RESUMEN
BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting 2% of the population aged over 65 years old. Mitochondrial defects and oxidative stress actively participate in degeneration of dopaminergic (DA) neurons in PD. Paeonolum, a main component isolated from Moutan cortex, has potent antioxidant ability. Here, we have examined the effects of paeonolum against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells. METHODS: The overall viability and neurodegeneration of DA neurons was assessed in ETvmat2:green fluorescent protein (GFP) transgenic zebrafish, in which most monoaminergic neurons are labeled by GFP. Damage to PC12 cells was measured using a cell viability assay and assessment of nuclear morphology. Intracellular reactive oxygen species (ROS) and the level of total GSH were assessed. The mitochondrial cell death pathway including mitochondrial membrane potential, cytochrome C release and caspase-3 activity were also examined in PC12 cells. RESULTS: Paeonolum protected against MPP(+)-induced DA neurodegeneration and locomotor dysfunction in zebrafish in a concentration-dependent manner. Similar neuroprotection was replicated in the PC12 cellular model of MPP(+) toxicity. Paeonolum attenuated MPP(+)-induced intracellular ROS accumulation and restored the level of total GSH in PC12 cells. Furthermore, paeonolum significantly inhibited the mitochondrial cell death pathway induced by MPP(+). CONCLUSIONS: Collectively, the present study demonstrates that paeonolum protects zebrafish and PC12 cells against MPP(+)-induced neurotoxicity.
Asunto(s)
Antioxidantes/uso terapéutico , Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Paeonia/química , Enfermedad de Parkinson/prevención & control , Fitoterapia , Extractos Vegetales/uso terapéutico , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Citocromos c/metabolismo , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Humanos , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/prevención & control , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fármacos Neuroprotectores/farmacología , Neurotoxinas/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Enfermedad de Parkinson/metabolismo , Extractos Vegetales/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Pez CebraRESUMEN
High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.
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Anticonvulsivantes/farmacología , Epilepsia/tratamiento farmacológico , Éteres Fenílicos/farmacología , Piranos/farmacología , Animales , Anticonvulsivantes/química , Anticonvulsivantes/aislamiento & purificación , Conducta Animal/efectos de los fármacos , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Hongos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Larva , Actividad Motora/efectos de los fármacos , Océanos y Mares , Pentilenotetrazol , Éteres Fenílicos/química , Éteres Fenílicos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-fos/genética , Piranos/química , Piranos/aislamiento & purificación , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Pez CebraRESUMEN
The effects of Salvianolic acid A (Sal A) on the treatment of Alzheimer's disease (AD) were investigated. Sal A significantly inhibits amyloid beta [Formula: see text] self-aggregation and disaggregates pre-formed [Formula: see text] fibrils, reduces metal-induced [Formula: see text] aggregation through chelating metal ions, and blocks the formation of reactive oxygen species (ROS) in SH-SY5Y cells. Sal A protects cells against [Formula: see text]-induced toxicity. Furthermore, Sal A, possibly because of the effects of decreasing toxicity effects of [Formula: see text] species, alleviates [Formula: see text]-induced paralysis in transgenic Caenorhabditis elegans. Circular dichroism (CD) experiments and Molecular dynamic (MD) simulations demonstrate that Sal A inhibits [Formula: see text] self-aggregation through binding to the C-terminus of [Formula: see text], and therefore stabilizing the [Formula: see text]-helical conformations. Altogether, our data show that Sal A, as the multifunctional agent, is likely to be promising therapeutics for AD.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/efectos de los fármacos , Ácidos Cafeicos/farmacología , Lactatos/farmacología , Placa Amiloide/tratamiento farmacológico , Proteína Amiloide A Sérica/efectos de los fármacos , Animales , Caenorhabditis elegans/efectos de los fármacos , Línea Celular Tumoral , Dicroismo Circular , Cobre/química , Evaluación Preclínica de Medicamentos , Humanos , Hierro/química , Quelantes del Hierro , Simulación de Dinámica Molecular , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Salvia miltiorrhiza , Proteína Amiloide A Sérica/metabolismo , Zinc/químicaRESUMEN
We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.
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Células Endoteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Piranos/farmacología , Pez Cebra , Animales , Elementos de Respuesta Antioxidante , Células Endoteliales/metabolismo , Hemo-Oxigenasa 1/genética , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , Piranos/química , Piranos/metabolismo , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.
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Benzofuranos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Benzofuranos/administración & dosificación , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pez Cebra/embriologíaRESUMEN
Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.
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Inductores de la Angiogénesis/farmacología , Organismos Acuáticos/química , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Inductores de la Angiogénesis/síntesis química , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/aislamiento & purificación , Animales , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Línea Celular , Cromonas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Hongos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Morfolinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pez Cebra/metabolismoRESUMEN
OBJECTIVE: To investigate the neuroprotective effects of Lycium barbarum extract against MPP(+) -induced neurotoxicity in Caenorhabditis elegans and PC12 cells and its mechanism. METHODS: Pretreated MPP(+) -induced nearotoxicity in C. elegans and PC12 cells with Lycium barbarum at different dosages. The viability and DA neurodegeneration was assessed in C. elegans selectively expressing green fluorescent protein (GFP) in DA neurons. PC12 cell damage was measured using MTT and nuclear morphology. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential and total GSH were assessed. RESULTS: Lycium barbarum extract protected against MPP(+) -induced loss of viability and DA neurodegeneration in C. elegans in a dose-dependent manner. Similar neuroprotection was replicated in MPP + PC12 cell model. Lycium barbarum extract attenuated MPP(+) -induced intracellular ROS accumulation, loss of mitochondrial membrane potential and restored total GSH levels in PCl2 cells. CONCLUSIONS: Lycium barbarum extract protects against MPP(+) -induced neurotoxicity in C. elegans and PC12 cells and its machanism may be related to its antioxidative property and restoration of total GSH level.
Asunto(s)
Lycium/química , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , 1-Metil-4-fenilpiridinio , Animales , Caenorhabditis elegans , Supervivencia Celular/efectos de los fármacos , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glutatión/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/administración & dosificación , Células PC12 , Extractos Vegetales/administración & dosificación , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients with coronary artery disease may undergo a variety of oxidative modifications, rendering it dysfunctional; whether the angiogenic effect is mitigated by such dysfunctional HDL (dHDL) is unknown. We hypothesized that dHDL compromises angiogenesis. The angiogenic effects of nHDL and dHDL were assessed using endothelial cell culture, endothelial sprouts from cardiac tissue from C57BL/6 mice, zebrafish model for vascular growth and a model of impaired vascular growth in hypercholesterolemic low-density lipoprotein receptor null(LDLr-/-)mice. MiRNA microarray and proteomic analyses were used to determine the mechanisms. Lipid hydroperoxides were greater in dHDL than in nHDL. While nHDL stimulated angiogenesis, dHDL attenuated these responses. Protein and miRNA profiles in endothelial cells differed between nHDL and dHDL treatments. Moreover, nHDL suppressed miR-24-3p expression to increase vinculin expression resulting in nitric oxide (NO) production, whereas dHDL delivered miR-24-3p to inhibit vinculin expression leading to superoxide anion (O2â¢-) generation via scavenger receptor class B type 1. Vinculin was required for endothelial nitric oxide synthase (eNOS) expression and activation and modulated the PI3K/AKT/eNOS and ERK1/2 signaling pathways to regulate nHDL- and VEGF-induced angiogenesis. Vinculin overexpression or miR-24-3p inhibition reversed dHDL-impaired angiogenesis. The expressions of vinculin and eNOS and angiogenesis were decreased, but the expression of miR-24-3p and lipid hydroperoxides in HDL were increased in the ischemic lower limbs of hypercholesterolemic LDLr-/- mice. Overexpression of vinculin or miR-24-3p antagomir restored the impaired-angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Collectively, nHDL stimulated vinculin and eNOS expression to increase NO production by suppressing miR-24-3p to induce angiogenesis, whereas dHDL inhibited vinculin and eNOS expression to enhance O2â¢- generation by delivering miR-24-3p to impair angiogenesis, and that vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis.
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Enfermedad de la Arteria Coronaria , MicroARNs , Animales , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/genética , Células Endoteliales , Voluntarios Sanos , Humanos , Lipoproteínas HDL , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/genética , Fosfatidilinositol 3-Quinasas , Proteómica , Pez CebraRESUMEN
Amniotic fluid-derived stem cells have attracted considerable attention in the field of regenerative medicine. Approach of genetic modification probably enhances their regenerative potential. In this work, we wanted to determine whether baculovirus as a new gene vector could efficiently and safely transduce mouse amniotic fluid-derived stem cells (mAFSs). Cells were isolated from mouse amniotic fluid and cultured in vitro. These cells were analyzed by examining phenotypes and differentiation potential. They were further transduced with baculovirus. Baculovirus-transduced mAFSs were induced to differentiate into adipogenic, osteogenic, myogenic, and neurogenic lineages. Mouse amniotic fluid-derived stem cells were successfully isolated and cultured in vitro. They were positive for CD29 and Sca-1, but negative for CD34, CD45, or CD11b. Furthermore, they could differentiate into adipocytes, osteocytes, myocytes, and neurocytes in vitro. Baculovirus could efficiently transduced mAFSs. More importantly, baculovirus-transduced mAFSs retained differentiation potential. Thus, baculovirus vector effective and safe transduction is an attractive promise for genetic modification of mAFSs. Baculovirus genetically modified mAFSs will probably be more suitable as vehicles for regenerative medicine.
Asunto(s)
Líquido Amniótico/citología , Baculoviridae/genética , Diferenciación Celular , Células Madre/citología , Células Madre/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Separación Celular , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos , Neuronas/citología , Osteogénesis , FenotipoRESUMEN
The use of stem cells will lead to novel treatments for a wide range of diseases due to their properties of self-renewing, pluripotent, and undifferentiated state, and the stem cells are usually genetically modified for cell and gene therapy. If the baculovirus, as a new gene vector, can be effectively transduced into various mammalian bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it will be a better gene vector to genetically modify the stem cells. The aim of the present study is to investigate the transduction efficiency of recombinant baculovirus (BacV-CMV-EGFP), which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter, into various mammalian BMSCs. The BMSCs of mouse, rat, porcine, rhesus, and human were cultured primarily in vitro. After more than three passages, the mammalian BMSCs were seeded into dishes and cultured in a humidified incubator at 37 °C with 5% CO(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with recombinant baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 µL PBS at 25 °C for 4 h. At the end of baculovirus transduction, cells were washed and incubated with 2 mL complete medium, and baculovirus-transduced mammalian BMSCs were cultured in a humidified incubator for 2 d. Then, the inverted fluorescent microscope was used to observe GFP expressions in different mammalian BMSCs, and flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs of mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs of mouse, rat and porcine, the inverted fluorescent microscope observations showed that there were more BMSCs expressing GFP and greater mean fluorescence intensity in rhesus and human transduced with baculovirus. The baculovirus could efficiently transduce into the BMSCs of mouse, rat, porcine, rhesus and human, and the transduction efficiency was (20.21±3.02)%, (22.51±4.48)%, (39.13±5.79)%, (71.16±5.36)% and (70.67±3.74)%, respectively. In conclusion, baculovirus displays different transduction efficiency into various mammalian BMSCs. Due to the high transduction efficiency for primate and human BMSCs, baculovirus is possibly a more suitable gene vector to genetically modify BMSCs of human and primates.
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Baculoviridae , Vectores Genéticos , Células Madre Mesenquimatosas/citología , Transducción Genética , Animales , Células de la Médula Ósea/citología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Macaca mulatta , Ratones , Regiones Promotoras Genéticas , Ratas , PorcinosRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0127775.].
RESUMEN
OBJECTIVE: To detect the female carriers from the intron and/or exon-deletion Duchenne/Becker musclular dystrophy (DMD) familial members for prenatal or preimplantation genetic diagnosis. METHODS: Using method of PCR to five microsatellite markers (located in 5' terminus and intron 44, 45, 49, 50), analysing of the short tandem repeat sequence polymorphism with the genescan and binding with the quantitative polymerase chain reaction, we detected the DMD carriers from 1 intron and exon -deletion family and 1 intron-deletion family. RESULTS: The STR-50 genotype of II 2 in family 5 was 245/245, so II3 is DMD gene carrier. The STR-45 genotype of II6 and II8 were del/172, III19 was del/178, so they were all DMD gene carriers. CONCLUSION: The STR haploid linkage analysis combined with quantitative polymerase chain reaction is accurate and efficient to detect the female carriers from the intron and/or exon-deletion DMD familial members.
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Eliminación de Gen , Repeticiones de Microsatélite/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Reacción en Cadena de la Polimerasa/métodos , Exones/genética , Femenino , Heterocigoto , Humanos , Intrones/genética , Masculino , LinajeRESUMEN
OBJECTIVE: To explore the association between angiotensin-converting enzyme (ACE) and the polymorphisms of N5, N10-methylenetetrahydrofolic acid reductase (MTHFR) gene in patients with ischemic stroke (IS). METHODS: Totally 454 patients with IS (IS group) and 334 controls (control group) were recruited in our study. Their I/D polymorphisms of ACE gene and C677T polymorphisms of MTHFR gene were detected by PCR and denaturing high performance liquid chromatography. RESULTS: The frequencies of DD, ID, II and CC, CT, TT genotype in IS group were 22.5%, 43.4%, 34.1%, and 51.8%, 40.5%, 7.7%, respectively, and were 17.4%, 45.5%, 37.1% and 56.9%, 38.3%, 4.8% in the control group, respectively. DD genotype was associated with large-artery atherosclerosis (LAA), and TT genotype and T allele were associated with LAA and cardioembolism. Synergistic effects were found between TT and DD/ID DD genotypes in the pathogenesis of ischemic stroke. CONCLUSION: DD, TT genotype and T allele are risk factors of IS, and ACE gene and MTHFR gene have synergistic effects in the pathogenesis of IS.
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Isquemia Encefálica/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Renina/genética , Accidente Cerebrovascular/genética , Isquemia Encefálica/complicaciones , Predisposición Genética a la Enfermedad , Humanos , Polimorfismo Genético , Accidente Cerebrovascular/etiologíaRESUMEN
OBJECTIVE: To investigate the clinical and lab features of sibling brother and sister both with Duchenne muscular dystrophy (DMD). METHODS: We conducted comprehensive clinical and lab investigations including the test of serum enzymes, electromyography (EMG), electrocardiography, color Doppler echocardiography, HE staining of skeletal muscles, immunohistochemical study of dystrophin and utrophin, multiple ligation probe amplification (MLPA) on exon 1-79 of dystrophin gene, and short tandem repeat-poly- merase chain reaction of CA repeats located in dystrophin gene. RESULTS: These two patients were confirmed to suffer from DMD. They were characterized by typical features of DMD including typical clinical manifestations, increased serum enzymes, EMG presenting myogenic impairment, HE staining presentation belonging to DMD, negative dystrophin in brother, and inconstantly positive on the sarcolemma of sister. Furthermore, no deletion or duplication was found in the 1-79 exons of dystrophin gene. The suffering brother and sister carried the same maternal X chromosome. CONCLUSIONS: Carriers of DMD gene show typical clinical and laboratory manifestations of DMD. Comprehensive examinations should be performed for such carriers.
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Distrofia Muscular de Duchenne , Distrofina/genética , Femenino , Ligamiento Genético , Heterocigoto , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , HermanosRESUMEN
OBJECTIVE: To study relationship between angiotensin converting enzyme (ACE) gene and ischemic stroke (IS). METHODS: (1)Four hundred and fifty-four patients and 334 controls were recruited in our study, their I/D polymorphisms of ACE gene were detected by polymerase chain reaction (PCR) and denaturing high performance liquid chromatogram, and their risk factors of IS were recorded at the same time. (2)In addition, 29 stroke-prone spontaneously hypertensive rats (SHR-SP) and 40 Sprague-Dawley (SD) rats were enrolled, and hypoxia- apnoea animal models and simple apnoea animal models were used at the same time. Their plasma angiotensin II (Ang II) levels were determined. RESULTS: The frequencies of DD, ID and II genotype in IS patients were 22.5%, 43.4% and 34.1%, respectively, and 17.4%, 45.5% and 37.1%, respectively in controls. DD genotype was associated with large artery arteriosclerosis (LAA). Plasma Ang II level in SHR-SP group was (164.49+/-34.58) ng/L, and it was higher than that in control group [(150.92+/-24.92)ng/L] with no significant difference (P>0.05). Ang II levels in apnoea and hypoxia-apnoea group were (382.84+/-62.75) ng/L and (295.90+/-55.07) ng/L, respectively, and they were significantly higher than that in control group (all P<0.01). The relative risks of DD genotype and D alleles in IS patients with smoking, alcohol abuse, or with diabetes mellitus were higher than those in controls, but II genotype and I alleles were lower than those in controls. CONCLUSION: DD genotype is a risk factor for IS, Ang II takes part in the course of hypoxia-stress, and it is correlated with smoking, alcohol abuse and diabetes mellitus in the pathogenesis of IS.
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Isquemia Encefálica/genética , Predisposición Genética a la Enfermedad , Peptidil-Dipeptidasa A/genética , Polimorfismo Genético , Accidente Cerebrovascular/genética , Adulto , Anciano , Anciano de 80 o más Años , Angiotensina II/sangre , Animales , Asfixia/sangre , Modelos Animales de Enfermedad , Femenino , Genotipo , Humanos , Hipoxia/sangre , Masculino , Persona de Mediana Edad , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Factores de RiesgoRESUMEN
OBJECTIVE: To establish a method for detecting the polymorphism of methylenetetrahydrofolate reductase gene (MTHFR). METHODS: The MTHFR was amplified, and the amplified products were detected by denaturing high performance liquid chromatography (DHPLC), and the amplified MTHFR was confirmed by sequencing and restriction enzyme digesting. RESULTS: A total of 334 individuals of Han people in southern China were recruited in our study, and their polymorphisms of MTHFR were detected. The accurate rate of the DHPLC method, that was very sensitive with 100% detection rate available, was over 99%. The frequencies of CC, CT and TT genotypes were 56.9%, 38.3% and 4.8% individually, and the frequencies of T and C alleles were 23.95% and 76.05% individually. CONCLUSION: The DHPLC method can detect polymorphism of MTHFR rapidly, effectively and economically. And there is the existence of different MTHFR polymorphisms in area and race.
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Cromatografía Líquida de Alta Presión/métodos , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético , Adulto , Anciano , Anciano de 80 o más Años , Alelos , China/etnología , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Metilenotetrahidrofolato Deshidrogenasa (NAD+)/genética , Persona de Mediana Edad , Técnicas de Amplificación de Ácido NucleicoRESUMEN
OBJECTIVE: To detect the disease-causing point mutations in the dystrophin gene of Duchenne muscular dystrophy (DMD) patients. METHODS: The approach of denaturing high performance liquid chromatography (DHPLC) coupling with sequencing was used to screen the point mutations of 79 exons and the untranslated regions of dystrophin gene without large deletions/duplications, which was in 6 unrelated DMD probands from 6 DMD families. RESULTS: Five disease-causing mutations, 697-698insGT, C616T, G1255T, C4279T, and C2302T, were ides created the new stop codons in downstream sites of mutations, respectively. In addition to the disease-causing point mutations, a point mutation T5586+61A in intron 39 was also found at patient 3, and a missense mutation A694T in exon 8 was detected at patient 5. Four point mutations, C2168+13T, 5740-13dupG, G5234A and C5280T, were also detected at patient 6 whose causative point mutation was unavailable. Seven point mutations have not been reported previously. Bi-directional PCR amplification of specific alleles (Bi-PASA) method was established to distinguish the haplotypes of heterozygote or homozygote in a single PCR reaction. CONCLUSION: Via automated DHPLC screening or detecting the subexonic mutations in dystrophin gene is feasible to clinical laboratories, and also is a superior method in terms of sensitivity and efficiency.
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Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutación Puntual , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Duplicación de Gen , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Eliminación de SecuenciaRESUMEN
OBJECTIVE: To construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection. METHODS: Retroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction. RESULTS: Micro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.
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Células de la Médula Ósea/metabolismo , Distrofina/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Transfección , Animales , Células de la Médula Ósea/citología , Distrofina/genética , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos mdx , Distrofia Muscular Animal/metabolismo , Infecciones por RetroviridaeRESUMEN
OBJECTIVE: To investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation. METHODS: The bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation. RESULTS: At week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased. CONCLUSIONS: After having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.
Asunto(s)
Células de la Médula Ósea/metabolismo , Distrofina/biosíntesis , Trasplante de Células Madre Hematopoyéticas , Distrofia Muscular de Duchenne/cirugía , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/metabolismo , Trasplante HomólogoRESUMEN
OBJECTIVE: To study the relationship between methylenetrahydrofolate reductase (MTHFR) gene and ischemic stroke. METHODS: Four hundred and fifty four ischemic stroke patients were enrolled in the study. They were divided into large artery atherosclerosis (LAA), cardioembolism (CE), small artery occlusion (SAA), stroke of other determined etiology (SOE) and stroke of undetermined etiology (SUE) according to TOAST (Trail of ORG 10172 in Acute Stroke Treatment) criteria; and they were divided into mild, moderate and severe types ischemic stroke according to their scores of neurologic impairment. Three hundred and thirty four subjects, in whom hypertension, coronary heart disease, cerebral vascular disease, diabetes mellitus, cancer, renal failure etc. were excluded, served as controls in the study. Their C677T polymorphisms of MTHFR gene were determined with polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC), and their risk factors of ischemic stroke were recorded at the same time. RESULTS: The frequencies of CC, CT and TT genotype in ischemic stroke were 51.8%, 40.5% and 7.7%, respectively, and they were 56.9%, 38.3% and 4.8% respectively in controls. TT genotype and T allele were associated with LAA and CE, moderate type and severe type of ischemic stroke. The frequencies of TT genotype and T allele in ischemic stroke patients were significantly higher in those with smoking, alcohol abuse or diabetes mellitus than those in controls (all P<0.10), but CC genotype and C allele were significantly lower in them than those in controls (all P<0.05). On the other hand, all of genotypes and alleles in ischemic stroke patients with no history of smoking, alcohol abuse or diabetes mellitus were not significantly different from those in controls. CONCLUSION: TT genotype and T allele are risk factors for ischemic stroke. It exists interactions between smoking, alcohol abuse, diabetes mellitus and MTHFR gene in the pathogenesis of ischemic stroke.