RESUMO
The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.
Assuntos
Proteínas de Bactérias/metabolismo , Fatores Corda/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Decanoatos/metabolismo , Escherichia coli , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/ultraestrutura , Trealose/metabolismoRESUMO
Transient permeability enhancers (PEs), such as caprylate, caprate, and salcaprozate sodium (SNAC), improve the bioavailability of poorly permeable macromolecular drugs. However, the effects are variable across individuals and classes of macromolecular drugs and biologics. Here, we examined the influence of bile compositions on the ability of membrane incorporation of three transient PEs-caprylate, caprate, and SNAC-using coarse-grained molecular dynamics (CG-MD). The availability of free PE monomers, which are important near the absorption site, to become incorporated into the membrane was higher in fasted-state fluids than that in fed-state fluids. The simulations also showed that transmembrane perturbation, i.e., insertion of PEs into the membrane, is a key mechanism by which caprylate and caprate increase permeability. In contrast, SNAC was mainly adsorbed onto the membrane surface, indicating a different mode of action. Membrane incorporation of caprylate and caprate was also influenced by bile composition, with more incorporation into fasted- than fed-state fluids. The simulations of transient PE interaction with membranes were further evaluated using two experimental techniques: the quartz crystal microbalance with dissipation technique and total internal reflection fluorescence microscopy. The experimental results were in good agreement with the computational simulations. Finally, the kinetics of membrane insertion was studied with CG-MD. Variation in micelle composition affected the insertion rates of caprate monomer insertion and expulsion from the micelle surface. In conclusion, this study suggests that the bile composition and the luminal composition of the intestinal fluid are important factors contributing to the interindividual variability in the absorption of macromolecular drugs administered with transient PEs.
Assuntos
Bile/química , Caprilatos/administração & dosagem , Caprilatos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Decanoatos/administração & dosagem , Decanoatos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Substâncias Macromoleculares/administração & dosagem , Ácidos e Sais Biliares/metabolismo , Disponibilidade Biológica , Voluntários Saudáveis , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismoRESUMO
Microbially derived surfactants, so-called biosurfactants, have attracted significant attention as an environmentally friendly alternative to their chemically synthesized counterparts. Particularly, rhamnolipids offer a large potential with their outstanding surfactant properties such as complete biodegradability, low toxicity, and stability. Rhamnolipids are naturally synthesized by the opportunistic human pathogen Pseudomonas aeruginosa under the tight regulation of a highly complex quorum-sensing system. The heterologous production of mono-rhamnolipids by a newly isolated nonpathogenic strain of the genus Pantoea was investigated. Analysis of the genome obtained by a chimeric assembly of Nanopore long reads and high-quality Illumina reads suggested that the strain has evolved to an epiphytic rather than a pathogenic lifestyle. Functional heterologous expression of the mono-rhamnolipid operon rhlAB derived from a P. aeruginosa strain was established and confirmed by HPLC analysis. Transcriptome analysis indicated destabilizing effects of the produced rhamnolipids on the cell envelope of the host resulting in the induction of molecular stress responses. After integration of the rmlBCDA operon, extracellular rhamnolipids in amounts up to 0.4 g/L could be detected and were identified as a mono-rhamnolipid Rha-C10 -C10 by MALDI-TOF mass spectrometry.
Assuntos
Decanoatos/metabolismo , Glicolipídeos/biossíntese , Pantoea/genética , Pantoea/metabolismo , Pseudomonas aeruginosa/genética , Ramnose/análogos & derivados , Farmacorresistência Bacteriana Múltipla/genética , Perfilação da Expressão Gênica , Genes Bacterianos , Espectrometria de Massas , Engenharia Metabólica , Óperon , Pantoea/isolamento & purificação , Plasmídeos , Ramnose/metabolismo , Tensoativos/metabolismoRESUMO
Pseudomonas strains produce rhamnolipid mixtures (RLs) that generally consist of one or two molecules of rhamnose linked to one or two molecules of 3-hydroxyalkanoic acid. This study evaluates carbon source effects (glycerol, glucose, myristic acid, and Brassica carinata oil) on the synthesis of monorhamnolipids (mono-RLs) versus dirhamnolipids (di-RLs) in a human isolate of Pseudomonas aeruginosa PAL05. Spectrophotometry, an emulsifying index (E24) test, and an orcinol assay confirmed the production of RLs by PAL05. Purified RLs were characterized by 1H NMR analysis. PAL05 primarily produces mono-RLs when provided carbon sources containing long chain fatty acids (FAs) (myristic acid and B. carinata oil) and di-RLs when provided glycerol or glucose. qRT-PCR analysis showed that delayed expression of rhlC occurred when B. carinata oil was used, but not glycerol, glucose, or myristic acid. Our data show that the carbon source influenced the transcriptional expression of the rhlC gene and, consequently, the predominance of mono-RLs or di-RLs in PAL05 cultures.
Assuntos
Carbono/farmacologia , Decanoatos/metabolismo , Glicolipídeos/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/microbiologia , Ramnose/análogos & derivados , Emulsões/química , Glicolipídeos/isolamento & purificação , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ramnose/metabolismoRESUMO
The rationale of the present study was to evaluate the potential of microbial adjunct cultures including Kocuria varians and/or Yarrowia lipolytica strains in the recovery of the typical sensory profile of traditional (raw-milk) Tetilla cheese. Four batches of Tetilla cheese, a short ripened cows' milk cheese produced in Galicia (NW Spain), were made in duplicate from pasteurized milk inoculated with different microbial cultures. A control batch was manufactured by adding a mesophilic commercial D-starter only. The other three batches were made with the same starter after a cheese-milk pre-ripening step carried out with (i) an adjunct culture of K. varians, (ii) an adjunct culture of Y. lipolytica, or (iii) a combination of both adjunct cultures. The highest pH and water activity values, associated with softer textures were determined in the cheeses manufactured with the Y. lipolytica adjunct after 21days of ripening. The contents of the volatile compounds 3-methylbutanol, dimethyl disulfide and dimethyl trisulfide were higher in the cheeses made with only the K. varians adjunct than in the cheeses made with the only yeast adjunct and in the control cheeses. The contents of hexanoic and octanoic acids were highest in the cheeses made with the Y. lipolytica adjunct, and levels of ethyl hexanoate, ethyl octanoate and ethyl decanoate were higher in the cheeses made with only the yeast adjunct than in the other batches of cheese. The cheeses manufactured with both adjunct cultures were awarded the highest scores for flavour and overall sensory parameters (considering the standards of the traditional product) and were considered very similar to 'good quality' artisanal raw-milk cheeses. We conclude that use of selected Micrococcaceae and Y. lipolytica strains as adjunct cultures would differentiate the sensory properties and contribute to the quality and typicality of the short-ripened rennet-curd Galician Tetilla and Arzúa-Ulloa cheeses.
Assuntos
Queijo/microbiologia , Aromatizantes/química , Micrococcaceae/metabolismo , Leite/microbiologia , Yarrowia/metabolismo , Animais , Caproatos/metabolismo , Caprilatos/metabolismo , Bovinos , Queijo/análise , Decanoatos/metabolismo , Fermentação , Aromatizantes/análise , Microbiologia de Alimentos , Leite/química , Espanha , PaladarRESUMO
Canastra cheese is a cheese with geographical indication recognized by the Brazilian National Institute of Industrial Protection under number IG201002. It is produced in seven municipalities in the state of Minas Gerais in a region called Serra da Canastra. In this work, samples of milk, "pingo" (natural starter), whey and Canastra cheese were collected on a farm in Medeiros-MG/Brazil to evaluate the yeast microbiota and select yeasts for whey fermentation to produce ethanol and volatile aromatic compounds of relevance in the production of cheese. Thirty-nine isolates capable of fermenting lactose in a synthetic medium were identified by MALDI-TOF as Kluyveromyces lactis (29), Torulaspora delbrueckii (7) and Candida intermedia (3). Eleven isolates of K. lactis and three of T. delbrueckii efficiently fermented lactose until 4th day, and due to this reason were selected for cheese whey fermentation with Brix 12, 14 and 18. Generally, the isolates T. delbrueckii B14, B35, and B20 and K. lactis B10 were the most effective regardless of the initial Brix value. The identification of these four isolates by MALDI TOF was confirmed by sequencing of the ITS region. In the fermentation of cheese whey 14 Brix, T. delbrueckii B14 and B35, respectively yielded 24.06g/L and 16.45g/L of ethanol, while K. lactis B10 was more efficient in the consumption of lactose. In sequential culture with K. lactis B10 inoculated 48h after T. delbrueckii B14, 97.82% of the total sugars were consumed resulting in the production of 19.81g/L ethanol and 39 aromatic volatile compounds. The most abundant compounds were 3-methyl-1-butanol, octanoic acid and ethyl decanoate, which are reported as important for the aroma and flavor of cheeses. Based in our results, B10 isolate inoculated 48h after B14 isolate is a promising yeast inoculum to be used for fermentation of dairy substrates.
Assuntos
Queijo/microbiologia , Fermentação , Manipulação de Alimentos/métodos , Microbiologia de Alimentos/métodos , Soro do Leite/microbiologia , Leveduras/metabolismo , Brasil , Caprilatos/metabolismo , Decanoatos/metabolismo , Kluyveromyces/metabolismo , Lactose/metabolismo , Odorantes , Pentanóis/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Paladar , Torulaspora/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Leveduras/classificação , Leveduras/isolamento & purificaçãoRESUMO
The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.
Assuntos
Vias Biossintéticas/genética , Ácidos Graxos/metabolismo , Glicolipídeos/biossíntese , Glicolipídeos/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Decanoatos/metabolismo , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Mutação , Óperon , Pseudomonas aeruginosa/genética , Percepção de Quorum , Ramnose/análogos & derivados , Ramnose/metabolismo , TensoativosRESUMO
This study identified chemicals found on the eggs of two stink bug species, one native to western North America, Euschistus conspersus, and an invasive species from Asia, Halyomorpha halys. The responses of two scelionid egg parasitoids, Trissolcus erugatus and Telenomus podisi, toward natural stink bug egg volatiles, and synthetic reconstructions of egg volatiles, were tested in bioassays. A compound, methyl (2E,4Z)-2,4-decadienoate, previously identified as the major component of the male-produced aggregation pheromone of E. conspersus, was the major volatile identified from extracts of E. conspersus eggs. In contrast, for H. halys, the sesquiterpenoids that compose the male-produced aggregation pheromone of this species were not detected on eggs, whereas the presence of hexadecanal, octadecanal, and eicosanal was detected. In laboratory olfactometer tests, both Tr. erugatus and Te. podisi females were attracted to extracts of E. conspersus eggs, and to synthetic methyl (2E,4Z)-2,4-decadienoate. However, female Tr. erugatus and Te. podisi wasps were repelled, both by extracts of H. halys eggs and by a blend of the aldehydes identified from H. halys eggs. A follow-up field study, using hexane-washed and intact E. conspersus as sentinel eggs, showed that the parasitoids Trissolcus erugatus and Gryon obesum emerged from these eggs. Sentinel hexane-washed eggs treated with 3 ng of methyl (2E,4Z)-2,4-decadienoate were parasitized more by these two species than were hexane-washed or unwashed eggs, whereas hexane-washed eggs treated with a comparable dose of the C16,18,20 aldehyde mixture were avoided by these parasitoids. In a further field experiment, Trissolcus basalis was the primary parasitoid found in sticky traps baited with methyl (2E,4Z)-2,4-decadienoate, indicating that this species was attracted to, but either did not oviposit or develop in the E. conspersus sentinel eggs in the previous experiment.
Assuntos
Heterópteros/parasitologia , Óvulo/parasitologia , Vespas/fisiologia , Animais , Decanoatos/análise , Decanoatos/metabolismo , Feminino , Heterópteros/química , Heterópteros/fisiologia , Interações Hospedeiro-Parasita , Masculino , Oviposição , Óvulo/química , Óvulo/fisiologia , Controle de Pragas , Feromônios/análise , Feromônios/metabolismo , Olfato , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo , Vespas/anatomia & histologiaRESUMO
Mango kernel oil (MKO), derived from mango kernels, considered to be one of the highly generated agro-industrial waste, is assessed for its use as substrate for sustainable production of rhamnolipids. In the present study, MKO in combination with glucose gave maximum rhamnolipid yield of 2.8g/l which reduced the surface tension of water from 72 to 30mN/m, holding a CMC of 80mg/l and also showed high emulsification activity (73%) with diesel. Cell free broth was found to be stable even at high temperature (autoclaved at 121°C for 30min), pH value (up to pH 12) and salinity (up to 20% NaCl). The LC-MS data showed mono-rhamnolipid to be predominant congener followed by di-rhamnolipid in presence of MKO. Whereas, di-rhamnolipid was abundant when a combination of MKO with glucose was used. The produced rhamnolipid mixture showed good antifungal activity against various phytopathogens.
Assuntos
Antifúngicos/metabolismo , Glicolipídeos/biossíntese , Mangifera/metabolismo , Óleos de Plantas/química , Pseudomonas aeruginosa/metabolismo , Antifúngicos/farmacologia , Decanoatos/metabolismo , Resíduos Industriais , Mangifera/química , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Óleos de Plantas/metabolismo , Ramnose/análogos & derivados , Ramnose/metabolismo , Salinidade , Tensão Superficial , Tensoativos/metabolismoRESUMO
Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.
Assuntos
Decanoatos/metabolismo , Engenharia Metabólica , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Ramnose/análogos & derivados , Tensoativos/metabolismo , Animais , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Genoma Bacteriano , Redes e Vias Metabólicas/genética , Camundongos , Óperon , Plasmídeos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Ramnose/metabolismo , Análise de Sequência de DNA , VirulênciaRESUMO
A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Ácido Acético/metabolismo , Biofilmes/crescimento & desenvolvimento , Butileno Glicóis/metabolismo , Decanoatos/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Glicerol/metabolismo , Metabolômica , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Proteômica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Vinho/análiseRESUMO
The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified with octadecanol, glycerol, and dioleoylglycerol. These phenolic derivatives were treated in taurodeoxycholate microemulsion and unilamellar liposomes with ex vivo porcine skin and an aqueous extract of the skin. Extracted esterases hydrolyzed the microemulsions at rates in the order: tyrosyl lipoate > tyrosyl decanoate > hydroxytyrosyl lipoate > hydroxytyrosyl decanoate. The tyrosyl decanoate was subject to comparatively little hydrolysis (10-30% after 24h) when incorporated into liposomes, while hydroxytyrosyl decanoate in liposomes was not hydrolyzed at all by the skin extract. Ferulate esters were not hydrolyzed by the extract in aqueous buffer, microemulsion, nor liposomes. Tyrosyl decanoate applied topically to skin explants in microemulsion were readily hydrolyzed within 4h, while hydrolysis was minimal when applied in liposomes. These findings indicate that porcine skin displays a general esterase activity toward medium-chain esters of tyrosol and hydroxytyrosol, which can be moderated by the physiochemical properties of the lipid vehicle, but no feruloyl esterase activity.
Assuntos
Esterases/metabolismo , Fenol/metabolismo , Pele/enzimologia , Pele/metabolismo , Suínos/metabolismo , Animais , Decanoatos/química , Decanoatos/metabolismo , Emulsões/química , Emulsões/metabolismo , Esterases/química , Ésteres/química , Ésteres/metabolismo , Hidrólise , Lipossomos/química , Fenol/química , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/química , Álcool Feniletílico/metabolismo , Pele/químicaRESUMO
A mathematically based fed-batch bioprocess demonstrated the suitability of using a relatively cheap and renewable substrate (butyric acid) for Pseudomonas putida CA-3 high cell density cultivation. Butyric acid fine-tuned addition is critical to extend the fermentation run and avoid oxygen consumption while maximising the biomass volumetric productivity. A conservative submaximal growth rate (µ of 0.25 h(-1)) achieved 71.3 g L(-1) of biomass after 42 h of fed-batch growth. When a more ambitious feed rate was supplied in order to match a µ of 0.35 h(-1), the volumetric productivity was increased to 2.0 g L(-1) h(-1), corresponding to a run of 25 h and 50 g L(-1) of biomass. Both results represent the highest biomass and the best biomass volumetric productivity with butyrate as a sole carbon source. However, medium chain length polyhydroxyalkanoate (mcl-PHA) accumulation with butyrate grown cells is low (4 %). To achieve a higher mcl-PHA volumetric productivity, decanoate was supplied to butyrate grown cells. This strategy resulted in a PHA volumetric productivity of 4.57 g L(-1) h(-1) in the PHA production phase and 1.63 g L(-1) h(-1)over the lifetime of the fermentation, with a maximum mcl-PHA accumulation of 65 % of the cell dry weight.
Assuntos
Butiratos/metabolismo , Enzimas , Pseudomonas putida/enzimologia , Pseudomonas putida/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Biomassa , Biotransformação , Carbono/metabolismo , Decanoatos/metabolismo , Modelos Teóricos , Poli-Hidroxialcanoatos/metabolismoRESUMO
Poly(glycerol sebacate) (PGS) and poly(xylitol sebacate) (PXS) are biodegradable elastomers with tremendous potential in soft tissue engineering. This study was aimed at exploring the enzymatic degradation mechanisms of these polyesters, using biochemical conditions similar to those occurring in vivo. To this end, PGS and PXS (crosslinked at 130 for 2 or 7 (PGS)/12 days (PXS)) were incubated in vitro under physiological conditions in tissue culture media supplemented with either a biodegrading enzyme (esterase), an oxidant species (FeSO4/H2O2 with 0.11 molar ratio of Fe(2+/)H2O2), an oxidant generating enzyme (xanthine oxidase and xanthine) or combinations of these (FeSO4/H2O2 and esterase, or (v) xanthine oxidase/xanthine and esterase), based on their independent effects on polymer degradation. Testing was performed over 35 days of continuous incubation, during which mechanical properties, mass loss, biomaterial thickness and pH value of the culture medium were determined. Degradation kinetics of both PGS and PXS samples were primarily determined by the degree of crosslink density. Esterase and FeSO4/H2O2 accelerated the degradation of both polymers, by promoting hydrolysis and free-radical degradation, although this action was not affected by the presence of xanthine oxidase and xanthine. Degradation of PGS and PXS is primarily mediated by the action of esterase, with free-radical oxidation playing a secondary role, suggesting that both could synergistically affect the biodegradability of biomaterial implants, under more complex biological conditions.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Decanoatos/química , Decanoatos/metabolismo , Glicerol/análogos & derivados , Poliésteres/química , Poliésteres/metabolismo , Polímeros/química , Polímeros/metabolismo , Implantes Absorvíveis , Animais , Fenômenos Biomecânicos , Elastômeros/química , Elastômeros/metabolismo , Esterases/metabolismo , Glicerol/química , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Oxidantes/metabolismo , Oxirredução , Engenharia Tecidual , Xantina Oxidase/metabolismoRESUMO
This work describes the structural elucidation of the sex pheromone of the soybean stink bug, Pallantia macunaima. The biological activity of the synthetic pheromone was demonstrated by behavioral and EAD experiments. Furthermore, the absolute configuration of the natural pheromone was determined as (6R,10S)-6,10,13-trimethyltetradecan-2-one. This is the first ketone identified as a male-produced sex pheromone in Pentatomidae, and the trivial name, pallantione, was assigned to this novel pheromone molecule.
Assuntos
Decanoatos/isolamento & purificação , Hemípteros/química , Atrativos Sexuais/isolamento & purificação , Animais , Decanoatos/química , Decanoatos/metabolismo , Hemípteros/fisiologia , Masculino , Estrutura Molecular , Feromônios/química , Atrativos Sexuais/química , Atrativos Sexuais/metabolismo , EstereoisomerismoAssuntos
Materiais Biocompatíveis/química , Elastômeros/química , Animais , Materiais Biocompatíveis/toxicidade , Decanoatos/química , Decanoatos/metabolismo , Decanoatos/toxicidade , Glicerol/análogos & derivados , Glicerol/química , Glicerol/metabolismo , Glicerol/toxicidade , Humanos , Ácido Láctico/química , Ácido Láctico/metabolismo , Ácido Láctico/toxicidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácido Poliglicólico/química , Ácido Poliglicólico/metabolismo , Ácido Poliglicólico/toxicidade , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Polímeros/metabolismo , Polímeros/toxicidade , Engenharia TecidualRESUMO
Bacteria possess multiple mechanisms to survive exposure to various chemical stresses and antimicrobial compounds. In the enteric bacterium Escherichia coli, three homologous transcription factors-MarA, SoxS, and Rob-play a central role in coordinating this response. Three separate systems are known to regulate the expression and activities of MarA, SoxS, and Rob. However, a number of studies have shown that the three do not function in isolation but rather are coregulated through transcriptional cross talk. In this work, we systematically investigated the extent of transcriptional cross talk in the mar-sox-rob regulon. While the three transcription factors were found to have the potential to regulate each other's expression when ectopically expressed, the only significant interactions observed under physiological conditions were between mar and rob systems. MarA, SoxS, and Rob all activate the marRAB promoter, more so when they are induced by their respective inducers: salicylate, paraquat, and decanoate. None of the three proteins affects the soxS promoter, though unexpectedly, it was mildly repressed by decanoate by an unknown mechanism. SoxS is the only one of the three proteins to repress the rob promoter. Surprisingly, salicylate somewhat activates transcription of rob, while decanoate represses it a bit. Rob, in turn, activates not only its downstream promoters in response to salicylate but also the marRAB promoter. These results demonstrate that the mar and rob systems function together in response to salicylate.
Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regulon , Transcrição Gênica , Decanoatos/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Paraquat/metabolismo , Salicilatos/metabolismoRESUMO
Codling moth, Cydia pomonella (L.), larvae cause severe internal feeding damage to apples, pears, and walnuts worldwide. Research has demonstrated that codling moth neonate first instar larvae are attracted to a pear-derived kairomone, ethyl (2E,4Z)-2,4-decadienoate, the pear ester (PE). Reported here are the behavioral activities of neonate codling moth larvae to microencapsulated pear ester (MEC-PE) applied in aqueous solutions to both filter paper and apple leaf surfaces that were evaluated over a period of up to 20 d of aging. In dual-choice tests the MEC-PE treatment elicited attraction to and longer time spent on treated zones of filter papers relative to water-treated control zones for up to 14 d of aging. A higher concentration of MEC-PE caused no preferential response to the treated zone for the first 5 d of aging followed by significant responses through day 20 of aging, suggesting sensory adaptation as an initial concentration factor. Estimated emission levels of PE from treated filter papers were experimentally calculated for the observed behavioral thresholds evident over the aging period. When applied to apple leaves, MEC-PE changed neonate walking behavior by eliciting more frequent and longer time periods of arrestment and affected their ability to find the leaf base and stem or petiole. Effects of MEC-PE on extended walking time and arrestment by codling moth larvae would increase temporal and spatial exposure of neonates while on leaves; thereby potentially disrupting fruit or nut finding and enhancing mortality by increasing the exposure to insecticides, predation, and abiotic factors.
Assuntos
Decanoatos/metabolismo , Mariposas/fisiologia , Feromônios/metabolismo , Pyrus/química , Envelhecimento , Animais , Composição de Medicamentos , Comportamento Alimentar , Controle de Insetos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Malus , Mariposas/crescimento & desenvolvimento , Orientação , Tamanho da Partícula , Folhas de PlantaRESUMO
The accumulation of octanoic (OA) and decanoic (DA) acids in tissue is the common finding in medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), the most frequent defect of fatty acid oxidation. Affected patients present hypoketotic hypoglycemia, rhabdomyolysis, hepatomegaly, seizures and lethargy, which may progress to coma and death. At present, the pathophysiological mechanisms underlying hepatic and skeletal muscle alterations in affected patients are poorly known. Therefore, in the present work, we investigated the in vitro effects of OA and DA, the accumulating metabolites in MCADD, on various bioenergetics and oxidative stress parameters. It was verified that OA and DA decreased complexes I-III, II-III and IV activities in liver and also inhibit complex IV activity in skeletal muscle. In addition, DA decreased complexes II-III activity in skeletal muscle. We also verified that OA and DA increased TBA-RS levels and carbonyl content in both tissues. Finally, DA, but not OA, significantly decreased GSH levels in rat skeletal muscle. Our present data show that the medium-chain fatty acids that accumulate in MCADD impair electron transfer through respiratory chain and elicit oxidative damage in rat liver and skeletal muscle. It may be therefore presumed that these mechanisms are involved in the pathophysiology of the hepatopathy and rhabdomyolysis presented by MCADD-affected patients.
Assuntos
Caprilatos/metabolismo , Decanoatos/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/metabolismo , Animais , Caprilatos/farmacologia , Creatina Quinase/metabolismo , Decanoatos/farmacologia , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutationa/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Mitocôndrias/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Oxirredução , Carbonilação Proteica , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The biosurfactant produced by Pseudomonas desmolyticum NCIM 2112 (Pd 2112) was confirmed as rhamnolipid based on the formation of dark blue halos around the colonies in CTAB-methylene blue agar plates and the content of rhamnose sugar. The average yield of rhamnolipid was 0.398 g/l/day when grown on hexadecane as sole carbon source. Pd 2112 emulsification potential associated with cell free culture broth was stable for 72 h using various hydrocarbons and vegetable oils. Chemical structure of the biosurfactant was identified as mono-rhamnolipid (Rha-C(6) -C(8) ) using HPTLC, fourier transform infrared spectroscopy, (1) H and (13) C NMR and gas chromatography-mass spectroscopy analysis. Pd 2112 mono-rhamnolipid (1 mg/ml) had increased permeabilization of Bacillus sp VUS NCIM 5342 and increased decolorization rate of textile dye Brown 3REL by 50%. Extracellular activities of lignin peroxidase and veratryl alcohol oxidase, enzymes involved in dye degradation, were significantly increased in the presence of mono-rhamnolipid by 324.52% and 100% respectively. Scanning electron micro-scopy observations revealed that rhamnolipid did not exert any disruptive action on Bacillus cells as compared to Tween 80. The mono-rhamnolipid of Pd 2112 has potential for its application in biodegradation of textile dyes.