido com as 10 aves inoculadas. Com base nos resultados observados, foi possível atribuir um valor de patogenicidade a cada uma das cepas, permitindo classificá-las em grupos de baixa (27/200), intermediária (95/200) e alta patogenicidade (78/200). A utilização de critérios, como tempo de morte e presença de lesões septicêmicas, permitiu a determinação de um índice de patogenicidade para cada cepa. Além disso, o modelo proposto apresentou diferença significativa entre os grupos de alta, intermediária e baixa patogenicidade, permitindo, assim, a sua aplicação para classificação futura das cepas isoladas em granjas avícolas.
Assuntos
Animais , Aves Domésticas , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/patogenicidade , Salmonelose Animal/patologia , Interações Hospedeiro-Patógeno , Fatores de VirulênciaRESUMO
Salmonella Enteritidis and Salmonella Typhimurium are responsible for causing huge economic loses in aviculture, as they lead young broiler chicks to develop clinical disease and thus increase mortality. Salmonella's pathogenicity is considered complex and multifactorial, demanding more studies that could elucidate the interaction between host and pathogen. The present study aims to evaluate the virulence of 130S. Enteritidis isolates and 70S. Typhimurium inoculated in one-day-old chicks through the establishment of a pathogenicity index. For each strain, 10 commercial chicks from the Cobb lineage were used. Then, 200µL of a solution containing 2x108 CFU of S. Enteritidis or S. Typhimurium were inoculated in the birds by intraperitoneal via. Mortality and presence of lesions such as aerosaculitis (A), perihepatitis (Ph), pericarditis (Pc), peritonitis (Pt), onfalitis (O) and cellulitis (C) were registered daily for seven days. From the second to the seventh day there was a proportional decrease in the punctuation of the time of death (TD) for each day that the bird had survived. The pathogenicity index was calculated using the following formula: PI = (TD x 5) + A + Ph + Pc + Pt + O + C. The obtainment of the PI of each bacterial sample was achieved by calculating the rate of the ten inoculated birds. Based on the obtained results, it was possible to attribute the pathogenicity value for each strain, which enabled us to classify them in groups of low (27/200), intermediate (95/200) and high (78/200) pathogenicity. The utilization of standards like time of death and presence of septicemic lesions made it possible to determine the pathogenicity rate for each strain. Besides that, the proposed model has presented dramatic differences between the high, intermediate and low pathogenicity groups, which makes this mechanism useful for further classification of strains isolated in poultry farms.(AU)
Salmonella Enteritidis e Salmonella Typhimurium são responsáveis por imensos prejuízos econômicos ao setor avícola, podendo levar ao desenvolvimento de doença clínica e ao aumento da mortalidade em aves jovens. A patogenicidade de Salmonella é considerada complexa e multifatorial, necessitando de estudos que possam esclarecer a interação entre patógeno e hospedeiro. O presente trabalho teve por objetivo avaliar a virulência de 130 isolados de S. Enteritidis e 70 de S.Typhimurium, inoculadas em pintos de um dia de idade, por meio do estabelecimento de um índice de patogenicidade. Para cada cepa, foram utilizados 10 pintos comerciais da linhagem Cobb. As aves foram inoculadas com 200µL de uma solução contendo 2x108 UFC de S. Enteritidis ou S. Typhimurium, por via intraperitoneal. A mortalidade e a presença de lesões como aerossaculite (A), peri-hepatite (Ph), pericardite (Pc), peritonite (Pt), onfalite (O) e celulite (C) foram registradas diariamente durante sete dias. Do segundo ao sétimo dia, houve uma diminuição proporcional da pontuação no tempo de morte (TM) a cada dia em que o animal sobrevivia. O cálculo do índice de patogenicidade de cada pintinho inoculado (IP) obedeceu à seguinte fórmula: IP = (TMx5) + A + Ph + Pc + Pt + O + C. Para obtenção do IP de cada amostra, foi realizada a média do IP obtido com as 10 aves inoculadas. Com base nos resultados observados, foi possível atribuir um valor de patogenicidade a cada uma das cepas, permitindo classificá-las em grupos de baixa (27/200), intermediária (95/200) e alta patogenicidade (78/200). A utilização de critérios, como tempo de morte e presença de lesões septicêmicas, permitiu a determinação de um índice de patogenicidade para cada cepa. Além disso, o modelo proposto apresentou diferença significativa entre os grupos de alta, intermediária e baixa patogenicidade, permitindo, assim, a sua aplicação para classificação futura das cepas isoladas em granjas avícolas.(AU)
Assuntos
Animais , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/patogenicidade , Salmonelose Animal/patologia , Aves Domésticas , Fatores de Virulência , Interações Hospedeiro-PatógenoRESUMO
Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.
Assuntos
Nucleotídeos de Adenina/sangue , Dermatite de Contato/genética , Dermatite Irritante/genética , Receptores Purinérgicos P2X7/genética , Animais , Antígenos CD/sangue , Apirase/sangue , Óleo de Cróton/toxicidade , Dermatite de Contato/sangue , Dermatite de Contato/patologia , Dermatite Irritante/sangue , Dermatite Irritante/patologia , Modelos Animais de Doenças , Humanos , Hidrólise , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Nucleotídeo Desaminases/sangue , Antagonistas do Receptor Purinérgico P2X/administração & dosagem , Receptores Purinérgicos P2X7/sangueRESUMO
Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.
Assuntos
Alcaloides/análise , Animais , Argentina , Artrópodes , Bufonidae , Dieta , Especificidade da EspécieRESUMO
OBJECTIVES: The aim of this study was to verify the prevalence of peri-implant disease and analyse possible risk variables associated with peri-implant mucositis and peri-implantitis. The study group consisted of 212 partially edentulous subjects rehabilitated with osseointegrated implants. MATERIAL AND METHODS: The implants placed were examined clinically and radiographically to assess the peri-implant status. The degree of association between peri-implant disease and various independent variables was investigated using a multinomial regression analysis. RESULTS: The prevalence of peri-implant mucositis and peri-implantitis were 64.6% and 8.9%, respectively. In univariate modelling, healthy peri-implant subjects presented lower plaque scores, less periodontal bleeding on probing, and less time elapsed since placement of supra-structures. In multivariate analyses, the risk variables associated with increased odds for having peri-implant disease included: gender, plaque scores, and periodontal bleeding on probing. Presence of periodontitis and diabetes were statistically associated with increased risk of peri-implantitis. The only two factors, which did not contribute to the presence of the disease, were the time elapsed since placement of supra-structures and the frequency of visits for maintenance care. CONCLUSION: Our data suggest that subjects with periodontitis, diabetes, and poor oral hygiene were more prone to develop peri-implantitis.
Assuntos
Implantes Dentários/estatística & dados numéricos , Doenças Periodontais/epidemiologia , Fatores Etários , Perda do Osso Alveolar/epidemiologia , Brasil/epidemiologia , Estudos Transversais , Placa Dentária/epidemiologia , Complicações do Diabetes/epidemiologia , Feminino , Hemorragia Gengival/epidemiologia , Humanos , Arcada Parcialmente Edêntula/reabilitação , Masculino , Pessoa de Meia-Idade , Osseointegração/fisiologia , Perda da Inserção Periodontal/epidemiologia , Bolsa Periodontal/epidemiologia , Periodontite/epidemiologia , Prevalência , Fatores de Risco , Fatores Sexuais , Fatores de TempoRESUMO
The in vitro antioxidant and free radical scavenging properties in bark extracts of South American tree Copaifera reticulata Ducke. (Caesalpinaceae) were studied using different bioassays. Lipid peroxidation was assessed by means of the production of thiobarbituric acid reactive substances (TBARS) in rat liver homogenate. All the extracts tested were effective in this method. The highest activity was observed in the aqueous extract, showing an IC50 of 30 micrograms/ml. DNA sugar damage induced by Fe (II) salts was also used to determine the capacity of the samples to suppress hydroxyl radical-mediated degradation of DNA. Although all the extracts tested were effective in reducing oxidation of DNA, the highest activity was observed in the methanol extract, showing an IC50 of 2 micrograms/ml. Bioassay-guided fractionation of a total methanol extract monitored by luminol-enhanced chemiluminescence, together with structural elucidation using 13C NMR and FABMS, led to the identification of profisetinidin type tannins in a semi-pure fraction. The fraction containing the active compounds also reduced the production of TBARS in rat liver homogenates (IC50 = 530 micrograms/ml) and DNA damage (IC50 = 1 microgram/ml), suggesting that profisetinidins could be responsible for the free radical scavenging and antioxidant activities observed in the extracts.
Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Plantas Medicinais/química , Taninos/química , Taninos/farmacologia , Animais , Brasil , Dano ao DNA , Radicais Livres , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Ratos , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The bioactivity-guided fractionation of an active chloroform extract of Conyza albida led to the isolation of three alkenynes, deca-4,6-diyn-2-(Z)-enoic methyl ester (1), deca-4,6-diyn-2-(Z)-enoic ethyl ester (2) and deca-2,4-diene-4-hydroxy-6-yn-1,4-olide (3), and the terpenoid spathulenol (4), as the active toxic metabolites in the Artemia sp. lethality test. When tested in the KB cell cytotoxicity assay, compounds 1-4 demonstrated IC50 values of 52.2, 38.4, 117.9, and 83.8 microM, respectively. All compounds studied were inactive in the DNA methyl green and DNA strand scission assays, while compounds 3 and 4 showed moderate activity as inhibitors of human topoisomerase I. Compound 2 is reported here for the first time.