RESUMO
Differential rootstock tolerance to Fusarium spp. supports viticulture worldwide. However, how plants stand against the fungus still needs to be explored. We hypothesize it involves a differential metabolite modulation. Thus, we performed a gas chromatography coupled with mass spectrometry (GC-MS) analysis of Paulsen P1103 and BDMG573 rootstocks, co-cultured with Fusarium oxysporum (FUS) for short, medium, and long time (0, 4, and 8 days after treatment [DAT]). In shoots, principal component analysis (PCA) showed a complete overlap between BDMG573 non-co-cultivated and FUS at 0 DAT, and P1103 treatments showed a slight overlap at both 4 and 8 DAT. In roots, PCA exhibited overlapping between BDMG573 treatments at 0 DAT, while P1103 treatments showed overlapping at 0 and 4 DAT. Further, there is a complete overlapping between BDMG573 and P1103 FUS profiles at 8 DAT. In shoots, 1,3-dihydroxyacetone at 0 and 4 DAT and maltose at 4 and 8 DAT were biomarkers for BDMG573. For P1103, glyceric acid, proline, and sorbitol stood out at 0, 4, and 8 DAT, respectively. In BDMG573 roots, the biomarkers were ß-alanine at 0 DAT, cellobiose and sorbitol at both 4 and 8 DAT. While in P1103 roots, they were galactose at 0 and 4 DAT and 1,3-dihydroxyacetone at 8 DAT. Overall, there is an increase in amino acids, glycolysis, and tricarboxylic acid components in tolerant Paulsen P1103 shoots. Thus, it provides a new perspective on the primary metabolism of grapevine rootstocks to F. oxysporum that may contribute to strategies for genotype tolerance and early disease identification.
Assuntos
Fusarium , Vitis , Vitis/metabolismo , Di-Hidroxiacetona/metabolismo , Doenças das Plantas/microbiologia , Sorbitol/metabolismoRESUMO
Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Chlorocebus aethiops , Di-Hidroxiacetona/metabolismo , Di-Hidroxiacetona/toxicidade , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Osmorregulação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
In this work, several immobilization strategies for Gluconobacter oxydans NBRC 14819 (Gox) were tested in the bioconversion of crude glycerol to dihydroxyacetone (DHA). Agar, agarose and polyacrylamide were evaluated as immobilization matrixes. Glutaraldehyde crosslinked versions of the agar and agarose preparations were also tested. Agar immobilized Gox proved to be the best heterogeneous biocatalyst in the bioconversion of crude glycerol reaching a quantitative production of 50 g/L glycerol into DHA solely in water. Immobilization allowed reutilization for at least eight cycles, reaching four times more DHA than the amount obtained by a single batch of free cells which cannot be reutilized. An increase in scale of 34 times had no impact on DHA productivity. The results obtained herein constitute a contribution to the microbiological production of DHA as they not only attain unprecedented productivities for the reaction with immobilized biocatalysts but also proved that it is feasible to do it in a clean background of solely water that alleviates the cost of downstream processing.
Assuntos
Di-Hidroxiacetona , Gluconobacter oxydans , Biotransformação , GlicerolRESUMO
In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.
Assuntos
Biotransformação , Gluconobacter/metabolismo , Glicerol/metabolismo , Metabolismo dos Carboidratos , Cromatografia Líquida de Alta Pressão , Di-Hidroxiacetona/metabolismo , Ácidos Glicéricos/metabolismo , Cinética , Espectroscopia de Ressonância MagnéticaRESUMO
Background: Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels into the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problemthe waste glycerol generated during biodiesel production at a concentration of approximately 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results: Utilization of the waste fraction at 48 h with an initial glycerol concentration of 30 g·L-1 and proceeding with 62% efficiency enabled the production of 9 g·L-1 dihydroxyacetone at 50% substrate consumption. The re-use of the immobilized biocatalyst resulted in a similar concentration of dihydroxyacetone (8.7 g·L-1) in two-fold shorter time, with an efficiency of 85% and lower substrate consumption (35%). Conclusions: The method proposed in this work is based on the conversion of waste glycerol to dihydroxyacetone in a reaction catalyzed by immobilized Gluconobacter oxydans cell extract with glycerol dehydrogenase activity, and it could be an effective way to convert waste glycerol into a valuable product.
Assuntos
Células Imobilizadas/metabolismo , Di-Hidroxiacetona/metabolismo , Glicerol/metabolismo , Resíduos , Extratos Celulares , Células Imobilizadas/química , Gluconobacter oxydans , Biocombustíveis , Reciclagem , Energia Renovável , Glicerol/químicaRESUMO
Abstract The principal objective of this study was to evaluate the kinetics of dihydroxyacetone production by Gluconobacter frateurii CGMCC 5397 under different oxygen volumetric mass transfer coefficient (kLa) conditions in submerged bioreactors using biodiesel-derived crude glycerol as the carbon source. kLa is a key fermentation parameter for the production of dihydroxyacetone. Cultivations were conducted in baffled- and unbaffled-flask cultures (the kLa values were 24.32 h−1 and 52.05 h−1, respectively) and fed-batch cultures (the kLa values were held at 18.21 h−1, 46.03 h−1, and 82.14 h−1) to achieve high dihydroxyacetone concentration and productivity. The results showed that a high kLa could dramatically increase dihydroxyacetone concentrations and productivities. The baffled-flask culture (with a kLa of 52.05 h−1) favored glycerol utilization and dihydroxyacetone production, and a dihydroxyacetone concentration as high as 131.16 g/L was achieved. When the kLa was set to 82.14 h−1 in the fed-batch culture, the dihydroxyacetone concentration, productivity and yield were 175.44 g/L, 7.96 g/L/h and 0.89 g/g, respectively, all of which were significantly higher than those in previous studies and will benefit dihydroxyacetone industrial production.
Assuntos
Di-Hidroxiacetona/metabolismo , Gluconobacter/metabolismo , Glicerol/metabolismo , Oxigênio/metabolismo , Biotransformação , Reatores Biológicos/microbiologia , Carbono/metabolismoRESUMO
The principal objective of this study was to evaluate the kinetics of dihydroxyacetone production by Gluconobacter frateurii CGMCC 5397 under different oxygen volumetric mass transfer coefficient (kLa) conditions in submerged bioreactors using biodiesel-derived crude glycerol as the carbon source. kLa is a key fermentation parameter for the production of dihydroxyacetone. Cultivations were conducted in baffled- and unbaffled-flask cultures (the kLa values were 24.32h(-1) and 52.05h(-1), respectively) and fed-batch cultures (the kLa values were held at 18.21h(-1), 46.03h(-1), and 82.14h(-1)) to achieve high dihydroxyacetone concentration and productivity. The results showed that a high kLa could dramatically increase dihydroxyacetone concentrations and productivities. The baffled-flask culture (with a kLa of 52.05h(-1)) favored glycerol utilization and dihydroxyacetone production, and a dihydroxyacetone concentration as high as 131.16g/L was achieved. When the kLa was set to 82.14h(-1) in the fed-batch culture, the dihydroxyacetone concentration, productivity and yield were 175.44g/L, 7.96g/L/h and 0.89g/g, respectively, all of which were significantly higher than those in previous studies and will benefit dihydroxyacetone industrial production.
Assuntos
Di-Hidroxiacetona/metabolismo , Gluconobacter/metabolismo , Glicerol/metabolismo , Oxigênio/metabolismo , Reatores Biológicos/microbiologia , Biotransformação , Carbono/metabolismoRESUMO
The principal objective of this study was to evaluate the kinetics of dihydroxyacetone production by Gluconobacter frateurii CGMCC 5397 under different oxygen volumetric mass transfer coefficient (kLa) conditions in submerged bioreactors using biodiesel-derived crude glycerol as the carbon source. kLa is a key fermentation parameter for the production of dihydroxyacetone. Cultivations were conducted in baffled- and unbaffled-flask cultures (the kLa values were 24.32 h1 and 52.05 h1, respectively) and fed-batch cultures (the kLa values were held at 18.21 h1, 46.03 h1, and 82.14 h1) to achieve high dihydroxyacetone concentration and productivity. The results showed that a high kLa could dramatically increase dihydroxyacetone concentrations and productivities. The baffled-flask culture (with a kLa of 52.05 h1) favored glycerol utilization and dihydroxyacetone production, and a dihydroxyacetone concentration as high as 131.16 g/L was achieved. When the kLa was set to 82.14 h1 in the fed-batch culture, the dihydroxyacetone concentration, productivity and yield were 175.44 g/L, 7.96 g/L/h and 0.89 g/g, respectively, all of which were significantly higher than those in previous studies and will benefit dihydroxyacetone industrial production. (AU)
Assuntos
Transferência de Oxigênio , Di-Hidroxiacetona , Glicerol , Biocombustíveis , GluconobacterRESUMO
Dihydroxyacetone (DHA), a three-carbon sugar, is the browning ingredient in commercial sunless tanning formulations. DHA preparations have been used for more than 50 years and are currently highly popular for producing temporary pigmentation resembling an ultraviolet-induced tan. In this work, the in vitro antifungal activity of dihydroxyacetone was tested against causative agents of dermatomycosis, more specifically against dermatophytes and Candida spp. The antifungal activity was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines for yeasts and filamentous fungi. The data obtained show that the fungicidal activity varied from 1.6 to 50 mg ml(-1). DHA seems to be a promising substance for the treatment of dermatomycosis because it has antifungal properties at the same concentration used in artificial suntan lotions. Therefore, it is a potential low-toxicity antifungal agent that may be used topically because of its penetration into the corneal layers of the skin.
Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Dermatomicoses/microbiologia , Di-Hidroxiacetona/farmacologia , Arthrodermataceae/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacosRESUMO
Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas' disease, have shown side effects and variable efficiency. With the aim to describe parasite enzymes involved in the mechanisms of action of trypanocidal drugs and since it has been reported that reductases are crucial in their metabolism, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi. The percolation of a soluble fraction of epimastigote lysates through a Cibacron Blue-Sepharose column followed by elution by NADPH yielded a predominant protein with an apparent molecular weight of 32 kDa. This protein was identified by MALDI-TOF as an aldo-keto reductase (AKR) and hence denominated TcAKR. TcAKR was mainly localized in the cytosol and was also present in trypomastigote and amastigote lysates. The recombinant TcAKR (recTcAKR) showed NADPH-dependent reductase activity with the AKR substrates 4-nitrobenzaldehyde and 2-dihydroxyacetone. The saturation curves for both substrates were consistent with the Michaelis-Menten model. We also tested whether recTcAKR may reduce naphthoquinones (NQ), since many of these compounds have displayed important trypanocidal activity. recTcAKR reduced o-NQ (1,2-naphthoquinone, 9,10-phenanthrenquinone and beta-lapachone) with concomitant generation of free radicals but did not exhibit affinity for p-NQ (5-hydroxy-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, alpha-lapachone and menadione). The substrate saturation curve with o-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In addition, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. TcAKR is the first member of the AKR family described in T. cruzi. Our results indicate that this enzyme may participate in the mechanisms of action of trypanocidal drugs.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Benzoquinonas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Aldeído Redutase , Aldo-Ceto Redutases , Animais , Benzaldeídos/metabolismo , Cromatografia em Gel , Cromatografia Líquida/métodos , Clonagem Molecular , Coenzimas/metabolismo , DNA de Protozoário/química , DNA de Protozoário/genética , Di-Hidroxiacetona/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , NADP/metabolismo , Oxirredução , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Dihydroxyacetone (DHAT) is a color additive that is added to sunless tanning products to produce an artificial tan. Although this agent has been used extensively as safe sunless tanning, no published data are available to judge whether the abuse of DHAT causes a potential hazard to the human skin. The purpose of this study was to clarify whether frequent treatment with DHAT solutions had a deleterious effect on the wide skin surface of hairless descendants of Mexican hairless dogs. The skin reactions to the DHAT-treatment were investigated by daily clinical observations and histopathological examinations (21 and 42 days after the beginning of the DHAT-treatment). Clinical observations showed that skin color changes were apparent within 6 h after the first treatment with 5% DHAT solutions, with maximal darkening between 12 and 24 h. Twenty-one days after the beginning of the treatment with 5% DHAT solutions, the skin developed irritant dermatitis, and then the skin lesions gradually became severe during this study. Histopathological examinations showed entire epidermal thickening, 21 days after the beginning of the treatment with 5% DHAT solutions. Forty-two days after the beginning of the treatment with 5% DHAT solutions, the skin exhibited remarkable epidermal degeneration (hyperplastic and dyskeratotic changes) and moderate inflammatory reactions in the dermis. In severe dermatitic sites, I found focal epidermal necrosis or interepidermal blister formation beneath the thickened parakeatotic corneum. Throughout this study, there were no clinical and histopathological changes in the sites treated with vehicle alone. These results revealed that the skin coloring generated by frequent wide treatments with DHAT caused severe contact dermatitis which was associated with the damaged stratum corneum.
Assuntos
Dermatite Irritante/patologia , Di-Hidroxiacetona/toxicidade , Pele/efeitos dos fármacos , Animais , Cor , Modelos Animais de Doenças , Cães , Feminino , Masculino , Pele/patologia , Testes de ToxicidadeRESUMO
In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co-localized with GS. Interestingly, the synthesis of Glu-6-P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.
Assuntos
Frutose-Bifosfatase/metabolismo , Hepatócitos/enzimologia , Frações Subcelulares/enzimologia , Animais , Di-Hidroxiacetona/fisiologia , Imunofluorescência , Glucose/fisiologia , Insulina/fisiologia , Masculino , RatosRESUMO
Pancreas pieces of Bufo arenarum were incubated with several sugars at basal and stimulatory concentrations, and with inhibitors of their metabolism, measuring the insulin released by radioimmunoassay. Glucose, mannose, fructose, glyceraldehyde and dihydroxyacetone all at 8 mM, significantly enhanced the release of insulin elicited by basal concentrations of these carbohydrates (2 mM). The nonmetabolizable sugars galactose and 2-deoxyglucose failed to enhance insulin secretion. N-Acetyl-glucosamine at 8 mM did not significantly affect the release of insulin. D-Glucose, but not L-glucose, at 8 mM stimulated insulin secretion above baseline (2 mM glucose). At 8 mM, the D-glucose alpha-anomer significantly increased insulin release, while this effect was not observed using the beta-anomer. Insulin release elicited by 2 mM of the alpha-anomer was significantly higher than that triggered by the beta-anomer. Iodoacetate (5 mM), and dinitrophenol (0.3 mM) exerted an inhibitory effect upon glucose-induced insulin secretion. The effect of these carbohydrates and metabolic inhibitors--tested for the first time in amphibians--was similar to that described in the mammalian pancreas, thus suggesting that such compounds play an important role in the metabolic control of insulin secretion in amphibians.
Assuntos
Bufo arenarum/fisiologia , Carboidratos/farmacologia , Insulina/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Animais , Carboidratos/química , Di-Hidroxiacetona/farmacologia , Frutose/farmacologia , Galactose/farmacologia , Glucose/química , Glucose/farmacologia , Gliceraldeído/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Mamíferos , Manose/farmacologia , Especificidade da EspécieRESUMO
El envejecimiento induce marcados cambios de la superficie cutánea y particularmente de la capa córnea. La aplicación de lipo-hidroxi-ácido, un derivado lipofílico del ácido salicílico, aporta una mejoría de ciertas alteraciones asociadas al envejecimiento. Produce una eliminación más uniforme de los corneocitos y un aumento del espesor de las capas basal y de Malpighi, dentro de los límites fisiológicos (AU)
Assuntos
Humanos , Idoso , Envelhecimento da Pele/efeitos dos fármacos , Hidroxiácidos/uso terapêutico , Hidroxiácidos/administração & dosagem , Hidroxiácidos/farmacologia , Di-Hidroxiacetona/diagnóstico , Ceratolíticos/uso terapêutico , Envelhecimento da Pele/fisiologia , Envelhecimento da Pele/patologiaRESUMO
El envejecimiento induce marcados cambios de la superficie cutánea y particularmente de la capa córnea. La aplicación de lipo-hidroxi-ácido, un derivado lipofílico del ácido salicílico, aporta una mejoría de ciertas alteraciones asociadas al envejecimiento. Produce una eliminación más uniforme de los corneocitos y un aumento del espesor de las capas basal y de Malpighi, dentro de los límites fisiológicos
Assuntos
Humanos , Idoso , Hidroxiácidos/uso terapêutico , Envelhecimento da Pele/efeitos dos fármacos , Ceratolíticos/uso terapêutico , Di-Hidroxiacetona , Hidroxiácidos/administração & dosagem , Hidroxiácidos/farmacologia , Envelhecimento da Pele/patologia , Envelhecimento da Pele/fisiologiaRESUMO
The effect of blood glucose extraction on the perception of exertion was examined during prolonged arm exercise. Eight male subjects consumed in counter-balanced order a standard daily diet containing either (1) 75 g dihydroxyacetone and 25 g sodium pyruvate (DHAP) or (2) an isocaloric amount of placebo, to manipulate blood glucose extraction. Following each 7-day diet, subjects exercised to exhaustion at 60% of peak arm oxygen consumption. Ratings of perceived exertion (Borg, CR-10 scale) were obtained for the arms (RPE-A), legs (RPE-L), chest (RPE-C) and overall body (RPE-O) every 10 min of exercise. After 60 min of continuous exercise, blood samples were drawn from the radial artery and axillary vein. Ratings of perceived exertion did not differ between trials during the first 50 min of exercise. At the 60-min time point, perceived exertion was lower (P less than 0.01) in the DHAP than placebo trials for the arms (RPE-A: 4.25 vs 5.50) and overall body (RPE-O: 3.25 vs 4.00). These differences persisted throughout exercise. RPE-L and RPE-C did not differ between trials. Whole-arm arterial-venous glucose difference was higher (P less than 0.05) in the DHAP (1.00 mmol.l-1) than placebo (0.36 mmol.l-1) trials, as was fractional extraction of glucose (22.5 vs 9.0%). Respiratory exchange ratio was the same between trials. Triceps muscle glycogen was (1) higher in the DHAP than placebo trial at pre-exercise (P less than 0.05), (2) decreased during exercise and (3) did not differ between trials at exercise termination.(ABSTRACT TRUNCATED AT 250 WORDS)