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1.
Int J Nanomedicine ; 14: 4045-4057, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31213814

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Colatos/química , Lipídeos/química , Nanopartículas/química , Polímeros/química , Quercetina/administração & dosagem , Administração Oral , Animais , Área Sob a Curva , Células CACO-2 , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Humanos , Leucemia/patologia , Camundongos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Quercetina/farmacocinética , Quercetina/farmacologia , Ratos Sprague-Dawley
2.
Environ Microbiol ; 21(2): 800-813, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30680854

RESUMO

Bile salts are steroid compounds from the digestive tract of vertebrates and enter the environment via defecation. Many aerobic bile-salt degrading bacteria are known but no bacteria that completely degrade bile salts under anoxic conditions have been isolated so far. In this study, the facultatively anaerobic Betaproteobacterium Azoarcus sp. strain Aa7 was isolated that grew with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. Phenotypic and genomic characterization revealed that strain Aa7 used the 2,3-seco pathway for the degradation of bile salts as found in other denitrifying steroid-degrading bacteria such as Sterolibacterium denitrificans. Under oxic conditions strain Aa7 used the 9,10-seco pathway as found in, for example, Pseudomonas stutzeri Chol1. Metabolite analysis during anaerobic growth indicated a reductive dehydroxylation of 7α-hydroxyl bile salts. Deletion of the gene hsh2 Aa7 encoding a 7-hydroxysteroid dehydratase led to strongly impaired growth with cholate and chenodeoxycholate but not with deoxycholate lacking a hydroxyl group at C7. The hsh2 Aa7 deletion mutant degraded cholate and chenodeoxycholate to the corresponding C19 -androstadienediones only while no phenotype change was observed during aerobic degradation of cholate. These results showed that removal of the 7α-hydroxyl group was essential for cleavage of the steroid skeleton under anoxic conditions.


Assuntos
Azoarcus/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Anaerobiose , Azoarcus/enzimologia , Azoarcus/genética , Proteínas de Bactérias/genética , Ácidos e Sais Biliares/química , Colatos/metabolismo , Desnitrificação , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroides/metabolismo , Rhodocyclaceae/enzimologia , Rhodocyclaceae/genética , Rhodocyclaceae/metabolismo , Esteroides/química , Esteroides/metabolismo
3.
NPJ Biofilms Microbiomes ; 5(1): 14, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-32382020

RESUMO

Clostridium difficile is a major cause of nosocomial infections. Bacterial persistence in the gut is responsible for infection relapse; sporulation and other unidentified mechanisms contribute to this process. Intestinal bile salts cholate and deoxycholate stimulate spore germination, while deoxycholate kills vegetative cells. Here, we report that sub-lethal concentrations of deoxycholate stimulate biofilm formation, which protects C. difficile from antimicrobial compounds. The biofilm matrix is composed of extracellular DNA and proteinaceous factors that promote biofilm stability. Transcriptomic analysis indicates that deoxycholate induces metabolic pathways and cell envelope reorganization, and represses toxin and spore production. In support of the transcriptomic analysis, we show that global metabolic regulators and an uncharacterized lipoprotein contribute to deoxycholate-induced biofilm formation. Finally, Clostridium scindens enhances biofilm formation of C. difficile by converting cholate into deoxycholate. Together, our results suggest that deoxycholate is an intestinal signal that induces C. difficile persistence and may increase the risk of relapse.


Assuntos
Ácidos e Sais Biliares/metabolismo , Biofilmes/crescimento & desenvolvimento , Clostridium difficile/fisiologia , Microbiota , Ácidos e Sais Biliares/farmacologia , Biofilmes/efeitos dos fármacos , Colatos/metabolismo , Clostridiales/metabolismo , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/genética , Ácido Desoxicólico/farmacologia , Perfilação da Expressão Gênica , Redes e Vias Metabólicas , Interações Microbianas
4.
Appl Environ Microbiol ; 84(1)2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29054875

RESUMO

Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria (Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl-coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria.IMPORTANCE This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors.


Assuntos
Proteínas de Bactérias/genética , Colatos/metabolismo , Coenzima A Ligases/genética , Sphingomonadaceae/genética , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/metabolismo , Redes e Vias Metabólicas , Sphingomonadaceae/metabolismo , Esteroides/química
5.
FEBS Lett ; 591(10): 1419-1428, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28423182

RESUMO

Bacteria sense and respond to osmolarity through the EnvZ-OmpR two-component system. The structure of the periplasmic sensor domain of EnvZ (EnvZ-PD) is not available yet. Here, we present the crystal structure of EnvZ-PD in the presence of CHAPS detergent. The structure of EnvZ-PD shows similar folding topology to the PDC domains of PhoQ, DcuS, and CitA, but distinct orientations of helices and ß-hairpin structures. The CD and NMR spectra of EnvZ-PD in the presence of cholate, a major component of bile salts, are similar to those with CHAPS. Chemical cross-linking shows that the dimerization of EnvZ-PD is significantly inhibited by the CHAPS and cholate. Together with ß-galactosidase assay, these results suggest that bile salts may affect the EnvZ structure and function in Escherichia coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Colatos/farmacologia , Ácidos Cólicos/farmacologia , Detergentes/farmacologia , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Complexos Multienzimáticos/química , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Dicroísmo Circular , Cristalografia por Raios X , Proteínas de Escherichia coli/efeitos dos fármacos , Modelos Moleculares , Complexos Multienzimáticos/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos
6.
Gastroenterology ; 152(6): 1521-1535.e8, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28088462

RESUMO

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.


Assuntos
Colesterol/metabolismo , Cálculos Biliares/genética , Cálculos Biliares/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Colatos/administração & dosagem , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/metabolismo , Regulação para Baixo/genética , Feminino , Vesícula Biliar/patologia , Cálculos Biliares/patologia , Heme Oxigenase-1/genética , Hepatócitos/metabolismo , Humanos , Hipóxia/metabolismo , Inflamação/etiologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mucinas/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Água/metabolismo
7.
Environ Microbiol ; 18(12): 5187-5203, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27648822

RESUMO

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ1,4 -diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ4,6 -structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ4,6 -metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ1,4 -diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to the formation of a dead-end steroid with a 3-keto-7-deoxy-Δ4,6 -diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria.


Assuntos
Alphaproteobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/metabolismo , Colatos/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Animais , Bactérias/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroides/metabolismo , Redes e Vias Metabólicas , Pseudomonas/genética , Pseudomonas/metabolismo
8.
Chirality ; 28(7): 525-33, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27300496

RESUMO

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.


Assuntos
Colatos/química , Ácido Desoxicólico/química , Naftalenos/química , Organofosfatos/química , Cromatografia Capilar Eletrocinética Micelar , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Naftóis/química , Solubilidade , Estereoisomerismo
9.
Infect Immun ; 84(8): 2198-2208, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27185788

RESUMO

Salmonella spp. are carried by and can acutely infect agricultural animals and humans. After ingestion, salmonellae traverse the upper digestive tract and initiate tissue invasion of the distal ileum, a virulence process carried out by the type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). Salmonellae coordinate SPI-1 expression with anatomical location via environmental cues, one of which is bile, a complex digestive fluid that causes potent repression of SPI-1 genes. The individual components of bile responsible for SPI-1 repression have not been previously characterized, nor have the bacterial signaling processes that modulate their effects been determined. Here, we characterize the mechanism by which bile represses SPI-1 expression. Individual bile acids exhibit repressive activity on SPI-1-regulated genes that requires neither passive diffusion nor OmpF-mediated entry. By using genetic methods, the effects of bile and bile acids were shown to require the invasion gene transcriptional activator hilD and to function independently of known upstream signaling pathways. Protein analysis techniques showed that SPI-1 repression by bile acids is mediated by posttranslational destabilization of HilD. Finally, we found that bile acids function synergistically to achieve the overall repressive activity of bile. These studies demonstrate a common mechanism by which diverse environmental cues (e.g., certain short-chain fatty acids and bile acids) inhibit SPI-1 expression. These data provide information relevant to Salmonella pathogenesis during acute infection in the intestine and during chronic infection of the gallbladder and inform the basis for development of therapeutics to inhibit invasion as a means of repressing Salmonella pathogenicity.


Assuntos
Ácidos e Sais Biliares/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Salmonella/efeitos dos fármacos , Salmonella/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/metabolismo , Colatos/farmacologia , Sinergismo Farmacológico , Porinas/genética , Porinas/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Virulência/genética
10.
Environ Microbiol ; 18(10): 3373-3389, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26691005

RESUMO

The bile salts cholate, deoxycholate, chenodeoxycholate and lithocholate are released from vertebrates into soil and water where environmental bacteria degrade these widespread steroid compounds. It was investigated whether different enzymes are required for the degradation of these tri-, di- and monohydroxylated bile salts in the model organism Pseudomonas sp. strain Chol1. Experiments with available and novel mutants showed that the degradation of the C5 -carboxylic side chain attached to the steroid skeleton is catalysed by the same set of enzymes. A difference was found for the degradation of partially degraded bile salts consisting of H-methylhexahydroindanone-propanoates (HIPs). With deoxycholate and lithocholate, which lack a hydroxy group at C7 of the steroid skeleton, an additional acyl-coenzyme A (CoA) dehydrogenase was required for ß-oxidation of the C3 -carboxylic side chain attached to the methylhexahydroindanone moiety. The ß-oxidation of this side chain could be measured in vitro. With cholate and deoxycholate, a reductive dehydroxylation of the C12-hydroxy group of HIP was required. Deletion of candidate genes for this reaction step revealed that a so-far unknown steroid dehydratase and a steroid oxidoreductase were responsible for this CoA-dependent reaction. These results showed that all bile salts are channelled into a common pathway via bypass reactions with 3'-hydroxy-HIP-CoA as central intermediate.


Assuntos
Ácidos e Sais Biliares/metabolismo , Pseudomonas/metabolismo , Esteroides/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/química , Catálise , Colatos/metabolismo , Hidroliases/genética , Hidroliases/metabolismo , Pseudomonas/genética , Esteroides/química
11.
Pharmacol Rep ; 67(3): 553-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25933969

RESUMO

BACKGROUND: 2,4,5-Trimethoxycinnamic acid (2,4,5-TMC) is the major and non-toxic metabolite of α-asarone, which retains hypocholesterolemic and choleretic activities. We compared the activities of 2,4,5-TMC with those of 2,4-dimethoxycinnamic acid (2,4-DMC), 3,4-DMC and 3,5-DMC, to understand the role of the methoxyls on carbons 2, 4 and 5 on the pharmacologic properties of these compounds. METHODS: The methoxycinnamic acids were administered to high-cholesterol/cholate-fed rats. We measured bile flow, and quantified bile acids, phospholipids and cholesterol in bile, and cholesterol and cholesterol-lipoproteins in serum. The inhibition of HMG-CoA reductase by the methoxycinnamic acids was evaluated in vitro. RESULTS: The four methoxycinnamic acids decreased serum cholesterol, without affecting the concentration of HDL-cholesterol. 2,4,5-TMC produced the highest decrease in LDL-cholesterol, 73.5%, which exceeds the range of statins (20-40%), and produced the highest inhibition of the activity of HMG-CoA reductase. 3,4-DMC produced the highest increase in bile flow, bile acids and phospholipids concentrations, and reduction in bile cholesterol, which led to a decrease in the biliary cholesterol saturation index. CONCLUSIONS: 2,4,5-TMC (which has three methoxyls) had the highest hypocholesterolemic activity, while 3,4-DMC, which lacks the methoxyl in carbon 2 but conserves the two other methoxyls in an adjacent position, had the highest choleretic activity and a probable cholelitholytic activity. In methoxycinnamic acids with two methoxyls in non-adjacent positions (2,4-DMC and 3,5-DMC), the hypocholesterolemic and choleretic activities were not as evident. 2,4,5-TMC and 3,4-DMC, which did not cause liver damage during the treatment period, should be further explored as a hypocholesterolemic and choleretic compounds in humans.


Assuntos
Anticolesterolemiantes/uso terapêutico , Colagogos e Coleréticos/uso terapêutico , Colatos/efeitos adversos , Colesterol na Dieta/efeitos adversos , Cinamatos/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Animais , Colatos/administração & dosagem , Colesterol na Dieta/administração & dosagem , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Cinamatos/química , Hipercolesterolemia/sangue , Hipercolesterolemia/induzido quimicamente , Masculino , Ratos , Ratos Wistar
12.
Infect Immun ; 83(6): 2350-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25824834

RESUMO

Resistance to the innate defenses of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common colonizer of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent antimicrobial activity. The mechanisms by which S. aureus is able to resist such defenses in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates the efflux of radiolabeled cholic acid both in S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated the survival of S. aureus in an anaerobic three-stage continuous-culture model of the human colon (gut model), which represents different anatomical areas of the large intestine.


Assuntos
Proteínas de Bactérias/metabolismo , Colatos/metabolismo , Colo/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Colo/microbiologia , Humanos , Modelos Biológicos , Staphylococcus aureus/genética
13.
J Chem Inf Model ; 55(4): 747-59, 2015 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-25760928

RESUMO

Identifying physiological ligands is necessary for annotating new protein structures, yet this presents a significant challenge to biologists and pharmaceutical chemists. Here we develop a predictor of cholesterol and cholate binding that works across diverse protein families, extending beyond sequence motif-based prediction. This approach combines SimSite3D site comparison with the detection of conserved interactions in cholesterol/cholate bound crystal structures to define three-dimensional interaction motifs. The resulting predictor identifies cholesterol sites with an ∼82% unbiased true positive rate in both membrane and soluble proteins, with a very low false positive rate relative to other predictors. The CholMine Web server can analyze users' structures, detect those likely to bind cholesterol/cholate, and predict the binding mode and key interactions. By deciphering the determinants of binding for these important steroids, CholMine may also aid in the design of selective inhibitors and detergents for targets such as G protein coupled receptors and bile acid receptors.


Assuntos
Colatos/metabolismo , Colesterol/metabolismo , Biologia Computacional/métodos , Proteínas/química , Proteínas/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Humanos , Ligantes , Aprendizado de Máquina , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica
14.
Langmuir ; 31(13): 3919-25, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25782344

RESUMO

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.


Assuntos
Colatos/química , Lipídeos de Membrana/química , Água/química , Solubilidade
15.
J Control Release ; 208: 93-105, 2015 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-25813888

RESUMO

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.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Polímeros/química , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colatos/química , Preparações de Ação Retardada , Dioxanos , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Nanopartículas , Cimento de Policarboxilato , Polietilenoglicóis , Polimerização , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Curr Opin Gastroenterol ; 31(3): 199-208, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25714706

RESUMO

PURPOSE OF REVIEW: It is our opinion that there is an unmet need in hepatology for a minimally or noninvasive test of liver function and physiology. Quantitative liver function tests define the severity and prognosis of liver disease by measuring the clearance of substrates whose uptake or metabolism is dependent upon liver perfusion or hepatocyte function. Substrates with high-affinity hepatic transporters exhibit high 'first-pass' hepatic extraction and their clearance measures hepatic perfusion. In contrast, substrates metabolized by the liver have low first-pass extraction and their clearance measures specific drug metabolizing pathways. RECENT FINDINGS: We highlight one quantitative liver function test, the dual cholate test, and introduce the concept of a disease severity index linked to clinical outcome that quantifies the simultaneous processes of hepatocyte uptake, clearance from the systemic circulation, clearance from the portal circulation, and portal-systemic shunting. SUMMARY: It is our opinion that dual cholate is a relevant test for defining disease severity, monitoring the natural course of disease progression, and quantifying the response to therapy.


Assuntos
Colatos/metabolismo , Hepatócitos/metabolismo , Hepatopatias/metabolismo , Testes de Função Hepática , Fígado/metabolismo , Necessidades e Demandas de Serviços de Saúde , Humanos , Fígado/fisiopatologia , Hepatopatias/diagnóstico , Hepatopatias/fisiopatologia , Testes de Função Hepática/métodos , Taxa de Depuração Metabólica , Valor Preditivo dos Testes , Índice de Gravidade de Doença
17.
Biochemistry ; 54(7): 1441-3, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25635829

RESUMO

Translocator protein 18 kDa (TSPO) in the mitochondrial outer membrane has been implicated in cholesterol transport regulating steroidogenesis. A human single polymorphism associated with anxiety disorders (A147T) and reduced pregnenolone production is adjacent to TSPO's cholesterol binding motif. In a mutant mimicking this polymorphism, we observe a lower level of binding of cholesterol. Further, three residues preceding A147 are more hydrophilic in a bacterial TSPO that has an affinity for cholesterol 1000-fold lower than that of the human form. Converting these residues to the human form in the bacterial homologue strikingly increases the affinity for cholesterol. An important role for this extended motif is further supported by covariance analysis.


Assuntos
Colesterol/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Receptores de GABA/química , Receptores de GABA/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Colatos/metabolismo , Bases de Dados de Proteínas , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Software
18.
Magn Reson Chem ; 53(4): 256-60, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25631685

RESUMO

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.


Assuntos
Colatos/síntese química , Géis/síntese química , Colatos/química , Géis/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Peso Molecular
19.
Chem Biol ; 22(2): 175-85, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25619932

RESUMO

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.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridium difficile/metabolismo , Bibliotecas de Moléculas Pequenas/química , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Biflavonoides/química , Biflavonoides/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Colatos/química , Colatos/metabolismo , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/metabolismo , Humanos , Cinética , Necrose , Floretina/química , Floretina/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Células Vero
20.
Fiziol Zh ; 61(6): 86-95, 2015.
Artigo em Ucraniano | MEDLINE | ID: mdl-27025049

RESUMO

Chronic pancreatitis is an inflammatory disease of the pancreas, which is characterized by destruction of pancreatic secretory parenchyma and progressing exocrine and endocrine insufficiency. Usually these patients have complications as cardiovascular, renal, respiratory and liver failure, and various gastric dysfunctions. The data of clinical observations do not reveal fully the functional state of the stomach and liver in chronic pancreatitis also remains an open question about the quality of the gastric juices and bile by this pathology. Therefore our aim was to investigate the secretory functions of the stomach and liver features in rats at the experimental chronic pancreatitis. This pathology modeled using L-arginine. Basal gastric secretion was investigated in chronic experiment by aspiration method for 10th and 63rd days, and pancreas and liver--in acute experiments at 13th and 68th days after the last administration of L-arginine. It was established that the character of the secretory response of the digestive tract depends on the duration of the pathology course. On the 10th day the functional state of the gastric secretory glands in rats with chronic pancreatitis characterized by twice increase of gastric acid production but decrease the level of hexosamines on 23.8% (P < 0.001) that indicate a increase of gastric content aggressiveness and mucus producing cells secretory insufficiency. In these animals the rate of total protein decreased on 61.7% (P < 0.05). On the 13th day observed the increase of pancreatic juice on 332% (P < 0.01), hepatic secret volume on 74.9% (P < 0.001) and redistribution in the cholates spectrum: glycocholates level increased but tauro-, free and total dehydroxylated bile acids decreased. These changes suggest deterioration of bile detergent properties, inhibition of acidic pathway of bile acids biosynthesis and conjugation of cholates with taurine. In two months total deficit of amino acids in gastric juice correlated with exocrine pancreatic insufficiency. Herein the acidity of gastric content partially restored, while the level of protein and mucus secretion proceed to decline. Consequently gastric mucosa is more vulnerable. In these rats the rates of free bile acids greatly increased while tauro- and glycocholates significantly decreased. Thus the processes of hydroxylation and conjugation of bile acids with amino acids inhibited suggesting interruption of synthetic and detoxification functions of the liver. The present work is important for comprehension the pathophysiological aspects of chronic pancreatitis particularly the digestive system functioning features at this pathology. These data could be considered in the appointment of treatment to avoid complications.


Assuntos
Bile/química , Suco Gástrico/química , Mucosa Gástrica/metabolismo , Pancreatite Crônica/metabolismo , Animais , Animais não Endogâmicos , Arginina , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/química , Colatos/metabolismo , Mucosa Gástrica/patologia , Hexosaminas/biossíntese , Hidroxilação , Fígado/metabolismo , Fígado/patologia , Masculino , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/patologia , Biossíntese de Proteínas , Proteínas/metabolismo , Ratos , Estômago/patologia , Taurina/metabolismo
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