RESUMO
It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~SrfâGS+Srf correlated to apparent K d values of 6-9 µM in gas-phase and 1 µM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low µM K dapp values of GS~SrfâGS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract á .
Assuntos
Anti-Infecciosos/química , Gramicidina/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Sítios de Ligação , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Quebracho (Schinopsis lorentzii and Schinopsis balansae) extract is an important source of natural polymers for leather tanning and adhesive manufacturing. We combined established phyto- and synthetic chemistry perspectives with electrospray mass spectrometry experiments to prove that quebracho proanthocyanidin polymers consist of an homologous series of flavan-3-ol based oligomers. The starter unit is always catechin which is angularly bonded to fisetinidol extender units. By comparison of the MS(2) fragmentation spectra of the oligomer with product ion scans of authentic catechin and robinetinidol samples, we proved that quebracho extract contains no robinetinidol, as is often reported. Quebracho proanthocyanidins have acid resistant interflavanyl bonds, due to the absence of 5-OH groups in fisetinidol, and the aDP cannot be determined via conventional thiolysis and phloroglucinolysis. We used the MS data to estimate a conservative (minimum value) aDP of 3.1.
Assuntos
Anacardiaceae/química , Flavonoides/análise , Proantocianidinas/análise , Catequina/análise , Catequina/química , Flavonoides/química , Flavonoides/isolamento & purificação , Estrutura Molecular , Proantocianidinas/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
During the development of a method for quantitative determination of venlafaxine and its major metabolite O-desmethylvenlafaxine, elevated concentrations of the analyte as well as co-eluting matrix compounds caused ion suppression. This ion suppression was inconsistent and therefore influenced the reproducibility of detection. The use of atmospheric pressure photoionization (APPI) in the positive mode was investigated as a tool to circumvent this problem. Employing APPI resulted in negligible ion suppression and increased linearity of the concentration range. A selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of venlafaxine and its major metabolite O-desmethylvenlafaxine in human plasma was developed. The analyte was extracted from plasma into tert-butyl methyl ether followed by back extraction into 2% formic acid. An Agilent 1100 high-performance liquid chromatography (HPLC) system, employing reversed-phase chromatography on a cyano column, coupled to an Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, was used for separation and detection of the analytes. The method was validated between 2.36-605 ng per mL with a mean recovery of approximately 88% for both parent compound and metabolite analytes. APPI technology was employed to improve the reproducibility of detection enabling rapid, selective and sensitive quantification of venlafaxine and O-desmethylvenlafaxine in human plasma samples.
Assuntos
Antidepressivos de Segunda Geração/análise , Cicloexanóis/análise , Antidepressivos de Segunda Geração/sangue , Antidepressivos de Segunda Geração/farmacocinética , Biotransformação , Calibragem , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cicloexanóis/sangue , Cicloexanóis/farmacocinética , Succinato de Desvenlafaxina , Análise de Injeção de Fluxo , Humanos , Indicadores e Reagentes , Modelos Lineares , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Cloridrato de VenlafaxinaRESUMO
Cytochrome b5 (cyt b5) is an ubiquitous hemoprotein also associated with microsomal cytochromes P450. It has been reported that cyt b5 influences cytochrome P450-dependent catalyses through electron transport as well as direct protein-protein interactions. To investigate the influence of cyt b5 on ovine adrenal steroidogenesis, we isolated and characterized cyt b5 from ovine liver. The molecular mass of the purified protein was 15,260 as determined by electrospray mass spectrometry. SDS-Polyacrylamide gel electrophoresis, even after stringent detergent and mercaptoethanol pretreatment, indicated multimeric forms of the protein, the most prominent being the tetramer (+/-60 kDa) with minor bands corresponding to the monomer (+/-16 kDa) and dimer (+/-30 kDa). Trypsin treatment of cyt b5 resulted in a truncated enzyme with a molecular mass of +/-10 kDa. The aggregation of cytochrome b5 was abolished by the tryptic removal of the membrane binding region. In Western blot analyses antibodies against the truncated protein recognised only this low molecular mass form and not the full length cyt b5, or any of the higher molecular complexes, showing the involvement of the membrane binding domain of the protein, not only in aggregation, but also in the quaternary structure which determines epitope presentation for antibody production. Immunoblot analyses of sheep adrenal microsomes with the anti-truncated cyt b5 antibody were also negative. Immunoblot analyses and immunocytochemistry of adrenal tissue with antibodies against the full length cyt b5 indicated that the tetrameric form of the protein was in all probability the dominant specie in vivo.
Assuntos
Glândulas Suprarrenais/metabolismo , Citocromos b5/química , Citocromos b5/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Citocromos b5/efeitos dos fármacos , Soros Imunes , Microssomos/metabolismo , Peso Molecular , Testes de Precipitina , Progesterona/metabolismo , Isoformas de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Ovinos , Espectrometria de Massas por Ionização por Electrospray , Tripsina/farmacologiaRESUMO
A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L. monocytogenes strains. Two wild-type strains, L. monocytogenes B73 and 412, two spontaneous mutants of L. monocytogenes B73 with intermediate resistance to leucocin A (+/-2.4 and +/-4 times the 50% inhibitory concentrations [IC50] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC50 for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant L. monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (L. monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L. monocytogenes strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.
Assuntos
Bacteriocinas/farmacologia , Membrana Celular/metabolismo , Farmacorresistência Bacteriana/fisiologia , Listeria monocytogenes/metabolismo , Fosfatidilgliceróis/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Concentração Inibidora 50 , Listeria monocytogenes/química , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/química , Fosfolipídeos/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.