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The original version of this article (Bartolo-Aguilar et al. 2017) was written and published including the first construction strategy of pLGC09, but not the final one. This error was pointed out by a reader and an analysis of sequences of parts of the plasmid corroborated this. The final construction strategy was reanalysed and confirmed the error. This error affected the text, Table 2, Fig. 1 and Additional files, but did not affect the results and conclusions stated in the paper. The authors regret that this error occurred in the original publication of the article. The corrected text, Table 2 and Fig. 1, and Additional files (Additional file 1. Construction strategy of pLGC09 and Additional file 2. Plasmid pLGC09) are given in this correction.
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BACKGROUND: Growth conditions that bring about stress on Phaffia rhodozyma cells encourage the synthesis of astaxanthin, an antioxidant carotenoid, which protects cells against oxidative damage. Using P. rhodozyma cultures performed with and without copper limitation, we examined the kinetics of astaxanthin synthesis along with the expression of asy, the key astaxanthin synthesis gene, as well as aox, which encodes an alternative oxidase protein. RESULTS: Copper deficiency had a detrimental effect on the rates of oxygen consumption and ethanol reassimilation at the diauxic shift. In contrast, copper deficiency prompted alcoholic fermentation under aerobic conditions and had a favorable effect on the astaxanthin content of cells, as well as on aox expression. Both cultures exhibited strong aox expression while consuming ethanol, but particularly when copper was absent. CONCLUSION: We show that the induction of either astaxanthin production, aox expression, or aerobic fermentation exemplifies the crucial role that redox imbalance plays in triggering any of these phenomena. Based on our own results and data from others, we propose a mechanism that rationalizes the central role played by changes of respiratory activity, which lead to redox imbalances, in triggering both the short-term antioxidant response as well as fermentation in yeasts and other cell types.
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Antioxidantes/metabolismo , Basidiomycota/metabolismo , Fermentação , Aerobiose , Cobre/química , Meios de Cultura/química , Glicólise , Cinética , Oxirredução , Oxigênio/química , Xantofilas/biossínteseRESUMO
2,4-Diacetylphloroglucinol (DAPG) (1) is a phenolic polyketide produced by some plant-associated Pseudomonas species, with many biological activities and ecological functions. Here, we aimed at reconstructing the natural history of DAPG using phylogenomics focused at its biosynthetic gene cluster or phl genes. In addition to around 1500 publically available genomes, we obtained and analyzed the sequences of nine novel Pseudomonas endophytes isolated from the antidiabetic medicinal plant Piper auritum. We found that 29 organisms belonging to six Pseudomonas species contain the phl genes at different frequencies depending on the species. The evolution of the phl genes was then reconstructed, leading to at least two clades postulated to correlate with the known chemical diversity surrounding DAPG biosynthesis. Moreover, two of the newly obtained Pseudomonas endophytes with high antiglycation activity were shown to exert their inhibitory activity against the formation of advanced glycation end-products via DAPG and related congeners. Its isomer, 5-hydroxyferulic acid (2), detected during bioactivity-guided fractionation, together with other DAPG congeners, were found to enhance the detected inhibitory activity. This report provides evidence of a link between the evolution and chemical diversity of DAPG and congeners.
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Endófitos/química , Floroglucinol/análogos & derivados , Piper/microbiologia , Plantas Medicinais/microbiologia , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , Pseudomonas/química , Ácidos Cumáricos/química , Ácidos Cumáricos/isolamento & purificação , México , Estrutura Molecular , Família Multigênica , Floroglucinol/química , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Piper/genética , Componentes Aéreos da Planta/química , Plantas Medicinais/genética , Policetídeos/química , EstereoisomerismoRESUMO
The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-ß-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.
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Membranous vesicular organelles (MVOs), such as secretory vesicles and exosomes, perform a variety of biological functions ranging from secretion to cellular communication in eukaryotic cells. Exosomes, particularly those of mammalian cells, have been widely studied as potential carriers in human therapeutic applications. However, no study has yet demonstrated the use of yeast secretory vesicles for such applications. Therefore, we explore here the current state of knowledge on yeast secretory vesicles and their potential use in therapeutic delivery systems. We focus on the characteristics shared by exosomes and yeast secretory vesicles to provide insights into the use of the latter as delivery vehicles. From this perspective, we speculate on the potential application of post-Golgi vesicles (PGVs) in the biomedical field.
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Portadores de Fármacos , Sistemas de Liberação de Medicamentos/métodos , Exossomos/metabolismo , Técnicas de Transferência de Genes , Saccharomyces cerevisiae/fisiologia , Vesículas Secretórias/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Química Farmacêutica , Genótipo , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
CONTEXT: Byrsonima crassifolia is a tropical tree, commonly known as nance and distributed widely in Mexico and Central and South America. Since pre- Hispanic times, the seeds of the fruits have been used in folklore medicine as an anti-inflammatory; however, currently no researchers have examined its potential pharmacological properties in scientific studies. OBJECTIVE: This study investigated the anti-inflammatory activity of extracts obtained with the solvents n-hexane, chloroform, and methanol from seeds of B crassifolia. DESIGN: The research team induced edemas in Wistar rats with 12-O-tetradecanoylphorbol (TPA), formaldehyde, carrageenan, and histamine to study the anti-inflammatory activity of the three organic extracts of seeds from B crassifolia. The team also used the cotton-pellet granuloma method to induce edemas in Wistar rats and study the inhibitory effect of the three extracts from B crassifolia. Finally, the team examined the participation of the nitric oxide (NO) system in the anti-inflammatory activity of the hexane extract of nance seeds (NS), diclorofenac, and L-NAME as well as the effects of L-arginine and D-arginine on the antiinflammatory actions of the compounds. SETTING: This research was conducted in the Laboratory of Research of Natural Products, School of Chemical Engineering, National Polytechnic Institute (IPNESIQIE) and Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. IPN 2508, Col. San Pedro Zacatenco, Mexico D.F., CP 07360, Mexico. OUTCOME MEASURES: The research team measured the edema that the solvents caused, either in the ears of rats for tetradecanoylphorbol or in the paws for formaldehyde, carrageenan, and histamine. To study the antiproliferative effects of the extracts after implantation of the cotton-pellet granuloma, the team determined the wet and dry weights of the pellets, after drying at 70°C for 1 hour in the second case. To study the participation of the NO system in the anti-inflammatory activity of the hexane extract of NS, diclofenac, and L-NAME, the research team measured paw edema. RESULTS: Among the extracts tested, NS showed the most significant anti-inflammatory activity. That extract decreased the paw edema that carrageenan, formaldehyde, histamine, and cotton pellet-induced, either by oral or topical administration at doses of 200 mg/kg, with 31%, 66%, 83%, and 58.2% inhibition respectively. In addition, NS inhibited the ear edema that TPA induced by 62%. Methanol and chloroform extracts produced a small effect, so the team does not present the results in this article. L-arginine, a precursor of NO, significantly inhibited the anti-inflammatory effects of NS and L-NAME, an anti-inflammatory drug, on mouse paw edema, but D-arginine did not. In contrast, neither D-arginine nor L-arginine inhibited the anti-inflammatory effects that diclofenac produced. These results indicate that the anti-inflammatory effect of NS on mouse paw edema occurs via the inhibition of NO production, as does the anti-inflammatory effect of L-NAME but not the anti-inflammatory effect of diclofenac. The anti-inflammatory activity of NS was comparable to standard anti-inflammatory drugs such as indomethacin, dexamethasone, and sodium diclofenac. CONCLUSIONS: The hexane extract from seeds of B crassifolia exhibited significant anti-inflammatory activity in both acute and chronic inflammatory models with a partial contribution of inhibitory actions on some cellular inflammatory responses. The anti-inflammatory mechanism of NS may be related to the other isoform (iNOS).
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Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Edema/prevenção & controle , Granuloma de Corpo Estranho/prevenção & controle , Malpighiaceae/química , Extratos Vegetais/farmacologia , Sementes , Animais , Anti-Inflamatórios/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Feminino , Granuloma de Corpo Estranho/induzido quimicamente , Hexanos , Masculino , Extratos Vegetais/administração & dosagem , Plantas Medicinais , Ratos , Ratos WistarRESUMO
The aim of this study was to investigate the antioxidant activity of hexane extracts from leaves of Piper auritum (HS). Eight complementary in vitro test methods were used, including inhibition of DPPH· radicals, nitric oxide, superoxide anion, ion-chelating, ABTS, oxygen radical absorbance capacity, ß-carotene bleaching and peroxy radical scavenging. The results indicated that HS possesses high antioxidant activity. To add to these finding we tested the effect against oxidative stress in liver, pancreas and kidney in diabetic rats. Low levels of SOD, CAT, GPx and GSH in diabetic rats were reverted to near normal values after treatment with HS. These results suggest that P. auritum prevents oxidative stress, acting as a suppressor of liver cell damage. Given the link between glycation and oxidation, we proposed that HS might possess significant in vitro antiglycation activity. Our data confirmed the inhibitory effect of HS on bovine serum albumin, serum glycosylated protein, glycation of LDL, and glycation hemoglobin. The effect of HS on diabetic renal damage was investigated using streptozotocin-induced diabetic rats. The oral administration of HS at a dose of 200 and 400 mg/kg body weight/day for 28 days significantly reduced advanced glycation endproduct (AGE) formation, elevated renal glucose and thiobarbituric acid-reactive substance levels in the kidneys of diabetic rats. This implies that HS would alleviate the oxidative stress under diabetes through the inhibition of lipid peroxidation. These findings indicate that oxidative stress is increased in the diabetic rat kidney and that HS can prevent renal damage associated with diabetes by attenuating the oxidative stress.
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Antioxidantes/farmacologia , Nefropatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Piper/química , Extratos Vegetais/farmacologia , Animais , Antioxidantes/administração & dosagem , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/etiologia , Sequestradores de Radicais Livres/farmacologia , Glicosilação/efeitos dos fármacos , Hexanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Quelantes de Ferro/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oxirredutases/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Fenóis/química , Extratos Vegetais/administração & dosagem , Folhas de Planta/química , Ratos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.
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Fungos/classificação , Fungos/isolamento & purificação , Paclitaxel/metabolismo , Filogenia , Taxus/microbiologia , DNA Fúngico/genética , DNA Ribossômico/genética , Fungos/genética , Fungos/fisiologia , Dados de Sequência Molecular , Simbiose , Taxus/fisiologiaRESUMO
Gibberellic acid has been known since 1954 but its effect on rice still remains very important in the agricultural world. Gibberellic acid (GA3) is the main secondary metabolite produced by the Gibberella fujikuroi fungus. This hormone is of great importance in agriculture and the brewing industry, due to its fast and strong effects at low concentrations (µg) on the processes of growth stimulation, flowering, stem elongation, and germination of seeds, among others. Plant promoters of growth production such as the gibberellins, especially the GA3 are a priority in obtaining better harvests in the agricultural area and by extension, improving the food industry. Three routes to obtaining GA3 have been reported: extraction from plants, chemical synthesis and microbial fermentation. The latter being the most common method used to produce GA3. In this investigation, glucose-corn oil mixture was used as a carbon source on the basis of 40 g of carbon in a 7 L stirred tank bioreactor. A pH of 3.5, 29°C, 600 min(-1) agitation and 1 vvm aeration were maintained and controlled with a biocontroller connected to the bioreactor, throughout the entire culture time. The carbon source mixture affected the fermentation time as well as the production of the GAs. The production of 380 mg GA3L(-1) after 288 h of fermentation was obtained when the glucose-corn oil mixture was employed contrasting the 136 mg GA3L(-1) at 264 h of culture when only glucose was used.
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A concentric draft tube jet loop bioreactor (10.5 m3) was used to study the influence of aerated liquid height (above the draft tube) on the amount of surfactant addition allowable without foaming. Sodium lauryl sulfate (SLS) and defatted soybean flour in tap water were used as model artificial media. The amount of surfactant required to develop foaming and the maximum gas holdup achieved prior to foaming were notably influenced by aerated liquid height. Decreasing the aerated liquid height from 1.50 to 0.05 m increased the amount of SLS allowed without foaming from 2.2 to 12.1 g, the gas holdup in the riser from 0.18 to 0.31 and the gas holdup in the downcomer from 0.12 to 0.25. Similar behavior was observed for soybean flour. Decreasing the aerated liquid height from 1.45 to 0.05 m increased the amount of soybean flour allowed without foaming from 822 to 3200 g, the gas holdup in the riser from 0.17 to 0.26 and the gas holdup in the downcomer from 0.10 to 0.19. Data from a representative continuous yeast culture are reported to show that operation at low aerated liquid heights (0.5 m) can also be used to produce a culture broth with large gas holdup and oxygen transfer but without foaming.