RESUMO
Penicillium roqueforti is used in the production of several kinds of ripened blue-veined cheeses. In addition, this fungus produces interesting secondary metabolites such as roquefortine C, andrastin A and mycophenolic acid. To date, there is scarce information concerning the regulation of the production of these secondary metabolites. Recently, the gene named pcz1 (Penicillium C6 zinc domain protein 1) was described in P. roqueforti, which encodes for a Zn(II)2Cys6 protein that controls growth and developmental processes in this fungus. However, its effect on secondary metabolism is currently unknown. In this work, we have analyzed how the overexpression and down-regulation of pcz1 affect the production of roquefortine C, andrastin A and mycophenolic acid in P. roqueforti. The three metabolites were drastically reduced in the pcz1 down-regulated strains. However, when pcz1 was overexpressed, only mycophenolic acid was overproduced while, on the contrary, levels of roquefortine C and andrastin A were diminished. Importantly, these results match the expression pattern of key genes involved in the biosynthesis of these metabolites. Taken together, our results suggest that Pcz1 plays a key role in regulating secondary metabolism in the fungus Penicillium roqueforti.
Assuntos
Proteínas Fúngicas/fisiologia , Fungos/genética , Fungos/metabolismo , Penicillium/genética , Penicillium/metabolismo , Metabolismo Secundário/genética , Androstadienos/metabolismo , Queijo/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Indóis/metabolismo , Ácido Micofenólico/metabolismo , Piperazinas/metabolismoRESUMO
Background: 3-Ketosteroid-∆¹-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd n) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.
Assuntos
Androstadienos/metabolismo , Corynebacterium/enzimologia , Mycobacterium/enzimologia , Oxirredutases/metabolismo , Códon , Proteínas RecombinantesRESUMO
BACKGROUND: The biotransformation of steroids by fungal biocatalysts has been recognized for many years. There are numerous fungi of the genus Aspergillus which have been shown to transform different steroid substances. The possibility of using filamentous fungi Aspergillus brasiliensis cells in the biotransformation of androsta-1,4-diene-3,17-dione, was evaluated. METHODS: The fungal strain was inoculated into the transformation medium which supplemented with androstadienedione as a substrate and fermentation continued for 5 days. The metabolites were extracted and isolated by thin layer chromatography. The structures of these metabolites were elucidated using (1)H-NMR, broadband decoupled (13)C-NMR, EI Mass and IR spectroscopies. RESULTS: The fermentation yielded one reduced product: 17ß-hydroxyandrost-1,4-dien-3-one and two hydroxylated metabolites: 11α-hydroxyandrost-1,4-diene-3,17-dione and 12ß-hydroxyandrost-1,4-diene-3,17-dione. CONCLUSIONS: The results obtained in this study show that A. brasiliendsis could be considered as a biocatalyst for producing important derivatives from androstadienedione.
Assuntos
Androstadienos/metabolismo , Aspergillus/metabolismo , Androstadienos/química , Aspergillus/classificação , Biocatálise , Biotransformação , Cromatografia em Camada Fina , Fermentação , Microbiologia Industrial , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de TempoRESUMO
Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17ß-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.
Assuntos
Androstadienos/metabolismo , Inibidores da Aromatase/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Androstadienos/urina , Inibidores da Aromatase/urina , Dopagem Esportivo , Humanos , MasculinoRESUMO
The aim of this study was to synthesize three different D-homoandrostadiene derivatives (2-4) and study their biological activity. We carried out in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. It was also determined the binding of these compounds to the progesterone receptors (PR). Since these steroids have a new D-homoandrostandienone skeleton in their molecular structure, it was of interest also to study their binding to the androgen receptors (AR). After LHRH treatment, the mice of the control group showed the presence of 14+/-4 corpus lutea in the ovary whereas the animals treated with steroids 2-4, with RBAs of 100%, exhibited 11+/-7, 12+/-2, and 10+/-4 respectively. As a result of this study, it is evident that these steroids did not inhibit the ovulation in these animals. The uterus of the control group, showed the typical progestational activity with an enlarged endometrial thickness with a secretory activity. However, the endometrium of the mice treated with steroids 2-4 did not show an enlargement of the endometrium and no secretory activity could be detected. This fact indicates that compounds 2-4 had antagonistic activity in this tissue. The overall data show that steroids 2-4 are antagonists of the PR. However, they do not bind to the AR. These results also demonstrate that 2-4 have an antiprogestational activity in vivo, but do not decrease the number of corpus lutea in the ovary of mice treated with LHRH.
Assuntos
Androstadienos/química , Androstadienos/farmacologia , Receptores de Progesterona/antagonistas & inibidores , Antagonistas de Receptores de Andrógenos , Androstadienos/metabolismo , Androstenodiona/análogos & derivados , Androstenodiona/química , Androstenodiona/farmacologia , Animais , Ligação Competitiva , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Homosteroides/química , Homosteroides/farmacologia , Antagonistas de Hormônios/química , Antagonistas de Hormônios/metabolismo , Antagonistas de Hormônios/farmacologia , Masculino , Camundongos , Mifepristona/química , Mifepristona/metabolismo , Mifepristona/farmacologia , Estrutura Molecular , Ovário/efeitos dos fármacos , Ovário/metabolismo , Fenilacetatos/química , Progesterona/química , Progesterona/metabolismo , Progesterona/farmacologia , Coelhos , Ratos , Receptores Androgênicos/metabolismo , Receptores de Progesterona/metabolismo , Relação Estrutura-Atividade , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Very little is known about the signaling pathways by which catecholamines exert anabolic effects on muscle protein metabolism, stimulating protein synthesis and suppressing proteolysis. The present work tested the hypothesis that epinephrine-induced inhibition of muscle proteolysis is mediated through the cAMP/Epac/PI3K-dependent pathway with the involvement of AKT and Foxo. The incubation of extensor digitorum longus (EDL) muscles from rats with epinephrine and/or insulin increased the phosphorylation of AKT and its downstream target Foxo3a, a well-known effect that prevents Foxo translocation to the nucleus and the activation of proteolysis. Similar effects on AKT/Foxo signaling were observed in muscles incubated with DBcAMP (cAMP analog). The stimulatory effect of epinephrine on AKT phosphorylation was completely blocked by wortmannin (selective PI3K inhibitor), suggesting that the epinephrine-induced activation of AKT is mediated through PI3K. As for epinephrine and DBcAMP, the incubation of muscles with 8CPT-2Me-cAMP (selective Epac agonist) reduced rates of proteolysis and increased phosphorylation levels of AKT and Foxo3a. The specific PKA agonist (N6BZ-cAMP) inhibited proteolysis and abolished the epinephrine-induced AKT and Foxo3a phosphorylation. On the other hand, inhibition of PKA by H89 further increased the phosphorylation levels of AKT and Foxo3a induced by epinephrine, DBcAMP or 8CPT-2Me-cAMP. These findings suggest that the antiproteolytic effect of the epinephrine on isolated skeletal muscle may occur through a cAMP/Epac/PI3K-dependent pathway, which leads to the phosphorylation of AKT and Foxo3a. The parallel activation of PKA-dependent pathway also inhibits proteolysis and seems to limit the stimulatory effect of cAMP on AKT/Foxo3a signaling.
Assuntos
AMP Cíclico/metabolismo , Epinefrina/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Músculo Esquelético , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Androstadienos/metabolismo , Animais , Bucladesina/metabolismo , AMP Cíclico/análogos & derivados , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Insulina/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/metabolismo , Ratos , Ratos Wistar , Ribonucleosídeos/metabolismo , WortmaninaRESUMO
Glycogen synthase kinase GSK-3beta has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer's disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3beta, have not been completely elucidated. GSK-3beta activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3beta, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3beta activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology.
Assuntos
Córtex Cerebral/citologia , Quinase 3 da Glicogênio Sintase/metabolismo , Neurônios , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Androstadienos/metabolismo , Animais , Forma Celular , Ativação Enzimática , Glicogênio Sintase Quinase 3 beta , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Wortmanina , Proteínas tau/genéticaRESUMO
The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.
Assuntos
Insulina/farmacologia , Receptor de Insulina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Androstadienos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Cromatografia , Cromonas/metabolismo , Cricetinae , Relação Dose-Resposta a Droga , Indóis/metabolismo , Fosfatos de Inositol/metabolismo , Insulina/metabolismo , Maleimidas/metabolismo , Morfolinas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Ratos , Estaurosporina/metabolismo , WortmaninaRESUMO
Our objective was to evaluate the efficacy and safety of two doses of fluticasone propionate (FP) in young children with recurrent wheezing and risk factors for asthma. Our study design was a randomized, double-blind, placebo-controlled comparison of inhaled FP 50 mcg twice daily (FP 100) and 125 mcg twice daily (FP 250), for 6 months. Outcome measures included number of wheezing episodes, days on albuterol, height standard deviation score (height SDS), osteocalcin (OC), bone alkaline phosphatase fraction (AKP), insulin-like growth factor-binding protein 3 (IGFBP-3), and serum levels of cortisol (SC). Our subjects were 30 patients, aged 7-24 months. Mean wheezing episodes were 6.0 +/- 1.9, 1.9 +/- 1.9, and 2.8 +/- 1.2; mean days of albuterol use were 24.3 +/- 1.3, 6.5 +/- 0.8, and 9.1 +/- 0.8, per patient for placebo, FP100, and FP250 groups, respectively. There was a significant reduction in clinical outcome in the two FP groups compared to placebo (P < 0.01). No significant correlations were found between FP dosage and height SDS, OC, AKP, IGFBP-3, and SC. In conclusion, in young children with asthmatic symptoms, FP at 50 and 125 mcg b.i.d. for 6 months significantly improved respiratory symptoms without causing significant side effects on growth and bone metabolism.
Assuntos
Androstadienos/administração & dosagem , Asma/tratamento farmacológico , Broncodilatadores/administração & dosagem , Administração por Inalação , Albuterol/uso terapêutico , Androstadienos/metabolismo , Androstadienos/uso terapêutico , Asma/complicações , Osso e Ossos/metabolismo , Broncodilatadores/metabolismo , Broncodilatadores/uso terapêutico , Pré-Escolar , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Fluticasona , Crescimento/efeitos dos fármacos , Humanos , Lactente , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Recidiva , Sons Respiratórios/etiologia , Resultado do TratamentoRESUMO
The resistance to androstandienedione (ADD) of industrial mycobacteria was demonstrated as a valuable approach to increasing ADD yield in sterol fermentations. Colonies growing at 1 mg/ml ADD in culture medium after nitrosoguanidine mutagenesis showed a differential behavior in respect to parentals in cholesterol biotransformation. In the presence of exogenous ADD, a substantial depletion of ADD production was observed in parental strains B3683 and Ex4, whereas it was unaffected, and even increased, in resistant colonies. An apparent reduction from ADD to androstandione and testosterone was also noticed. Furthermore, the ADD resistance phenotype may be related to the increase in steroid 1,2 dehydrogenase activity.
Assuntos
Androstadienos/metabolismo , Androstanos/metabolismo , Bactérias/genética , Bactérias/metabolismo , Microbiologia Industrial/métodos , Androstadienos/toxicidade , Androstanos/toxicidade , Bactérias/crescimento & desenvolvimento , Biotransformação , Fermentação , Mutagênese , Esteróis/metabolismoRESUMO
In this work, phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of beta-sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g beta-sitosterol/l as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotransformation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC) and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains MYcobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of beta-sitosterol into steroidal bases.