RESUMEN
The presence of lipopolysaccharide and emergence of drug resistance make the treatment of Gram-negative bacterial infections highly challenging. Herein, we present the synthesis and antibacterial activities of cholic acid-peptide conjugates (CAPs), demonstrating that valine-glycine dipeptide-derived CAP 3 is the most effective antimicrobial. Molecular dynamics simulations and structural analysis revealed that a precise intramolecular network of CAP 3 is maintained in the form of evolving edges, suggesting intramolecular connectivity. Further, we found high conformational rigidity in CAP 3 that confers maximum perturbations in bacterial membranes relative to other small molecules. Interestingly, CAP 3-coated catheters did not allow the formation of biofilms in mice, and treatment of wound infections with CAP 3 was able to clear the bacterial infection. Our results demonstrate that molecular conformation and internal connectivity are critical parameters to describe the antimicrobial nature of compounds, and the analysis presented here may serve as a general principle for the design of future antimicrobials.
Asunto(s)
Antibacterianos/uso terapéutico , Ácidos Cólicos/uso terapéutico , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Lipopolisacáridos/metabolismo , Péptidos/uso terapéutico , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Animales , Antibacterianos/síntesis química , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Ácidos Cólicos/síntesis química , Ácidos Cólicos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/fisiología , Bacterias Gramnegativas/metabolismo , Bacterias Gramnegativas/fisiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/metabolismo , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos/síntesis química , Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico-chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal-phase thin-layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure-activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α-hydroxy-12-oxo-5ß-cholanoic and 3α-hydroxy-7-oxo-5ß-cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α-hydroxy-12-oxo-5ß-cholanoic, 3α-hydroxy-7-oxo-5ß-cholanoic, 3,7,12-trioxo-5ß-cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Ácidos y Sales Biliares/farmacología , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica I/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Antagonistas de Receptores Androgénicos/química , Ácidos y Sales Biliares/química , Ácidos Cólicos/química , Ácidos Cólicos/farmacología , Cromatografía en Capa Delgada/métodos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Relación Estructura-Actividad CuantitativaRESUMEN
Bile acids are the endogenous modulators of the nuclear receptor FXR and the membrane receptor GPBAR1. FXR represents a promising pharmacological target for the treatment of cholestatic liver disorders. Currently available semisynthetic bile acid derivatives cover the same chemical space of bile acids and therefore they are poorly selective toward BA receptors, increasing patient risk for adverse side effects. In this report, we have investigated around the structure of CDCA describing the synthesis and the in vitro and in vivo pharmacological characterization of a novel family of compounds modified on the steroidal tetracyclic core and on the side chain. Pharmacological characterization resulted in the identification of several potent and selective FXR agonists. These novel agents might add utility in the treatment of cholestatic disorders by potentially mitigating side effects linked to unwanted activation of GPBAR1.
Asunto(s)
Ácidos Cólicos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Ácidos Cólicos/síntesis química , Evaluación Preclínica de Medicamentos , Células HEK293 , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/agonistas , Activación Transcripcional/efectos de los fármacosRESUMEN
Eph-ephrin system is emerging as a new potential target in several diseases including cancer, diabetes, neurodegenerative diseases and inflammation. In the last decade, several efforts have been made to develop small molecule antagonists of Eph receptors. Both natural and synthetic compounds were discovered with (poly) phenol and steroidal derivatives on one side and the α1 agonist doxazosin, 2,5-dimethylpyrrol- 1-yl-benzoic acids and amino acid conjugates of lithocholic acid on the other. In the present paper we critically present available data for these compounds and discuss their potential usefulness as pharmacological tools or as candidates for a lead-optimization program.
Asunto(s)
Descubrimiento de Drogas/métodos , Efrinas/metabolismo , Preparaciones de Plantas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Receptores de la Familia Eph/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Benzoatos/química , Benzoatos/farmacología , Ácidos Cólicos/química , Ácidos Cólicos/farmacología , Doxazosina/química , Doxazosina/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Preparaciones de Plantas/química , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , Receptores de la Familia Eph/agonistas , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
BACKGROUND: Amyloid-ß (Aß) 42 has been implicated as the initiating molecule in the pathogenesis of Alzheimer's disease (AD); thus, therapeutic strategies that target Aß42 are of great interest. γ-Secretase modulators (GSMs) are small molecules that selectively decrease Aß42. We have previously reported that many acidic steroids are GSMs with potencies ranging in the low to mid micromolar concentration with 5ß-cholanic acid being the most potent steroid identified GSM with half maximal effective concentration (EC50) of 5.7 µM. RESULTS: We find that the endogenous cholesterol metabolite, 3ß-hydroxy-5-cholestenoic acid (CA), is a steroid GSM with enhanced potency (EC50 of 250 nM) relative to 5ß-cholanic acid. CA i) is found in human plasma at ~100-300 nM concentrations ii) has the typical acidic GSM signature of decreasing Aß42 and increasing Aß38 levels iii) is active in in vitro γ-secretase assay iv) is made in the brain. To test if CA acts as an endogenous GSM, we used Cyp27a1 knockout (Cyp27a1-/-) and Cyp7b1 knockout (Cyp7b1-/-) mice to investigate if manipulation of cholesterol metabolism pathways relevant to CA formation would affect brain Aß42 levels. Our data show that Cyp27a1-/- had increased brain Aß42, whereas Cyp7b1-/- mice had decreased brain Aß42 levels; however, peripheral dosing of up to 100 mg/kg CA did not affect brain Aß levels. Structure-activity relationship (SAR) studies with multiple known and novel CA analogs studies failed to reveal CA analogs with increased potency. CONCLUSION: These data suggest that CA may act as an endogenous GSM within the brain. Although it is conceptually attractive to try and increase the levels of CA in the brain for prevention of AD, our data suggest that this will not be easily accomplished.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Colesterol/análogos & derivados , Fragmentos de Péptidos/metabolismo , Animales , Barrera Hematoencefálica , Células CHO , Células Cultivadas , Colestanotriol 26-Monooxigenasa/deficiencia , Colestanotriol 26-Monooxigenasa/genética , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacología , Ácidos Cólicos/farmacología , Técnicas de Cocultivo , Cricetinae , Cricetulus , Familia 7 del Citocromo P450 , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Neuroglía/metabolismo , Neuronas/metabolismo , Esteroide Hidroxilasas/deficiencia , Esteroide Hidroxilasas/genética , Relación Estructura-ActividadRESUMEN
From the organic extracts of the sponge Siphonochalina fortis, collected at Bahía Bustamante, Chubut, Argentina, three major compounds were isolated and identified as deoxycholic acid 3, 12-diacetate (1), cholic acid 3, 7, 12-triacetate (2) and cholic acid, 3, 7, 12-triacetate. (3). This is the first report of acetylated bile acids in sponges and the first isolation of compound 3 as a natural product. The potential induction of DNA lesions by the isolated compounds was investigated using the comet assay in lymphocytes of human peripheral blood as in vitro model. The results showed that the administration of the bile acid derivatives would not induce DNA damages, indicating that acetylated bile acids are nontoxic metabolites at the tested concentrations. Since the free bile acids were not detected, it is unlikely that the acetylated compounds may be part of the sponge cells detoxification mechanisms. These results may suggest a possible role of acetylated bile acids as a chemical defense mechanism, product of a symbiotic relationship with microorganisms, which would explain their seasonal and geographical variation, and their influence on the previously observed genotoxicity of the organic extract of S. fortis.
Asunto(s)
Ácidos Cólicos/aislamiento & purificación , Daño del ADN , Mutágenos/aislamiento & purificación , Poríferos/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácidos Cólicos/farmacología , Ensayo Cometa , Evaluación Preclínica de Medicamentos , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Mutágenos/farmacologíaRESUMEN
Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the ß-adrenergic receptor (ß-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.
Asunto(s)
Membrana Celular/metabolismo , Ácidos Cólicos/farmacología , Terapia Molecular Dirigida , Adenosina Trifosfatasas/análisis , Adenosina Trifosfatasas/fisiología , Agonistas Adrenérgicos beta/farmacología , Animales , Células CACO-2 , Proteínas Portadoras/fisiología , Ácidos Cólicos/química , Medicamentos Herbarios Chinos/metabolismo , Epinefrina/farmacología , Humanos , Íleon/efectos de los fármacos , Masculino , Fluidez de la Membrana/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , Receptores Adrenérgicos beta/fisiología , Receptores Muscarínicos/fisiologíaRESUMEN
The Ganoderma lucidum (G. lucidum) is one of the oriental fungi that has been reported to have immunomodulatory properties. Although effect of ß-glucans from G. lucidum has been well documented, little is known about how other major bioactive components, the triterpenes, contribute to the immunomodulatory function of G. lucidum. Here, we showed that triterpenes-rich extract of antlered form of G. lucidum (G. lucidum AF) induces TNFα production in monocytic THP-1 cells. Furthermore, the extract also synergized with lipopolysaccharide (LPS) to induce TNFα production in THP-1 cells, suggesting an immunostimulatory role of triterpenes-rich extract of G. lucidum AF. Notably, the extract enhanced LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), while it suppressed LPS-induced phosphorylation of c-Jun N-terminal kinase (JNK) MAPK. p38 Inhibitor suppressed TNFα production, while JNK inhibitor enhanced TNFα production, implying that synergistic effect of the extract may work by modulating p38 and JNK MAPKs. Moreover, we found that the triterpenes-rich extract of G. lucidum AF contains high amounts of lucidenic acids. Lucidenic acid-A, -F and -D(2), which seem to dominantly exist in the extract, were purified from the triterpenes-rich extract. We also identified Lucidenic acid-A and -F as modulators of JNK and p38, respectively. Thus, our data demonstrate that lucidenic acids-rich extract from G. lucidum AF enhances LPS-induced immune responses in monocytic THP-1 cells possibly via the modulation of p38 and JNK MAPKs activation.
Asunto(s)
Ácidos Cólicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Factores Inmunológicos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Reishi/química , Triterpenos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular , Ácidos Cólicos/aislamiento & purificación , Medicamentos Herbarios Chinos/aislamiento & purificación , Humanos , Factores Inmunológicos/aislamiento & purificación , Monocitos/enzimología , Monocitos/inmunología , Triterpenos/aislamiento & purificaciónRESUMEN
Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.
Asunto(s)
Bacteriorodopsinas/biosíntesis , Bacteriorodopsinas/aislamiento & purificación , Ácidos Cólicos/química , Detergentes/química , Fosforilcolina/análogos & derivados , Biosíntesis de Proteínas , Bacteriorodopsinas/antagonistas & inhibidores , Sistema Libre de Células , Ácidos Cólicos/farmacología , Detergentes/farmacología , Células Germinativas , Micelas , Fosforilcolina/química , Fosforilcolina/farmacología , TriticumRESUMEN
Previous studies have shown that Qing Kai Ling, a traditional Chinese medicine, was able to effectively prevent the inflammation from cerebral ischemia (Chen et al., 2002). The cholalic acid and hyodeoxycholalic acid (cholalic acid mixture) was major active components in Qing Kai Ling. To study the effects of cholalic acid mixture on the damage cascade of cerebral ischemia, rat model of focal cerebral ischemia was established by permanent occlusion of left middle cerebral artery. We found that the administration of cholalic acid mixture could reduce the ischemic infarct size after 24 h of ischemia, and cholalic acid mixture could be detected in cerebrospinal fluid after 2h of administration. We also found that the concentrations of tumor necrosis factor-alpha and interlukin-1beta in rat brain were significantly lower when compared to the untreated animals after 12 h and 24 h of ischemia. The concentrations of von Willebrand factor and neuron specific enolase in the plasma were remarkably decreased in cholalic acid mixture treated animals than in the untreated ones after 12h of ischemia. Our results suggested that cholalic acid mixture is able to decrease the expression of inflammation factors including tumor necrosis factor-alpha and interlukin-1beta after focal cerebral ischemia.
Asunto(s)
Antiinflamatorios/farmacología , Encéfalo/efectos de los fármacos , Ácidos Cólicos/farmacología , Ácido Desoxicólico/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Interleucina-1beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Infarto de la Arteria Cerebral Media/inmunología , Infarto de la Arteria Cerebral Media/patología , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de von Willebrand/metabolismoRESUMEN
Ganoderma lucidum is known as a medicinal mushroom used in traditional Chinese medicine. In the present study, the effect of lucidenic acids (A, B, C, and N) isolated from a new G. lucidum (YK-02) on induction of cell apoptosis and the apoptotic pathway in HL-60 cells were investigated. The results demonstrated that lucidenic acids decreased cell population growth of HL-60 cells, assessed with the MTT assay. The cell cycle assay indicated that treatment of HL-60 cells with lucidenic acid A, C, and N caused cell cycle arrest in the G 1 phase. Lucidenic acid B (LAB) did not affect the cell cycle profile; however, it increased the number of early and late apoptotic cells but not necrotic cells. Treatment of HL-60 cells with LAB caused loss of mitochondria membrane potential. Moreover, the ratio of expression levels of pro- and antiapoptotic Bcl-2 family members was changed by LAB treatment. LAB-induced apoptosis involved release of mitochondria cytochrome c and subsequently induced the activation of caspase-9 and caspase-3, which were followed by cleavage of poly(ADP-ribose) polymerase (PARP). Pretreatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented LAB from inhibiting cell viability in HL-60 cells. Our finding may be critical to the chemopreventive potential of lucidenic acid B.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Cólicos/farmacología , Mitocondrias/fisiología , Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/ultraestructuraRESUMEN
Oral drug delivery offers an attractive method of needle-free drug administration. Unfortunately, oral delivery is often hampered by the poor permeability of drugs across the intestinal epithelium. Although several single chemical permeation enhancers have been shown to alleviate permeability difficulties, this often occurs at the expense of safety. This in vitro study demonstrates the use of binary and ternary combinations of permeation enhancers to create synergistic enhancer formulations (SEFs) that offer a high level of potency while inducing very little toxicity in Caco-2 cells. Although relatively rare in the explored formulation space, SEFs were abundant enough to significantly increase the repertoire of permeation enhancers that are safe and effective in vitro. The most promising enhancers from the binary study led to easily identifiable ternary SEFs, thus increasing the efficiency of the discovery process. Some of the best performers of the study included binary combinations of hexylamine and chembetaine and ternary combinations of sodium laureth sulfate, decyltrimethyl ammonium bromide, and chembetaine, all at a total concentration of 0.1% (w/v). Furthermore, several SEFs were shown to be capable of increasing mannitol and 70 kDa dextran permeability across Caco-2 monolayers 15- and 8-fold, respectively. These results encourage further exploration of several leading formulations for in vivo applications in oral drug delivery.
Asunto(s)
Adyuvantes Farmacéuticos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Adyuvantes Farmacéuticos/administración & dosificación , Administración Oral , Aminas/administración & dosificación , Aminas/farmacología , Monoterpenos Bicíclicos , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Ácidos Cólicos/administración & dosificación , Ácidos Cólicos/farmacología , Ácidos Decanoicos/administración & dosificación , Ácidos Decanoicos/farmacología , Dextranos/metabolismo , Combinación de Medicamentos , Sinergismo Farmacológico , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Manitol/metabolismo , Pirrolidinonas/administración & dosificación , Pirrolidinonas/farmacología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/farmacología , Sarcosina/administración & dosificación , Sarcosina/análogos & derivados , Sarcosina/farmacología , Dodecil Sulfato de Sodio/administración & dosificación , Dodecil Sulfato de Sodio/análogos & derivados , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/administración & dosificación , Tensoactivos/farmacología , Terpenos/administración & dosificación , Terpenos/farmacologíaRESUMEN
Our research was aimed at establishing if and how selenium (Se) ion, N-acetylcysteine (NAC), sodium salt of monoketocholic acid (MKH) and superoxide-dismutase (SOD), administered in the experimental animal model, could affect the possible cytotoxicity associated with anthracycline-based combined chemotherapy with doxorubicin, vincristine and prednisolone (DVP). The following biochemical parameters were investigated: the extent of lipid peroxidation (LPx), and the activity of peroxidase (Px), catalase (CAT), glutathione-peroxidase (GSHPx), and xanthine-oxidase (XOD). A statistical increase in LPx activity was obtained by SOD, MKH, DVPSe and DVPMKH. All chemotherapeutic agents reduced Px activity in a statistically significant manner. There was no statistical significance for the results regarding the effects of the administered substances on GSHPx activity. The results for DVP, SOD, MKH, DVPSOD, DVPSe and DVPMKH showed reduced XOD activity which was statistically significant, which was lowest in the case of MKH, while NAC and Se reduced the activity of this enzyme but statistically non significant. NAC, Se, DVP, MKH and DVPMKH caused a reduction in CAT activity, while DVPSOD and DVPSe caused an increase of the latter.
Asunto(s)
Antraciclinas/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Doxorrubicina/efectos adversos , Acetilcisteína/farmacología , Análisis de Varianza , Animales , Antraciclinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Antioxidantes/farmacología , Catalasa/efectos de los fármacos , Ácidos Cólicos/farmacología , Doxorrubicina/administración & dosificación , Interacciones Farmacológicas , Depuradores de Radicales Libres/farmacología , Glutatión Peroxidasa/efectos de los fármacos , Corazón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Peroxidasa/efectos de los fármacos , Prednisolona/administración & dosificación , Prednisolona/efectos adversos , Selenio/farmacología , Superóxido Dismutasa/farmacología , Vincristina/administración & dosificación , Vincristina/efectos adversos , Xantina Oxidasa/efectos de los fármacosRESUMEN
Betaine serves as a methyl donor in a reaction converting homocysteine to methionine, catalysed by the enzyme betaine-homocysteine methyltransferase. It has been used for years to lower the concentration of plasma total homocysteine (tHcy) in patients with homocystinuria, and has recently been shown to reduce fasting and in particular post-methionine load (PML) tHcy in healthy subjects. Betaine exists in plasma at concentrations of about 30 micromol/L; it varies 10-fold (from 9 to 90 micromol/L) between individuals, but the intra-individual variability is small. Major determinants are choline, dimethylglycine and folate in plasma, folic acid intake and gender. Recent studies have demonstrated that plasma betaine is a stronger determinant of PML tHcy than are vitamin B6 and folate. The betaine-PML tHcy relationship is attenuated after supplementation with B-vitamins, and is most pronounced in subjects with low folate. Betaine shows a weaker association with fasting tHcy (than with PML tHcy), and also this association is most pronounced in subjects with low folate. In pregnancy, plasma betaine declines until gestational week 20, and thereafter remains constant. From gestational week 20 onwards, fasting tHcy shows a strong inverse association with plasma betaine, and betaine becomes a stronger predictor than folate of fasting tHcy. To conclude, betaine status is a component of an individual's biochemical make-up with ramifications to one-carbon metabolism. Betaine status should be investigated in pathologies related to altered metabolism of homocysteine and folate, including cardiovascular disease, cancer and neural tube defects.
Asunto(s)
Betaína/metabolismo , Carbono/metabolismo , Homocisteína/metabolismo , Animales , Betaína/sangre , Betaína/química , Betaína/orina , Carbono/química , Ácidos Cólicos/farmacología , Homocisteína/sangre , Humanos , Lípidos/sangre , Fenómenos Fisiológicos de la NutriciónRESUMEN
Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.
Asunto(s)
Ácidos Cólicos/farmacología , Proteínas de Unión al ADN/metabolismo , Hepatocitos/fisiología , Molécula 1 de Adhesión Intercelular/genética , Transducción de Señal/inmunología , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Hepatocitos/citología , Humanos , Isoxazoles/farmacología , Hígado/citología , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Receptores Citoplasmáticos y Nucleares , Elementos de Respuesta/genética , Transducción de Señal/genética , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin.
Asunto(s)
Detergentes/farmacología , Glucosiltransferasas/metabolismo , Proteínas de la Membrana , Nicotiana/enzimología , Plantas Tóxicas , Proteínas de Schizosaccharomyces pombe , Ácidos Cólicos/farmacología , Digitonina/farmacología , Activación Enzimática , Glucosiltransferasas/antagonistas & inhibidores , Polen , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Tripsina/farmacología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/farmacologíaRESUMEN
C57BL/6 mice are susceptible to diet-induced atherosclerosis, whereas BALB/c mice are resistant. The susceptibility of C57BL/6 mice has been linked to decreased plasma HDL cholesterol in response to a diet containing fat, cholesterol, and cholic acid. Feeding C57BL/6 mice a diet consisting of fat and cholesterol, but no cholic acid, increased plasma high density lipoprotein (HDL) cholesterol. The increase in HDL was associated with increases in both plasma apolipoprotein (apo)A-I and hepatic apoA-I mRNA. Supplementation of the cholesterol-rich diet with cholic acid inhibited the stimulatory effect of cholesterol on hepatic apoA-I mRNA expression, resulting in similar hepatic apoA-I mRNA levels compared to chow-fed mice. Atherosclerosis-resistant BALB/c mice were also resistant to diet-induced changes in plasma HDL, apoA-I, and hepatic apoA-I mRNA levels. Previous studies showed that the diets changed both the activity and mRNA encoding the liver specific enzyme 7alpha-hydroxylase (1993.J. Lipid Res. 34: 923-931). In both strains of mice, hepatic expression of apoA-I and 7alpha-hydroxylase mRNA varied in parallel. Whereas susceptible C57BL/6 mice also showed a significant correlation between HDL cholesterol and expression of 7alpha-hydroxylase, no such correlation was observed in BALB/c mice, suggesting that genetic differences in HDL metabolism, not hepatic apoA-I synthesis, are responsible for the strain specific differences in plasma HDL levels. The finding that lecithin: cholesterol acyltransferase (LCAT) activity was significantly decreased in C57BL/6 mice, but not in BALB/ c mice fed the atherogenic diet, further supports this conclusion. Additional studies show that McArdle hepatoma cells stably expressing plasmid-derived rat 7alpha-hydroxylase recapitulated the parallel linear relationship between 7alpha-hydroxylase and apoA-I mRNA expression observed in both strains of mice. These data link hepatic apoA-I mRNA expression to hepatic cholesterol/bile acid metabolism.
Asunto(s)
Apolipoproteína A-I/genética , Arteriosclerosis/genética , Colesterol 7-alfa-Hidroxilasa/metabolismo , Expresión Génica , Neoplasias Hepáticas Experimentales/genética , Hígado/metabolismo , Animales , Colesterol en la Dieta/administración & dosificación , HDL-Colesterol/sangre , Ácido Cólico , Ácidos Cólicos/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
BACKGROUND: Large intravenous bilirubin infusions in bile acid-depleted pigs (BADP) destroy hepatocyte canalicular membrane microvilli (CMV) and cause cholestasis. This study examines whether bilirubin photoconversion product infusions do the same. METHODS: The effects of systemic infusion of 135 mumol.kg-1 body weight bilirubin photoconversion products on CMV density and choleretic response to intraportal bile acid infusion were studied in BADP. Furthermore, the effects of 135 mumol.kg-1 b.w. bilirubin infusion, either through an arteriovenous bilirubin photoconversion shunt device (PCD) or intravenously, were measured in PCD-connected BADP. RESULTS: Intravenous bilirubin photoconversion product infusions affected neither the CMV density nor the choleretic response to cholic acid infusion, and neither did bilirubin infusion through the PCD. In contrast, intravenous bilirubin infusion caused canalicular injury and cholestasis in four of six PCD-connected BADP. CONCLUSION: Bilirubin photoconversion products do not destroy CMV or cause cholestasis in BADP. A bilirubin photoconversion shunt device can confer cholestasis protection to bilirubin-loaded BADP.
Asunto(s)
Ácidos y Sales Biliares/deficiencia , Ácidos y Sales Biliares/farmacología , Bilirrubina/farmacología , Colestasis Intrahepática/etiología , Animales , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/ultraestructura , Bilirrubina/administración & dosificación , Bilirrubina/efectos de la radiación , Colestasis Intrahepática/patología , Ácido Cólico , Ácidos Cólicos/farmacología , Infusiones Intravenosas , Microscopía Electrónica de Rastreo , Fototerapia/instrumentación , PorcinosRESUMEN
Promotion would suppose the selection of initiated cells. We tested the selection of aberrant crypt cells by cholic acid, a colon cancer promoter, and the effect of protectors, phytate and food restriction. After an azoxymethane injection, rats were allocated to a control diet, or to supplements of cholic acid, sodium phytate, or to a 50% food restriction. The proliferation and apoptosis of 1200 crypts were assessed, after immuno-staining for BrdU. Cholic acid increased the proliferation of aberrant crypts but not of normal crypts. Phytate and food restriction decreased the proliferation of normal crypts, but not of aberrant crypts. Apoptosis was not affected by diets. Results support the hypothesis that cholic acid can select initiated cells in the colon.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Cólicos/farmacología , Neoplasias del Colon/patología , Mucosa Intestinal/patología , Ácido Fítico/farmacología , Lesiones Precancerosas/patología , Análisis de Varianza , Animales , Peso Corporal , División Celular/efectos de los fármacos , Ácido Cólico , Colon/efectos de los fármacos , Colon/patología , Enfermedades del Colon/inducido químicamente , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/prevención & control , Ingestión de Alimentos/fisiología , Femenino , Mucosa Intestinal/efectos de los fármacos , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
The present study investigated the role of phytosterols in colonic cell proliferation and examined the possible role of protein kinase C (PKC) in this process. A total of 18 male Sprague-Dawley rats weighing 240-270 g were fed, for a period of 22 days, one of three experimental diets: a control diet, a diet supplemented with 0.2% cholic acid, or a diet supplemented with 0.2% cholic acid + 2% dietary phytosterols. Two hours before decapitation, animals were injected with 5'-bromo-2'-deoxyuridine (BrdU, 50 mg/kg body wt ip). Cell proliferation in the proximal colon was measured using a monoclonal antibody to BrdU. PKC activity in the proximal colonic mucosa was assayed using a myelin basic protein as a substrate. Cell proliferation was significantly increased by 276% with 0.2% cholic acid feeding compared with controls. The presence of 2% phytosterols in the diet abolished the cholic acid-induced hyperplasia. Cholic acid induced a 31% expansion of the proliferative zone. Only the cytosolic PKC was significantly lower in the phytosterol-fed group. Neither the total PKC nor the particulate PKC demonstrated an effect of phytosterols on enzyme activity. In conclusion, we found that dietary supplementation with 2% phytosterol has a significant protective effect on enhanced cell proliferation and that this effect is not mediated through the PKC system.