RESUMO
Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.
Assuntos
Hypericum , Hypericum/metabolismo , Hypericum/genética , Hypericum/química , Compostos Bicíclicos com Pontes/metabolismo , Compostos Bicíclicos com Pontes/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Simulação de Acoplamento Molecular , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Floroglucinol/química , Alcanos/metabolismo , Alcanos/química , Domínio Catalítico , Terpenos/metabolismo , Terpenos/química , Modelos MolecularesRESUMO
Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.
Assuntos
Carboxiliases , Polifenóis , Humanos , Polifenóis/metabolismo , Bactérias/metabolismo , Floroglucinol/metabolismo , Floretina/metabolismo , Carboxiliases/metabolismoRESUMO
Polyphenols are abundant in nature, and their anaerobic biodegradation by gut and soil bacteria is a topic of great interest. The O2 requirement of phenol oxidases is thought to explain the microbial inertness of phenolic compounds in anoxic environments, such as peatlands, termed the enzyme latch hypothesis. A caveat of this model is that certain phenols are known to be degraded by strict anaerobic bacteria, although the biochemical basis for this process is incompletely understood. Here, we report the discovery and characterization of a gene cluster in the environmental bacterium Clostridium scatologenes for the degradation phloroglucinol (1,3,5-trihydroxybenzene), a key intermediate in the anaerobic degradation of flavonoids and tannins, which constitute the most abundant polyphenols in nature. The gene cluster encodes the key C-C cleavage enzyme dihydrophloroglucinol cyclohydrolase, as well as (S)-3-hydroxy-5-oxo-hexanoate dehydrogenase and triacetate acetoacetate-lyase, which enable phloroglucinol to be utilized as a carbon and energy source. Bioinformatics studies revealed the presence of this gene cluster in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on human health and carbon preservation in peat soils and other anaerobic environmental niches. IMPORTANCE This study provides novel insights into the microbiota's anaerobic metabolism of phloroglucinol, a critical intermediate in the degradation of polyphenols in plants. Elucidation of this anaerobic pathway reveals enzymatic mechanisms for the degradation of phloroglucinol into short-chain fatty acids and acetyl-CoA, which are used as a carbon and energy source for bacterium growth. Bioinformatics studies suggested the prevalence of this pathway in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on carbon preservation in peat soils and human gut health.
Assuntos
Bactérias , Floroglucinol , Humanos , Floroglucinol/metabolismo , Anaerobiose , Bactérias/metabolismo , Bactérias Anaeróbias/metabolismo , Fenóis/metabolismo , Polifenóis/metabolismo , SoloRESUMO
Microtubules composed of α/ß tubulin heterodimers are an essential part of the cytoskeleton of eukaryotic cells and are widely regarded as targets for cancer chemotherapy. IC261, which is discovered as an ATP-competitive inhibitor of serine/threonine-specific casein kinase 1 (CK1), has shown its inhibitory activity on microtubule polymerization in recent studies. However, the structural information of the interaction between tubulin and IC261 is still unclear. Here, we provided a high-resolution (2.85 Å) crystal structure of tubulin and IC261 complex, revealed the intermolecular interaction between tubulin and IC261, and analyzed the structure-activity relationship (SAR). Subsequently, the structure of tubulin-IC261 complex was compared with tubulin-colchicine complex to further elucidate the novelty of IC261. Furthermore, eight optimal candidate compounds of new IC261-based microtubule inhibitors were obtained through molecular docking studies. In conclusion, the co-crystal structure of tubulin-IC261 complex paves a way for the design and development of microtubule inhibitor drugs.
Assuntos
Caseína Quinase I/antagonistas & inibidores , Desenho de Fármacos , Indóis/química , Microtúbulos/efeitos dos fármacos , Floroglucinol/análogos & derivados , Tubulina (Proteína)/química , Animais , Sítios de Ligação , Colchicina/química , Colchicina/metabolismo , Cristalografia por Raios X , Indóis/metabolismo , Simulação de Acoplamento Molecular , Floroglucinol/química , Floroglucinol/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Suínos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMO
BACKGROUND: The polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), produced by Pseudomonas fluorescens 2P24, is positively regulated by the GacS-GacA two-component system. RESULTS: Here we reported on the characterization of DsbA1 (disulfide oxidoreductase) as novel regulator of biocontrol activity in P. fluorescens. Our data showed that mutation of dsbA1 caused the accumulation of 2,4-DAPG in a GacA-independent manner. Further analysis indicated that DsbA1 interacts with membrane-bound glucose dehydrogenase Gcd, which positively regulates the production of 2,4-DAPG. Mutation of cysteine (C)-235, C275, and C578 of Gcd, significantly reduced the interaction with DsbA1, enhanced the activity of Gcd and increased 2,4-DAPG production. CONCLUSIONS: Our results suggest that DsbA1 regulates the 2,4-DAPG concentration via fine-tuning the function of Gcd in P. fluorescens 2P24.
Assuntos
Glucose Desidrogenase/metabolismo , Oxirredutases/genética , Floroglucinol/análogos & derivados , Pseudomonas fluorescens/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína , Regulação Bacteriana da Expressão Gênica , Glucose Desidrogenase/química , Glucose Desidrogenase/genética , Mutação , Oxirredutases/metabolismo , Floroglucinol/metabolismo , Ligação Proteica , Pseudomonas fluorescens/metabolismoRESUMO
Fifteen new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperforatones A-O (1-15), along with 3 structurally related analogues (16-18), were isolated from the stems and leaves of Hypericum perforatum. Their structures and absolute configurations were established by a combination of NMR spectroscopic analyses, experimental and calculated electronic circular dichroism (ECD), modified Mosher's methods, Rh2(OCOCF3)4- and [Mo2(OAc)4]-induced ECD, X-ray crystallography, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Compound 5 was found to be the first PPAP decorated by a rare 2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol moiety and an oxepane ring. Furthermore, the isolates were screened for their acetylcholinesterase (AChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities. Compounds 5, 10, 11, and 15 showed desirable AChE inhibitory activities (IC50 6.9-9.2 µM) and simultaneously inhibited BACE1 (at a concentration of 5 µM) with inhibition rates of 50.3%, 34.3%, 47.2%, and 34.6%, respectively. Interestingly, compound 5 showed the most balanced inhibitory activities against both AChE and BACE1 of all the tested compounds, which means that 5 could serve as the first valuable dual-targeted PPAP for the treatment of Alzheimer's disease. Preliminary molecular docking studies of 5 with BACE1 and AChE were also performed.
Assuntos
Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Hypericum/química , Floroglucinol/química , Floroglucinol/farmacologia , Compostos Policíclicos/química , Prenilação , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/uso terapêutico , Simulação de Acoplamento Molecular , Floroglucinol/metabolismo , Floroglucinol/uso terapêutico , Conformação ProteicaRESUMO
The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.
Assuntos
Encéfalo/enzimologia , Glicogênio Fosforilase/metabolismo , Floroglucinol/análogos & derivados , Pironas/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sítios de Ligação , Glicogênio Fosforilase/química , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Espectrometria de Massas , Simulação de Acoplamento Molecular , Floroglucinol/química , Floroglucinol/metabolismo , Estrutura Terciária de Proteína , Proteômica , Pironas/química , Ressonância de Plasmônio de SuperfícieRESUMO
Drug molecule interaction with human serum albumin (HSA) affects the distribution and elimination of the drug. The compound, 2,4-diacetylphloroglucinol (DAPG) has been known for its antimicrobial, antiviral, antihelminthic and anticancer properties. However, its interaction with HSA is not yet reported. In this study, the interaction between HSA and DAPG was investigated through steady-state fluorescence, time-resolved fluorescence (TRF), circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetry (ITC), molecular docking and molecular dynamics simulation (MDS). Fluorescence spectroscopy results showed the strong quenching of intrinsic fluorescence of HSA due to interaction with DAPG, through dynamic quenching mechanism. The compound bound to HSA with reversible and moderate affinity which explained its easy diffusion from circulatory system to target tissue. The thermodynamic parameters from fluorescence spectroscopic data clearly revealed the contribution of hydrophobic forces but, the role of hydrogen bonds was not negligible according to the ITC studies. The interaction was exothermic and spontaneous in nature. Binding with DAPG reduced the helical content of protein suggesting the unfolding of HSA. Site marker fluorescence experiments revealed the change in binding constant of DAPG in the presence of site I (warfarin) but not site II marker (ibuprofen) which confirmed that the DAPG bound to site I. ITC experiments also supported this as site I marker could not bind to HSA-DAPG complex while site II marker was accommodated in the complex. In silico studies further showed the lowest binding affinity and more stability of DAPG in site I than in site II. Thus the data presented in this study confirms the binding of DAPG to the site I of HSA which may help in further understanding of pharmacokinetic properties of DAPG.
Assuntos
Floroglucinol/análogos & derivados , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Calorimetria , Humanos , Simulação de Acoplamento Molecular , Floroglucinol/química , Floroglucinol/metabolismo , Ligação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Brown seaweeds such as Ascophyllum nodosum are a rich source of phlorotannins (oligomers and polymers of phloroglucinol units), a class of polyphenols that are unique to Phaeophyceae. At present, there is no information on the bioavailability of seaweed polyphenols and limited evidence on their bioactivity in vivo. Consequently, we investigated the gastrointestinal modifications in vitro of seaweed phlorotannins from A. nodosum and their bioavailability and effect on inflammatory markers in healthy participants. In vitro, some phlorotannin oligomers were identified after digestion and colonic fermentation. In addition, seven metabolites corresponding to in vitro-absorbed metabolites were identified. Urine and plasma samples contained a variety of metabolites attributed to both unconjugated and conjugated metabolites (glucuronides and/or sulphates). In both urine and plasma, the majority of the metabolites were found in samples collected at late time points (6-24 h), suggesting colonic metabolism of high-molecular-weight phlorotannins, with three phlorotannin oligomers (hydroxytrifuhalol A, 7-hydroxyeckol, C-O-C dimer of phloroglucinol) identified in urine samples. A significant increase of the cytokine IL-8 was also observed. Our study shows for the first time that seaweed phlorotannins are metabolised and absorbed, predominantly in the large intestine, and there is a large inter-individual variation in their metabolic profile. Three phlorotannin oligomers present in the capsule are excreted in urine. Our study is the first investigation of the metabolism and bioavailability of seaweed phlorotannins and the role of colonic biotransformation. In addition, IL-8 is a possible target for phlorotannin bioactivity.
Assuntos
Trato Gastrointestinal/metabolismo , Inflamação , Phaeophyceae/química , Floroglucinol/metabolismo , Floroglucinol/farmacocinética , Adolescente , Adulto , Idoso , Disponibilidade Biológica , Biomarcadores/sangue , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Citocinas/sangue , Digestão , Feminino , Humanos , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Fenóis/sangue , Fenóis/urina , Floroglucinol/farmacologia , Polímeros/metabolismo , Polímeros/farmacocinéticaRESUMO
The microbial polyketide, 2, 4-diacetylphloroglucinol (DAPG), exhibited a broad-spectrum of anti-leukemic, anti-lung, and anti-breast cancer properties. The aim of the present investigation was to study the interactive potentials of DAPG with the metastatic proteins such as MMP-2, MMP-9, and NF-κB and antiapoptotic Bcl-2 family proteins such as Bcl-2, Bcl-w, and Bcl-xL through in silico interaction and in vitro studies. The in silico modeling predicted high interactions of DAPG with the metastatic proteins, especially MMP-2, MMP-9, and NF-κB with the glide score of -7.028, -6.304, and -5.231, respectively. Similarly, the DAPG had weak interactions with the antiapoptotic Bcl-2, Bcl-w, and Bcl-xL with the glide score of -4.505, -3.839, and -4.003, respectively. The interaction studies further revealed the inhibition of MMP-2, MMP-9, and NF-κB activities with the low IC50 concentration of 5.82 ± 1.6, 6.74 ± 1.2, and 10.7 ± 1.5 µM respectively, in the presence of DAPG. Similarly, DAPG inhibited the Bcl-2, Bcl-xL and Bcl-w activities with the high IC50 concentration of 29.8 ± 1.9, 85.9 ± 2.7, and 97.4 ± 1.5 µM, respectively. These results correlate with the relatively high IC50 concentration of 16.3 ± 1.76, 7.67 ± 0.78, and 10.7 ± 0.96 µM in the Bcl-2-overexpressing HL-60, K562 and Raji leukemic cells than the metastatic A549 and MDA MB-231 cancer cells with the low IC50 concentration of 0.06 ± 0.02 and 0.08 ± 0.01 µM, respectively, compared to the healthy, human embryonic kidney (HEK-293) cells with the high IC50 concentration of 54.7 ± 1.43 µM. In summary, the affinity of DAPG with proteins are in the order of MMP-2 > MMP-9 > NF-κB > Bcl-2 > Bcl-xL > Bcl-w. Results presented in this study confirmed the high interaction of DAPG with the metastatic proteins than the antiapoptotic Bcl-2 family proteins.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/prevenção & controle , Leucemia/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Proteínas de Neoplasias/metabolismo , Floroglucinol/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Leucemia/patologia , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Metástase Neoplásica , Floroglucinol/metabolismo , Floroglucinol/farmacologiaRESUMO
The 2,4-diacetylphloroglucinol (DAPG), a polyketide metabolite extracted from Pseudomonas aeruginosa strain FP10, exhibited selective cytoxicity against lung (A549), breast (MDA MB-231), cervical (HeLa) and colon (HCT-15) cancer cells in differential and dose-dependent manner. The anticancer and antimetastatic activities of DAPG were mediated by the inhibition of ROS, NF-κB, Bcl-2, MMP-2, VEGF and primary inflammatory mediators such as TNF-α, IL-6, IL-1ß and NO. The DAPG induced apoptosis in cancer cells by intrinsic and extrinsic pathways via the release of cytochrome-C, upregulation of Bax and the activation of caspases and also, exhibited anti-inflammatory activity by the inhibition of LPS-inflammed cell proliferation of macrophage (Raw 264.7), monocytic cells (THP-1) and peripheral blood mononuclear cells (PBMCs). Results further confirmed that the DAPG inhibited the primary inflammatory mediators in cancer cells and inflammed immune cells through the down regulation of NF-κB. In the present study, for the first time, antiproliferative, proapoptotic, antimetastatic and anti-inflammatory activities of DAPG in various cancer cells and inflammation-induced immune cells have been reported.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inflamação/metabolismo , Invasividade Neoplásica/prevenção & controle , Floroglucinol/análogos & derivados , Policetídeos/farmacologia , Pseudomonas aeruginosa/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Camundongos , Floroglucinol/isolamento & purificação , Floroglucinol/metabolismo , Floroglucinol/farmacologia , Policetídeos/isolamento & purificação , Policetídeos/metabolismo , GravidezRESUMO
A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.
Assuntos
Fucus/metabolismo , Fucus/efeitos da radiação , Floroglucinol/metabolismo , Transcriptoma/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Fucus/genética , Fucus/fisiologia , Peso Molecular , Floroglucinol/químicaRESUMO
Tegula tridentata, is a common herbivore gastropod inhabiting the subtidal Lessonia trabeculata kelp forest, which tends to show higher densities after kelp harvesting. We investigated if harvested kelp beds may harbor higher densities of herbivore invertebrates, and the underlying mechanisms. Thus, we evaluated if the exudates of L. trabeculata change the seawater levels of soluble phenols, known to have a deterrent effect against the feeding behavior of some herbivore invertebrates. Finally we investigated whether the increase in T. tridentata densities in harvested kelp grounds could be related to a decrease in the seawater levels of soluble phenols. Our results showed that the density of invertebrate herbivores increased up to 32% in harvested kelp grounds. We provide the first estimate of the rate of phenolic exudation by L. trabeculata, and we demonstrate that T. tridentata changes its food dependent movement in the presence of exudates with synthetic phloroglucinol. We suggest that the recovery of harvested kelp ecosystems can be jeopardized by increased herbivory triggered by water-borne changes in the levels of herbivore deterrent compounds.
Assuntos
Cadeia Alimentar , Atividade Motora , Phaeophyceae/metabolismo , Fenóis/metabolismo , Caramujos/fisiologia , Animais , Quimiotaxia , Chile , Floroglucinol/metabolismo , Dinâmica Populacional , Água do Mar/análiseRESUMO
Among the most renowned natural products from brown algae, phlorotannins are phloroglucinol polymers that have been extensively studied, both for their biotechnological potential and their interest in chemical ecology. The accurate quantification of these compounds is a key point to understand their role as mediators of chemical defense. In recent years, the Folin-Ciocalteu assay has remained a classic protocol for phlorotannin quantification, even though it frequently leads to over-estimations. Furthermore, the quantification of the whole pool of phlorotannins may not be relevant in ecological surveys. In this study, we propose a rapid (1)H qNMR method for the quantification of phlorotannins. We identified phloroglucinol as the main phenolic compound produced by the brown macroalga Cystoseira tamariscifolia. This monomer was detected in vivo using (1)H HR-MAS spectroscopy. We quantified this molecule through (1)H qNMR experiments using TSP as internal standard. The results are discussed by comparison with a standard Folin-Ciocalteu assay performed on purified extracts. The accuracy and simplicity of qNMR makes this method a good candidate as a standard phlorotannin assay.
Assuntos
Floroglucinol/análise , França , Espectroscopia de Ressonância Magnética , Phaeophyceae/metabolismo , Floroglucinol/metabolismo , Taninos/análiseRESUMO
Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC50 of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.
Assuntos
Antineoplásicos/metabolismo , Ascomicetos/isolamento & purificação , Ascomicetos/metabolismo , Endófitos/isolamento & purificação , Endófitos/metabolismo , Myrtus/microbiologia , Floroglucinol/metabolismo , Ascomicetos/classificação , Ascomicetos/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Endófitos/classificação , Endófitos/genética , Humanos , Concentração Inibidora 50 , Masculino , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Análise de Sequência de DNARESUMO
Hyperforin is well-known for its anti-inflammatory, anti-tumor, anti-bacterial, and antioxidant properties. The application of a hyperforin-rich verum cream could strengthen the skin barrier function by reducing radical formation and stabilizing stratum corneum lipids. Here, it was investigated whether topical treatment with a hyperforin-rich cream increases the radical protection of the skin during VIS/NIR irradiation. Skin lipid profile was investigated applying HPTLC on skin lipid extracts. Furthermore, the absorption- and scattering coefficients, which influence radical formation, were determined. 11 volunteers were included in this study. After a single cream application, VIS/NIR-induced radical formation could be completely inhibited by both verum and placebo showing an immediate protection. After an application period of 4weeks, radical formation could be significantly reduced by 45% following placebo application and 78% after verum application showing a long-term protection. Furthermore, the skin lipids in both verum and placebo groups increased directly after a single cream application but only significantly for ceramide [AP], [NP1], and squalene. After long-term cream application, concentration of cholesterol and the ceramides increased, but no significance was observed. These results indicate that regular application of the hyperforin-rich cream can reduce radical formation and can stabilize skin lipids, which are responsible for the barrier function.
Assuntos
Anti-Inflamatórios/administração & dosagem , Sequestradores de Radicais Livres/administração & dosagem , Floroglucinol/análogos & derivados , Creme para a Pele/uso terapêutico , Pele/efeitos dos fármacos , Terpenos/administração & dosagem , Adulto , Anti-Inflamatórios/metabolismo , Ceramidas/metabolismo , Feminino , Sequestradores de Radicais Livres/metabolismo , Humanos , Lipídeos , Masculino , Floroglucinol/administração & dosagem , Floroglucinol/metabolismo , Pele/metabolismo , Absorção Cutânea , Creme para a Pele/metabolismo , Terpenos/metabolismo , Adulto JovemRESUMO
Pseudomonas strains producing 2,4-diacetylphloroglucinol (DAPG) can protect plants from soilborne phytopathogens and are considered the primary reason for suppressiveness of morainic Swiss soils to Thielaviopsis basicola-mediated black root-rot disease of tobacco, even though they also occur nearby in conducive sandstone soils. The underlying molecular mechanisms accounting for this discrepancy are not understood. In this study, we assessed the hypothesis that the presence of iron-rich vermiculite clay (dominant in suppressive soils) instead of illite (dominant in neighboring conducive soils) translates into higher levels of iron bioavailability and transcription of Pseudomonas DAPG synthetic genes in the tobacco rhizosphere. Rhizosphere monitoring of reporter gene systems pvd-inaZ and phlA-gfp in Pseudomonas protegens indicated that the level of iron bioavailability and the number of cells expressing phl genes (DAPG synthesis), respectively, were higher in vermiculitic than in illitic artificial soils. This was in accordance with the effect of iron on phlA-gfp expression in vitro and, indeed, iron addition to the illitic soil increased the number of cells expressing phlA-gfp. Similar findings were made in the presence of the pathogen T. basicola. Altogether, results substantiate the hypothesis that iron-releasing minerals may confer disease suppressiveness by modulating iron bioavailability in the rhizosphere and expression of biocontrol-relevant genes in antagonistic P. protegens.
Assuntos
Ferro/metabolismo , Minerais/química , Pseudomonas/efeitos dos fármacos , Pseudomonas/metabolismo , Minerais/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , RizosferaRESUMO
Anther formation and dehiscence are complex pivotal processes in reproductive development. The secondary wall thickening in endothecial cells of the anther is a known prerequisite for successful anther dehiscence. However, many gaps remain in our understanding of the regulatory mechanisms underlying anther dehiscence in planta, including a possible role for jasmonic acid (JA) and H(2)O(2) in secondary wall thickening of endothecial cells. Here, we report that the cystathionine ß-synthase domain-containing protein CBSX2 located in the chloroplast plays a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in Arabidopsis. A T-DNA insertion mutant of CBSX2 (cbsx2) showed increased secondary wall thickening of endothecial cells and early anther dehiscence. Consistently, overexpression of CBSX2 resulted in anther indehiscence. Exogenous JA application induced secondary wall thickening and caused flower infertility in the cbsx2 mutant, whereas it partially restored fertility in the CBSX2-overexpressing lines lacking the wall thickening. CBSX2 directly modulated thioredoxin (Trx) in chloroplasts, which affected the level of H(2)O(2) and, consequently, expression of the genes involved in secondary cell wall thickening. Our findings have revealed that CBSX2 modulates the H(2)O(2) status, which is linked to the JA response and in turn controls secondary wall thickening of the endothecial cells in anthers for dehiscence to occur.
Assuntos
Proteínas de Arabidopsis/metabolismo , Parede Celular/enzimologia , Cistationina beta-Sintase/metabolismo , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Cloroplastos/efeitos dos fármacos , Cloroplastos/enzimologia , Cloroplastos/genética , Ciclopentanos/farmacologia , Cistationina beta-Sintase/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Flores/enzimologia , Flores/genética , Flores/ultraestrutura , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Oxilipinas/farmacologia , Floroglucinol/metabolismo , Infertilidade das Plantas , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 µM).
Assuntos
Brassica/enzimologia , Catecol Oxidase/química , Catecol Oxidase/isolamento & purificação , Catecol Oxidase/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oxirredução , Floroglucinol/metabolismo , Especificidade por SubstratoRESUMO
Four new acylphloroglucinols with an unusual 6/6/5 spirocyclic skeleton, hyperbeanols A-D (1-4), were isolated from the methanol extract of Hypericum beanii along with 16 known compounds. Their structures were established on the basis of spectroscopic and X-ray diffraction analysis. Hyperbeanols A-C were three stereoisomers different only at the relative configuration of C-4 and C-13, which were distinguished by the nuclear Overhauser effect spectroscopy (NOESY) spectroscopic data in combination with the single X-ray analysis of hyperbeanol A (1). The cytotoxic activity of hyperbeanols A-D against the cancer cell lines SK-BR-3, HL-60, SMMC-7721, PANC-1, MCF-7, and K562 was also evaluated.