RESUMEN
The HepQuant SHUNT test quantifies liver function and blood flow using systemic and portal clearances of cholate. The test can identify the risk of well-compensated patients to develop complications of cirrhosis. To confirm the reliability of a single HepQuant SHUNT test we defined its within-individual reproducibility. Healthy subjects (nâ¯=â¯16), 16 with nonalcoholic steatohepatitis (NASH), and 16 with chronic hepatitis C virus (HCV) underwent 3 HepQuant SHUNT tests on 3 separate days within 30 days. The test involves simultaneous administration of 20 mg 13C-cholate IV and 40 mg d4-cholate PO, and subsequent collection of 3 mL blood samples at 5, 20, 45, 60, and 90 minutes. Clearances are expressed as systemic and portal hepatic filtration rate. Portal-systemic shunting (SHUNT), a disease severity index (DSI), and an estimate of DSI (STAT) are calculated from the clearances. Reproducibility was determined by the intraclass correlation coefficient (ICC > 0.70) and Bland-Altman analysis. Equal numbers of NASH and HCV patients had either early (F0-F2) or advanced (F3/F4) stages of fibrosis. All F3/F4 subjects were clinically compensated. The intraclass correlation coefficient (ICC) for DSI was 0.94 (0.90-0.96 95% confidence interval) indicating excellent reproducibility. The other test parameters had ICCs ranging from 0.74 (SHUNT) to 0.90 (STAT). In Bland-Altman analysis, the mean of differences between measurements of DSI was 0.13 with standard deviation 2.12. The excellent reproducibility of the HepQuant SHUNT test, particularly DSI, supports the use this minimally invasive, blood-based test as a reliable test of liver function and physiology.
Asunto(s)
Pruebas de Función Hepática/métodos , Hígado/fisiología , Adulto , Isótopos de Carbono , Colatos/administración & dosificación , Colatos/sangre , Colatos/química , Deuterio , Femenino , Voluntarios Sanos , Hepatitis C Crónica/fisiopatología , Humanos , Hígado/irrigación sanguínea , Circulación Hepática/fisiología , Pruebas de Función Hepática/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Valores de Referencia , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Investigación Biomédica Traslacional , Adulto JovenRESUMEN
In this article, capillary electrophoresis was applied to investigate the chiral recognition mechanism for the enantioseparation on a well-known second-generation photodynamic therapy drug of benzoporphyrin derivative monoacid ring A, that is, verteporfin. In our previous study, cholate salts have been studied as the chiral selectors, which can realize baseline separation of the four verteporfin isomers. Aiming to reveal the chiral recognition mechanism, the separation effect of several kinds of chiral selectors was discussed. According to the results and references, the chiral separation mechanism of this system was concluded: the analytes selectively combine with the chiral micelles, that is, dynamic H-bonds interactions occur between the hydroxyl groups on the outer side of the cholate micelles and the ester/carboxy groups of the four isomers. In addition, the role of dimethyl formamide as an organic modifier was also researched, including reducing the effective mobility of the analytes and mobility of electroosmotic flow, and preventing them from adsorbing to the capillary wall and self-aggregating of verteporfin, which are pretty beneficial for separation. The method used in this article provides a direct and reliable solution to study the mechanism of chiral separation.
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Colatos/química , Verteporfina/química , Electroforesis Capilar , Conformación Molecular , Sales (Química)/química , EstereoisomerismoRESUMEN
Background: Quercetin (QUE) shows a potential antileukemic activity, but possesses poor solubility and low bioavailability. Purpose: This article explored the bile salt transport pathway for oral deliver of QUE using cholate-modified polymer-lipid hybrid nanoparticles (cPLNs) aiming to enhance its antileukemic effect. Methods: QUE-loaded cPLNs (QUE-cPLNs) were developed through a nanoprecipitation technique and characterized by particle size, entrapment efficiency (EE), microscopic morphology and in vitro drug release. In vitro cellular uptake and cytotoxicity of QUE-cPLNs were examined on Caco-2 and P388 cells; in vivo pharmacokinetics and antileukemic effect were evaluated using Sprague Dawley rats and leukemic model mice, respectively. Results: The prepared QUE-cPLNs possessed a particle size of 110 nm around with an EE of 96.22%. QUE-cPLNs resulted in significantly enhanced bioavailability of QUE, up to 375.12% relative to the formulation of suspensions. In addition, QUE-cPLNs exhibited excellent cellular uptake and internalization capability compared to cholate-free QUE-PLNs. The in vitro cytotoxic and in vivo antileukemic effects of QUE-cPLNs were also signally superior to free QUE and QUE-PLNs. Conclusion: These findings indicate that cPLNs are a promising nanocarrier able to improve the oral bioavailability and therapeutic index of QUE.
Asunto(s)
Antineoplásicos/farmacología , Colatos/química , Lípidos/química , Nanopartículas/química , Polímeros/química , Quercetina/administración & dosificación , Administración Oral , Animales , Área Bajo la Curva , Células CACO-2 , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Liberación de Fármacos , Sinergismo Farmacológico , Endocitosis/efectos de los fármacos , Humanos , Leucemia/patología , Ratones , Nanopartículas/ultraestructura , Tamaño de la Partícula , Quercetina/farmacocinética , Quercetina/farmacología , Ratas Sprague-DawleyRESUMEN
Combining micellar electrokinetic capillary chromatography (MEKC) and nuclear magnetic resonance (NMR) experimentation, we shed light on the structural basis for the chirally selective solubilization of atropisomeric binaphthyl compounds by bile salt micelles comprised of cholate (NaC) or deoxycholate (NaDC). The model binaphthyl analyte R,S-BNDHP exhibits chirally selective interactions with primary micellar aggregates of cholate and deoxycholate, as does the closely related analyte binaphthol (R,S-BN). Chiral selectivity was localized, by NMR chemical shift analysis, to the proton at the C12 position of these bile acids. Correspondingly, MEKC results show that the 12α-OH group of either NaC or NaDC is necessary for chirally selective resolution of these model binaphthyl analytes by bile micelles, and the S isomer is more highly retained by the micelles. With NMR, the chemical shift of 12ß-H was perturbed more strongly in the presence of S-BNDHP than R-BNDHP. Intermolecular NOEs demonstrate that R,S-BNDHP and R,S-BN interact with a similar hydrophobic planar pocket lined with the methyl groups of the bile salts, and are best explained by the existence of an antiparallel dimeric unit of bile salts. Finally, chemical shift data and intermolecular NOEs support different interactions of the enantiomers with the edges of dimeric bile units, indicating that R,S-BNDHP enantiomers sample the same binding site preferentially from opposite edges of the dimeric bile unit. Chirality 28:525-533, 2016. © 2016 Wiley Periodicals, Inc.
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Colatos/química , Ácido Desoxicólico/química , Naftalenos/química , Organofosfatos/química , Cromatografía Capilar Electrocinética Micelar , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Naftoles/química , Solubilidad , EstereoisomerismoRESUMEN
Amphiphilic polycarbonate/PEG copolymer with a star-like architecture was designed to facilitate a unique supramolecular transformation of micelles to vesicles in aqueous solution for the efficient delivery of anticancer drugs. The star-shaped amphipilic block copolymer was synthesized by initiating the ring-opening polymerization of trimethylene carbonate (TMC) from methyl cholate through a combination of metal-free organo-catalytic living ring-opening polymerization and post-polymerization chain-end derivatization strategies. Subsequently, the self-assembly of the star-like polymer in aqueous solution into nanosized vesicles for anti-cancer drug delivery was studied. DOX was physically encapsulated into vesicles by dialysis and drug loading level was significant (22.5% in weight) for DOX. Importantly, DOX-loaded nanoparticles self-assembled from the star-like copolymer exhibited greater kinetic stability and higher DOX loading capacity than micelles prepared from cholesterol-initiated diblock analogue. The advantageous disparity is believed to be due to the transformation of micelles (diblock copolymer) to vesicles (star-like block copolymer) that possess greater core space for drug loading as well as the ability of such supramolecular structures to encapsulate DOX. DOX-loaded vesicles effectively inhibited the proliferation of 4T1, MDA-MB-231 and BT-474 cells, with IC50 values of 10, 1.5 and 1.0mg/L, respectively. DOX-loaded vesicles injected into 4T1 tumor-bearing mice exhibited enhanced accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect. Importantly, DOX-loaded vesicles demonstrated greater tumor growth inhibition than free DOX without causing significant body weight loss or cardiotoxicity. The unique ability of the star-like copolymer emanating from the methyl cholate core provided the requisite modification in the block copolymer interfacial curvature to generate vesicles of high loading capacity for DOX with significant kinetic stability that have potential for use as an anti-cancer drug delivery carrier for cancer therapy.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/química , Polímeros/química , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Línea Celular , Proliferación Celular/efectos de los fármacos , Colatos/química , Preparaciones de Acción Retardada , Dioxanos , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Conformación Molecular , Nanopartículas , Cemento de Policarboxilato , Polietilenglicoles , Polimerizacion , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Membrane curvature is an important parameter in biological processes such as cellular movement, division, and vesicle fusion and budding. Traditionally, only proteins and protein-derived peptides have been used as sensors for membrane curvature. Three water-soluble bischolate foldamers were synthesized, all labeled with an environmentally sensitive fluorophore to report their binding with lipid membranes. The orientation and ionic nature of the fluorescent label were found to be particularly important in their performance as membrane-curvature sensors. The bischolate with an NBD group in the hydrophilic α-face of the cholate outperformed the other two analogues as a membrane-curvature sensor and responded additionally to the lipid composition including the amounts of cholesterol and anionic lipids in the membranes.
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Colatos/química , Lípidos de la Membrana/química , Agua/química , SolubilidadRESUMEN
Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection.
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Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Biflavonoides/química , Biflavonoides/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Colatos/química , Colatos/metabolismo , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/metabolismo , Humanos , Cinética , Necrosis , Floretina/química , Floretina/metabolismo , Unión Proteica , Bibliotecas de Moléculas Pequeñas/metabolismo , Células VeroRESUMEN
Herein, we present the results obtained from our studies on supramolecular self-assembly and molecular mobility of low-molecular-weight gelators (LMWGs) in organic solvents using pulsed field gradient (PFG) diffusion ordered spectroscopy (DOSY) NMR. A series of concentration-dependent DOSY NMR experiments were performed on selected LMWGs to determine the critical gelation concentration (CGC) as well as to understand the behaviour of the gelator molecules in the gel state. In addition, variable-temperature DOSY NMR experiments were performed to determine the gel-to-sol transition. The PFG NMR experiments performed as a function of gradient strength were further analyzed using monoexponential DOSY processing, and the results were compared with the automated Bayesian DOSY transformation to obtain 2D plots. Our results provide useful information on the stepwise self-assembly of small molecules leading to gelation. We believe that the results obtained from these experiments are applicable in determining the CGC and gel melting temperatures of supramolecular gels.
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Colatos/síntesis química , Geles/síntesis química , Colatos/química , Geles/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Peso MolecularRESUMEN
We describe cholate-based cage amphiphiles with a unique architecture that combines elements of structural rigidity and flexibility. The cage compounds are built by extending and bridging three polar chains underneath the concave steroid rings of cholate and capping with another rigid, symmetrically trifunctionalized cyanuric acid moiety. The connecting chains are varied to include, for instance, oligo(ethylene glycol) or chains containing 1,2,3-triazole units to present flexibility in the chemical and structural space and potentially deliver functional molecules for molecular recognition applications.
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Colatos/química , Glicol de Etileno/química , Triazoles/química , Estructura MolecularRESUMEN
Hydrophobic derivatives of highly methylated citrus pectin, chitosan and cellulose were prepared and tested as potential cholesterol lowering agents. Elemental analysis and spectroscopic methods confirmed high substitution degree for all of them. Substitution with long alkyl/acyl groups led to significant changes in physical and thermal properties of modified polysaccharides. Sorption of cholate and cholesterol by these polysaccharide-based sorbents was estimated in comparison with the synthetic drug cholestyramine. It was found that modified polysaccharides have high affinity to cholesterol. By contrast, cholestyramine was effective only in cholate sorption.
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Colatos/química , Colesterol/química , Polisacáridos/química , Adsorción , Rastreo Diferencial de Calorimetría , Espectroscopía de Resonancia Magnética con Carbono-13 , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC < C ≈ TC < DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control < MKC < TC â¼ C < DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC < C < DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.
Asunto(s)
Ácidos y Sales Biliares/química , Colatos/química , Liposomas/química , Permeabilidad de la Membrana Celular , Ditionita/química , Concentración de Iones de Hidrógeno , Cinética , Fluidez de la MembranaRESUMEN
O-carboxymethyl chitosan (OCMC) was firstly decorated with cholic acid (CA) to acquire an amphiphilic polymer under alkaline condition. Then glycyrrhetinic acid (GA) was conjugated to the polymer via a succinate linker and finally treated with NaCO3 solution to obtain new conjugates for potential liver targeted delivery. These conjugates formed uniform aggregates with low critical aggregation concentrations (0.028-0.079 mg/mL) in PBS. The average diameter of cholic acid modified carboxymethyl chitosan (CMCA) aggregates (110-257 nm) decreased with the increase of CA substitution degree and became slightly larger after GA modification. Negative zeta potential (-15 mV) of GA decorated CMCA (GA-CMCA) revealed that the formation of negatively charged shells and spherical morphology was observed under transmission electron microscopy. Furthermore, hemolysis test, in vitro cytotoxicity assay and cellular uptake study all demonstrated the safety and feasibility of these conjugates as a promising carrier for liver targeted drug delivery.
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Quitosano/análogos & derivados , Colatos/química , Portadores de Fármacos/química , Ácido Glicirretínico/química , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/toxicidad , Línea Celular Tumoral , Quitosano/química , Quitosano/metabolismo , Quitosano/toxicidad , Colatos/metabolismo , Colatos/toxicidad , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidad , Sistemas de Liberación de Medicamentos , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/toxicidad , Hemólisis/efectos de los fármacos , Células Hep G2 , HumanosRESUMEN
The phosphorescence intensity of unilamellar DOPC vesicles with embedded Tb(3+)-cholate complexes depends on the concentration of dihydroxynaphthalene (DHN) as sensitizer in solution. This was used to monitor the enzymatic conversion of DHN esters or DHN glucosides by enzymes in aqueous buffered solution.
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Colatos/química , Luminiscencia , Naftalenos/metabolismo , Terbio/química , Liposomas Unilamelares/química , Lipasa , FosfatidilcolinasRESUMEN
We demonstrated label free ethidium bromide assisted characterization of DNA interaction with cholate capped AuNPs. Interactions between ss/ds DNA and AuNPs with two different lengths (0.5 and 0.85 kb) were analyzed through fluorescence spectrophotometer and agrose gel electrophoresis analysis. Further results were confirmed by UV-globally visible spectrophotometer, DLS and TEM. As 0.5 and 0.85 kb of ssDNA effectively interacted with AuNPs through the van der Waals interaction which consequently led to the prevention of salt induced aggregation, EtBr intercalations as well as fluorescence shift with less binding constant 0.098 and 0.108 µM, respectively. On the contrary, the same length of dsDNA (0.5 and 0.85 kb) not interacted with AuNPs which led to the NPs aggregation, EtBr intercalation as well as fluorescence shift with increased binding constant 0.166 and 0.599 µM, respectively. This approach helped to understand the mode of interactions of DNA with cholate capped AuNPs without any modifications in a simple method and the results could be readout through the naked eye under the UV transilluminator.
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Colatos/química , ADN/química , Etidio/química , Colorantes Fluorescentes/química , Oro/química , Nanopartículas del Metal/química , ADN/genética , Colorantes Fluorescentes/síntesis química , Fluorometría , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria.
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Ácidos y Sales Biliares/farmacología , Disulfuros/metabolismo , Desplegamiento Proteico/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Ácidos y Sales Biliares/química , Colatos/química , Colatos/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacología , Humanos , Oxidación-Reducción/efectos de los fármacos , Estructura Cuaternaria de ProteínaRESUMEN
Wulff-type boronic acids have been shown to act as ionophores at pH 8.2 by transporting Na(+) through phospholipid bilayers. A cholate-boronic acid conjugate was synthesised and shown to be an ionophore, although the hydroxyl-lined face of the cholate moiety did not enhance ion transport. Mechanistic studies suggested a carrier mechanism for Na(+) transport. The addition of fructose (>5 mM) strongly inhibited ionophoric activity of the cholate-boronic acid conjugate, mirrored by a strong decrease in the ability of this compound to partition into an organic phase. Modelling of the partitioning and ion transport data, using a fructose/boronic acid binding constant measured at pH 8.2, showed a good correlation with the extent of fructose/boronic acid complexation and suggested high polarity fructose/boronic acid complexes are poor ionophores. The sensitivity of ion transport to fructose implies that boronic acid-based antibiotic ionophores with activity modulated by polysaccharides in the surrounding environment may be accessible.
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Antibacterianos/química , Ácidos Borónicos/química , Colatos/química , Fructosa/química , Ionóforos/química , Estructura Molecular , Polisacáridos/químicaRESUMEN
The distribution and the metabolic pathways of bacteria degrading steroid compounds released by eukaryotic organisms were investigated using the bile salt cholate as model substrate. Cholate-degrading bacteria could be readily isolated from freshwater environments. All isolated strains transiently released steroid degradation intermediates into culture supernatants before their further degradation. Cholate degradation could be initiated via two different reaction sequences. Most strains degraded cholate via a reaction sequence known from the model organism Pseudomonas sp. strain Chol1 releasing intermediates with a 3-keto-Δ(1,4) -diene structure of the steroid skeleton. The actinobacterium Dietzia sp. strain Chol2 degraded cholate via a different and yet unexplored reaction sequence releasing intermediates with a 3-keto-Δ(4,6) -diene-7-deoxy structure of the steroid skeleton such as 3,12-dioxo-4,6-choldienoic acid (DOCDA). Using DOCDA as substrate, two Alphaproteobacteria, strains Chol10-11, were isolated that produced the same cholate degradation intermediates as strain Chol2. With DOCDA as substrate for Pseudomonas sp. strain Chol1 only the side chain was degraded while the ring system was transformed into novel steroid compounds accumulating as dead-end metabolites. These metabolites could be degraded by the DOCDA-producing strains Chol10-11. These results indicate that bacteria with potentially different pathways for cholate degradation coexist in natural habitats and may interact via interspecies cross-feeding.
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Bacterias/metabolismo , Colatos/metabolismo , Bacterias/aislamiento & purificación , Colatos/química , Agua Dulce/microbiología , Redes y Vías Metabólicas , Interacciones Microbianas , Pseudomonas/aislamiento & purificación , Pseudomonas/metabolismoRESUMEN
Oligocholate macrocycles self-assemble into transmembrane nanopores by the associative interactions of water molecules inside the amphiphilic macrocycles. Macrocycles functionalized with a terephthalic acid "side chain" displayed significantly higher transport activity for glucose across lipid bilayers than the corresponding methyl ester derivative. Changing the 1,4-substitution of the dicarboxylic acid to 1,3-substitution lowered the activity. Combining the hydrophobic interactions and the hydrogen-bond-based carboxylic acid dimerization was an effective strategy to tune the structure and activity of self-assembled nanopores in lipid membranes.
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Materiales Biomiméticos/química , Ácidos Carboxílicos/química , Colatos/química , Membrana Dobles de Lípidos/química , Compuestos Macrocíclicos/química , Nanoporos/ultraestructura , Transporte Biológico , Dimerización , Glucosa/química , Interacciones Hidrofóbicas e Hidrofílicas , Conformación MolecularRESUMEN
Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C(24) steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C(5) acyl side chain attached to C-17, with 7α,12ß-dihydroxy-androsta-1,4-diene-3,17-dione (12ß-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD(+) as cosubstrates. When the C(24) steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12α-DHADD and 7α-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C(22) steroid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12ß-DHADD in an NADPH-dependent reaction. When NAD(+) was omitted from assays with DHOCTO, a new product, identified as 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD(+), CoA, and ATP. These results revealed that degradation of the C(5) acyl side chain of cholate does not proceed via classical ß-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a ß-ketothiolase.
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Aldehídos/metabolismo , Colatos/metabolismo , Pseudomonas/metabolismo , Adenosina Trifosfato , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Aldehídos/química , Catálisis , Colatos/química , Coenzima A , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica , Estructura Molecular , NAD , NADP , Oxidación-Reducción , Pseudomonas/genéticaRESUMEN
Macrocycles made of cholate building blocks were previously found to transport glucose readily across lipid bilayers. In this study, an (15)N, (13)Cα-labeled glycine was inserted into a cyclic cholate trimer and attached at the end of a linear trimer, respectively. The isotopic labeling allowed us to use solid-state NMR spectroscopy to study the dynamics, aggregation, and depth of insertion of these compounds in lipid membranes. The cyclic compound was found to be mostly immobilized in DLPC, POPC/POPG, and POPC/POPG/cholesterol membranes, whereas the linear trimer displayed large-amplitude motion that depended on the membrane thickness and viscosity. (13)C-detected (1)H spin diffusion experiments revealed the depth of insertion of the compounds in the membranes, as well as their contact with water molecules. The data support a consistent stacking model for the cholate macrocycles in lipid membranes, driven by the hydrophobic interactions of the water molecules in the interior of the macrocycles. The study also shows a strong preference of the linear trimer for the membrane surface, consistent with its lack of transport activity in earlier liposome leakage assays.