RESUMO
BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.
Assuntos
Leite , Proinsulina , Feminino , Gravidez , Animais , Bovinos , Humanos , Animais Geneticamente Modificados/metabolismo , Proinsulina/análise , Proinsulina/metabolismo , Leite/química , Proteínas Recombinantes/metabolismo , Insulina/análise , Peptídeo Hidrolases/metabolismoRESUMO
Members of the genus Enterococcus are among the most relevant etiologic agents of bovine clinical and subclinical mastitis, a major problem for the dairy industry. In Brazil, clonal diversity, and multidrug resistance profiles related to bovine infections need further investigation. In this study, 11 bacterial strains recovered from mastitis subclinical cases detected in different farms of São Paulo, Brazil, were identified as Enterococcus faecalis (n = 8) and Enterococcus mundtii (n = 3) by biochemical testing and MALDI-TOF mass spectrometry. Pulsed-field gel electrophoresis categorized the enterococcal isolates into two main clusters (A and B) with similarity ranging from 85 to 100%. The isolates were shown to be resistant tetracycline (73%), erythromycin (73%), quinupristin-dalphopristin (64%), norfloxacin (9%), fosfomycin (9%) and linezolid (9%). Moreover, seven strains (64%) were considered multidrug-resistant. All the isolates were able to produce biofilms when grown in milk for 24 h: 54·54% were classified as moderate producers and 45·45% were weak producers. Interestingly, only two strains (Ef17 and Em42) remained as moderate biofilm producers after 48 h incubation. Moreover, all isolates showed no ability to form biofilm in tryptic soy broth (TSB) after 24 and 48 h incubation. In addition, cytoskeleton components were partially involved in E. faecalis and E. mundtii entry to epithelial cells as demonstrated by induction of actin stress fibre. In conclusion, enterococci isolates recovered from bovine subclinical mastitis were resistant to several classes of antibiotics, showing the ability to form biofilms in milk and invade mammary epithelial cells, suggesting an advantageous feature in mammary gland colonization during mastitis development. In addition, they can spread along the food chain by different routes and eventually constitute a possible threat for public health, including E. mundtii specie.
Assuntos
Mastite Bovina , Animais , Antibacterianos/farmacologia , Biofilmes , Brasil/epidemiologia , Bovinos , Farmacorresistência Bacteriana , Enterococcus , Enterococcus faecalis , Células Epiteliais , Feminino , Humanos , Mastite Bovina/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 µg/mL for samples collected during the spring, autumn, and winter seasons, but 280 µg/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 µg/mL. However, for 120 µg/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell death through apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological activities were determined to be harmful. Therefore, this propolis shows promise as an alternative to commercial antimicrobials in the control of bovine mastitis, offering support for organic milk production.(AU)
Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 µg/mL para as amostras de primavera, outono e inverno e 280 µg/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foi o mais tóxico, sendo a IC50 120 µg/mL. Entretanto, com 120 µg/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas atividades biológicas. Portanto, esta própolis mostra como uma alternativa promissora ao uso de antimicrobianos comerciais no controle da mastite bovina e uma opção para subsidiar a produção orgânica de leite.Palavras-chave: Células MAC-T. Mastite bovina. Propolis brasileira. Staphylococcus aureus.(AU)
Assuntos
Animais , Feminino , Bovinos , Staphylococcus aureus/patogenicidade , Mastite Bovina , Própole , Estações do AnoRESUMO
Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 g/mL for samples collected during the spring, autumn, and winter seasons, but 280 g/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 g/mL. However, for 120 g/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell deaththrough apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological
Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 g/mL para as amostras de primavera, outono e inverno e 280 g/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foio mais tóxico, sendo a IC50 120 g/mL. Entretanto, com 120 g/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas
RESUMO
Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 µg/mL for samples collected during the spring, autumn, and winter seasons, but 280 µg/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 µg/mL. However, for 120 µg/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell death through apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological activities were determined to be harmful. Therefore, this propolis shows promise as an alternative to commercial antimicrobials in the control of bovine mastitis, offering support for organic milk production.(AU)
Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 µg/mL para as amostras de primavera, outono e inverno e 280 µg/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foi o mais tóxico, sendo a IC50 120 µg/mL. Entretanto, com 120 µg/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas atividades biológicas. Portanto, esta própolis mostra como uma alternativa promissora ao uso de antimicrobianos comerciais no controle da mastite bovina e uma opção para subsidiar a produção orgânica de leite.Palavras-chave: Células MAC-T. Mastite bovina. Propolis brasileira. Staphylococcus aureus.(AU)
Assuntos
Animais , Feminino , Bovinos , Staphylococcus aureus/patogenicidade , Mastite Bovina , Própole , Estações do AnoRESUMO
Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 g/mL for samples collected during the spring, autumn, and winter seasons, but 280 g/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 g/mL. However, for 120 g/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell deaththrough apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological
Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 g/mL para as amostras de primavera, outono e inverno e 280 g/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foio mais tóxico, sendo a IC50 120 g/mL. Entretanto, com 120 g/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas
RESUMO
Mastitis is an inflammation of mammary gland parenchyma that adversely affects bovine health and dairy production worldwide despite significant efforts to eradicate it. The aim of this work was to characterize the antimicrobial activity of 7-epiclusianone (7-epi), a compound extracted from the Rheedia brasiliensis fruit, its complex with copper against Streptococcus spp. isolated from bovine mastitis, and to assess their cytotoxicity to bovine mammary alveolar cells (MAC-T). The complex 7-epiclusianone-Cu (7-epi-Cu) was an amorphous green solid with optical activity. Its vibrational spectrum in the infrared region showed absorption bands in the high-frequency region, as well as bands that can be attributed to the unconjugated and conjugated stretching of the free ligand. The complex was anhydrous. One of the tested bacterial strains was not sensitive to the compounds, while the other three had MIC values of 7.8 µg mL-1 and minimum bactericidal concentration (MBC) values between 15.6 and 31.3 µg mL-1. These two compounds are bacteriostatic, did not cause damage to the cell wall and, at sub-inhibitory concentrations, did not induce bacterial adhesion. The compounds were not cytotoxic. Based on these results, 7-epi and 7-epi-Cu exhibited desirable antimicrobial properties and could potentially be used in bovine mastitis treatment.
Assuntos
Antibacterianos/farmacologia , Benzofenonas/farmacologia , Benzoquinonas/farmacologia , Glândulas Mamárias Animais/citologia , Mastite Bovina/microbiologia , Streptococcus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Aderência Bacteriana , Benzofenonas/química , Benzofenonas/toxicidade , Benzoquinonas/química , Benzoquinonas/toxicidade , Bovinos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/toxicidade , Cobre/química , Feminino , Glândulas Mamárias Animais/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/isolamento & purificação , Streptococcus/fisiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/fisiologiaRESUMO
Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine ß-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.