RESUMEN
Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6 h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24 h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24 h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.
Asunto(s)
Colesterol , Hidroximetilglutaril-CoA Reductasas , Humanos , Células CACO-2 , Colesterol/metabolismo , Células Hep G2 , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Obesity presents a major risk factor in the development of cardiovascular diseases. Recent reports indicate that many kinds of polyphenols have the potential to prevent metabolic diseases. We hypothesized that rose polyphenols (ROSE) have the effect of improvement in lipid metabolism. In this study, we investigated whether rose polyphenols affected lipid metabolism and exerted antiobesity. To clarify the mechanism, C57BL/6J mice were fed a high-fat diet containing 0.25% ROSE for 35 days. Compared with the control group, body weight gain and adipose tissue weight in the 0.25% ROSE group were significantly decreased. Serum cholesterol and hepatic triglyceride concentrations significantly decreased, whereas fecal triglyceride was significantly increased in the 0.25% ROSE group. Liver stearoyl-CoA desaturase 1 (Scd1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and acyl-CoA:cholesterol acyltransferase 1 (Acat1) mRNA as well as protein stearoyl-CoA desaturase 1 concentrations were significantly lower in the 0.25% ROSE group than that in the control group. The mRNA and the protein concentrations of adipose triglyceride lipase, hormone-sensitive lipase, and peroxisomal acylcoenzyme A oxidase 1 in white adipose tissue were significantly higher in the 0.25% ROSE group than that in the control group. The components in rose polyphenols were quantified by liquid chromatography-tandem mass spectrometry, and we consider that ellagic acid plays an important role in an antiobesity effect because the ellagic acid content is the highest among polyphenols in rose polyphenols. In summary, rose polyphenols exhibit antiobesity effects by influencing lipid metabolism-related genes and proteins to promote lipolysis and suppress lipid synthesis.
Asunto(s)
Polifenoles , Estearoil-CoA Desaturasa , Ratones , Animales , Ratones Obesos , Polifenoles/farmacología , Polifenoles/uso terapéutico , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Ácido Elágico/metabolismo , Ácido Elágico/farmacología , Ratones Endogámicos C57BL , Tejido Adiposo/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Triglicéridos , ARN Mensajero/metabolismo , Expresión GénicaRESUMEN
SCOPE: Epigallocatechin gallate (EGCG), an active polyphenol in green tea, exhibits various physiological effects, including activation of low-density lipoprotein receptors (LDLR). The previous studies have suggested that EGCG activates LDLR via extracellular signal-regulated kinase (ERK) pathway in HepG2 cells. However, the detailed molecular mechanism remains unclear. Recently, 67 kDa laminin receptor (67LR) is identified as a receptor for EGCG. Therefore, this study aims to determine whether 67LR is involved in the mechanism of LDLR activation by EGCG. METHODS AND RESULTS: EGCG induces upregulation of LDLR when 67LR is knocked down in HepG2 cells. Similar effect is observed after the cells are treated with 67LR monoclonal antibody. The loss of antiallergic effect following 67LR siRNA knockdown and 67LR antibody treatment confirms the results since the antiallergic effect of EGCG is known to be mediated by 67LR. CONCLUSION: EGCG activates LDLR expression via 67LR-independent pathway in HepG2 cells.
Asunto(s)
Catequina/análogos & derivados , Receptores de LDL/metabolismo , Receptores de Laminina/metabolismo , Proteínas Ribosómicas/metabolismo , Anticuerpos/farmacología , Catequina/farmacología , Colesterol/metabolismo , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Receptores de LDL/genética , Receptores de Laminina/genética , Receptores de Laminina/inmunología , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an â¼200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5' and 3' junctions indicated that knock-in allele frequencies were higher at the 3' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.
RESUMEN
SCOPE: In animal studies, epigallocatechin gallate (EGCG), the dominant catechin in green tea, has been shown to improve cholesterol metabolism. However, the molecular mechanisms of EGCG underlying these functions have not been fully understood. In this study, we aimed to clarify the molecular mechanisms of the effect of EGCG on cholesterol metabolism mainly in HepG2 cells. METHODS AND RESULTS: We found that EGCG induced a reduction of the extracellular proprotein convertase subtilisin/kexin 9 (PCSK9) level accompanied by an up-regulation of the LDL receptor (LDLR) in HepG2 cells. The EGCG-induced up-regulation of LDLR occurred via the extracellular signal-regulated kinase (ERK) signaling pathway. Moreover, we showed that EGCG induced a significant early reduction of the extracellular PCSK9 protein level. However, there were no significant changes in the PCSK9 mRNA and the intracellular PCSK9 protein levels induced by EGCG. Annexin A2 knockdown affected the basal LDLR expression and did not affect the EGCG-induced reduction of the extracellular PCSK9 protein level or the up-regulation of LDLR. CONCLUSION: Annexin A2 possesses an essential function for the basal LDLR expression in HepG2 cells. But, EGCG induces the suppression of PCSK9 accompanied by an up-regulation of LDLR in an annexin A2-independent manner. EGCG attenuates the statin-induced an increase in PCSK9 level.
Asunto(s)
Anexina A2/metabolismo , Catequina/análogos & derivados , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Catequina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Lipoproteínas LDL/farmacocinética , Lovastatina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proproteína Convertasa 9/genética , Receptores de LDL/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites -3 and -2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.
Asunto(s)
Proteínas Aviares/química , Histidina/química , Muramidasa/química , Oligosacáridos/química , Trisacáridos/química , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Sitios de Unión , Clonación Molecular , Clara de Huevo/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Muramidasa/genética , Muramidasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/metabolismo , Unión Proteica , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Struthioniformes , Especificidad por Sustrato , Trisacáridos/metabolismo , Cigoto/químicaRESUMEN
To characterize the hydrogen-bonding network in lysozyme, we focused on the residue of Asp48 located at the active site in hen egg-white lysozyme. We constructed a mutant lysozyme (D48A) and analyzed using (GlcNAc)3 and chitin-affinity chromatography. The substrate binding of subsites D-F in D48A and the activity against (GlcNAc)5 were decreased. The parameters of substrate binding and rate constants obtained from computer simulations confirmed these changes. In the crystal structure, (GlcNAc)4 was located at the same position as wildtype. However, the side chains of Arg45 and Thr47 at subsites E-F were moved by the replacement. Further, the loss of the hydrogen bond between Asp48 and Ser50 changed the hydrogen-bonding network, and this resulted in an alteration of the side chain of Asn59. This result suggests that the hydrogen-bonding network plays a crucial in the function of Asp52 and of transglycosylation at subsites E-F.
Asunto(s)
Ácido Aspártico , Muramidasa/química , Muramidasa/metabolismo , Animales , Dominio Catalítico , Pollos , Cristalografía por Rayos X , Estabilidad de Enzimas/efectos de los fármacos , Guanidina/farmacología , Enlace de Hidrógeno , Modelos Moleculares , Muramidasa/genética , Mutagénesis Sitio-Dirigida , MutaciónRESUMEN
To evaluate the structure-function relationships of invertebrate lysozymes, a new invertebrate-type (i-type) lysozyme was isolated from the common orient clam (Meretrix lusoria) and the tertiary structure of this enzyme was determined. Comparison of the tertiary structure of this enzyme with those of chicken and Venerupi philippinarum lysozymes revealed that the location of the side chain of the second catalytic residue, an aspartic acid, and the N-acetylglucosamine trimer bound at subsites A-C were different. Furthermore, the amino acid electrostatically interacting with Asp30 in V. philippinarum lysozyme, Lys108, was substituted by Gly in M. lusoria lysozyme and no other possible amino acid that could contribute to this interaction was found in M. lusoria lysozyme. It therefore seems that the substitutions of the amino acids at the interface of the V. philippinarum lysozyme dimer are likely to change the oligomeric state of the M. lusoria lysozyme.
Asunto(s)
Bivalvos/enzimología , Muramidasa/química , Muramidasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Pollos , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
To determine the structure and functional relationships of invertebrate lysozymes, we isolated a new invertebrate (i)-type lysozyme from the common orient clam (Meretrix lusoria) and determined the complete amino acid sequence of two isozymes that differed by one amino acid. The determined sequence showed 65% similarity to a lysozyme from Venerupis philippinarum (Tapes japonica), and it was therefore classified as an i-type lysozyme. The lytic activities of this lysozyme were similar to those of previously reported bivalve i-type lysozymes, but unlike the V. philippinarum lysozyme, it did not exhibit an increase in activity in high ionic strength. Our data suggest that this lysozyme does not have a dimeric structure, due to the replacement of Lys108 which contributes to dimer formation in the V. philippinarum lysozyme. GlcNAc oligomer activities suggested an absence of transglycosylation activity and a higher number of subsites on this enzyme compared with hen egg lysozyme.
Asunto(s)
Bivalvos/enzimología , Muramidasa/genética , Filogenia , Secuencia de Aminoácidos , Animales , Bivalvos/química , Pollos/metabolismo , Estabilidad de Enzimas , Glicosilación , Concentración de Iones de Hidrógeno , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Muramidasa/clasificación , Muramidasa/aislamiento & purificación , Muramidasa/metabolismo , Concentración Osmolar , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
The larvicidal activity against Culex pipiens of all stereoisomers of dihydroguaiaretic acid (DGA) and secoisolariciresinol was measured, and these DGAs were found to be potent. Sixteen (-)-DGA derivatives were then newly synthesized to analyze their structure-activity relationship. Two derivatives monohydroxylated at the 3- or 4-position of the 7-phenyl group of DGA induced acute paralytic activity in the mosquitoes. Derivatives with several hydroxyl groups had lower activity than the natural compound, suggesting that hydrophobicity was probably an important factor for their insecticidal activity.
Asunto(s)
Butileno Glicoles , Culex/efectos de los fármacos , Guayacol/análogos & derivados , Control de Insectos/métodos , Insecticidas , Larva/efectos de los fármacos , Lignanos , Animales , Butileno Glicoles/síntesis química , Butileno Glicoles/farmacología , Culex/crecimiento & desarrollo , Vectores de Enfermedades , Guayacol/síntesis química , Guayacol/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Hidroxilación , Insecticidas/síntesis química , Insecticidas/farmacología , Larva/crecimiento & desarrollo , Dosificación Letal Mediana , Lignanos/síntesis química , Lignanos/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The relationship between the stereochemistry and antimicrobial activity of butane-type lignans was clarified. All stereoisomers of dihydroguaiaretic acid (DGA) showed both antibacterial and antifungal activity. The (+)- and (-)-7,7'-dioxodihydroguaiaretic acid (ODGA) also showed both antibacterial and antifungal activity, while meso-ODGA did not show antibacterial activity, but showed antifungal activity. No activity of any stereoisomer of secoisolariciresinol (SECO) was apparent.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Butanos/química , Guayacol/análogos & derivados , Lignanos/química , Lignanos/farmacología , Antiinfecciosos/síntesis química , Bacterias/efectos de los fármacos , Enfermedades Transmitidas por los Alimentos/microbiología , Hongos/efectos de los fármacos , Guayacol/síntesis química , Guayacol/química , Guayacol/farmacología , Lignanos/síntesis química , Enfermedades de las Plantas/microbiología , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The antioxidant activity of butane-type lignans was evaluated. Secoisolariciresinol (SECO) and dihydroguaiaretic acid (DGA) showed higher radical scavenging activity than that of 7,7'-dioxodihydroguaiaretic acid (ODGA). SECO and DGA inhibited the oxidation of unsaturated fatty acid. Both enantiomers of DGA were also lipoxygenase inhibitors, but neither enantiomer of SECO inhibited the lipoxygenase activity.