RESUMEN
BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low density lipoprotein receptor (LDLR) gene. Two novel LDLR mutations, D151Y and M391T, had been previously identified in unrelated Thai patients with heterozygous FH. To confirm that these mutations cause FH, the functional characteristics of D151Y and M391T, which are located in the fourth cysteine repeat of the ligand-binding domain and in the sixth YWTD repeat of the epidermal growth factor precursor homology domain, respectively, were studied. METHODS: CHO-ldlA7 cells were transfected with wild type and mutant LDLR cDNAs. Thereafter, the localization, expression, and ability of LDL uptake of LDLR were evaluated by confocal laser scanning microscope (CLSM), and flow cytometry. RESULTS: CLSM revealed both D151Y and M391T LDLR were partially retained in the endoplasmic reticulum, with the remaining residual activity observed by LDL uptake. Similarly, flow cytometric analysis showed a significant reduction of LDLR expression to 18% and 38% and of LDL uptake to 15% and 71% in D151Y and M391T LDLR, respectively. CONCLUSIONS: The transport defect of LDLR contributes to the pathology of FH. These data are useful for an insight inspires the development of novel lipid-lowering drugs with beneficial therapeutic value.
Asunto(s)
Hipercolesterolemia/genética , Mutación Missense , Receptores de LDL/genética , Animales , Transporte Biológico/genética , Línea Celular , Retículo Endoplásmico , Humanos , Lipoproteínas LDL/metabolismo , Receptores de LDL/análisis , Receptores de LDL/metabolismo , Tailandia , TransfecciónRESUMEN
The low-density lipoprotein receptor (LDLR) is a key regulator of cholesterol homeostasis, and defects in the function of LDLR result in familial hypercholesterolemia (FH). In the present study, we performed structural analyses of two novel LDLR mutations, D151Y and M391T. Both mutations occurred in conserved residues of LDLR. The D151Y mutation, in the ligand binding domain, caused an elimination of a hydrogen bond in the calcium binding site, higher solvent accessibility and a loss of negative charge in the Y151 residue. On the other hand, the M391T mutation, in the beta-propeller of the epidermal growth factor (EGF) precursor homology domain, caused an additional hydrogen bond to form, higher solvent accessibility and a distortion of the beta-strand. These data suggest that the irregular structures of the mutated LDLRs are likely to cause the functional defect that contributes to the pathology of FH.
Asunto(s)
Simulación por Computador , Modelos Moleculares , Receptores de LDL/química , Receptores de LDL/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia Conservada , Análisis Mutacional de ADN , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Ratas , Análisis de Secuencia de ProteínaRESUMEN
UNLABELLED: Moringa oleifera is used in Thai traditional medicine as cardiotonic. Recent studies demonstrated its hypocholesterolaemic effect. However, to be clinically useful, more scientific data are needed. AIM OF THE STUDY: We investigated the antioxidant, hypolipidaemic and antiatherosclerotic activities of Moringa oleifera leaf extract. MATERIALS AND METHODS: Scavenging activity of the extract on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), and the inhibitory effect on Cu(2+)-induced low-density lipoprotein (LDL) oxidation were determined in in vitro experiment. The effects of the extract on cholesterol levels, conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) and plaque formations in cholesterol-fed rabbits were investigated. RESULTS: We found that in scavenging DPPH radicals the extract and Trolox had IC(50) of 78.15+/-0.92 and 2.14+/-0.12microg/ml, respectively. The extract significantly (P<0.05) prolonged the lag-time of CD formation and inhibited TBARS formation in both in vitro and ex vivo experiments in a dose-dependent manner. In hypercholesterol-fed rabbits, at 12 weeks of treatment, it significantly (P<0.05) lowered the cholesterol levels and reduced the atherosclerotic plaque formation to about 50 and 86%, respectively. These effects were at degrees comparable to those of simvastatin. CONCLUSIONS: The results indicate that this plant possesses antioxidant, hypolipidaemic and antiatherosclerotic activities and has therapeutic potential for the prevention of cardiovascular diseases.
Asunto(s)
Antioxidantes/farmacología , Aterosclerosis/prevención & control , Hipolipemiantes/farmacología , Moringa oleifera/química , Extractos Vegetales/farmacología , Animales , Aterosclerosis/tratamiento farmacológico , Compuestos de Bifenilo , Peso Corporal/efectos de los fármacos , Colesterol/sangre , Colesterol en la Dieta/administración & dosificación , Colesterol en la Dieta/farmacología , Humanos , Lípidos/sangre , Lipoproteínas LDL/metabolismo , Masculino , Picratos/análisis , Hojas de la Planta/química , Conejos , Simvastatina/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Vitamina E/farmacología , Agua/químicaRESUMEN
BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant hypercholesterolemia caused by mutation in the LDL receptor gene. M412T mutation of the LDL receptor gene was previously observed in a single female patient diagnosed as having primary hypercholesterolemia. However, the analysis was incomplete and there was no confirmation of the M412T as the FH-causing mutation. We identified a mutation in the LDL receptor gene that underlines the severe FH phenotype in a new case, a female Chinese Thai patient. METHODS: Identification was made by PCR-SSCP, direct DNA sequencing and confirmed by allele specific amplification (ASA) originally designed for this current study. RESULTS: The entire LDL receptor gene screening revealed the genetic alteration that also caused M412T mutation in this new index patient. ASA analysis confirmed the DNA sequence in this patient and further identified three family members as M412T carriers. CONCLUSIONS: The finding of this mutation in 2 apparently unrelated index patients and the co-segregation of M412T and FH phenotype in the family of the present index case should provide evidence and confirm that the M412T was likely to be a disease-causing mutation. Whether M412T is common either as a founder or recurrent mutation among FH Chinese Thai population is unknown at present and remains to be clarified.