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1.
Sci Rep ; 7(1): 12378, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28959036

RESUMO

General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms. General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). DT40 or SH-SY5Y cells with only or over 99% expression of InsP3R-1 were treated with isoflurane or propofol. Cell viability was determined by MTT reduction or LDH release assays. Apoptosis was determined by measuring Caspase-3 or by TUNEL assay. Autophagy activity was determined by measuring LC3 II and P62. We evaluated mitochondrial integrity using MitoTracker Green and cytosolic ATP levels. Fura2-AM was used to measure the concentrations of cytosolic calcium ([Ca2+]c). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R. Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. Short exposure to propofol adequately activated InsP3-1 to provide sufficient autophagy for cytoprotection, while prolonged exposure to propofol induced cell apoptosis via impairment of autophagy flux through over activation of InsP3-1. Propofol damaged mitochondria and decreased cytosolic ATP. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R.


Assuntos
Anestésicos Gerais/farmacologia , Autofagia/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Síndromes Neurotóxicas/patologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Galinhas , Humanos , Linfócitos , Células-Tronco Neurais , Síndromes Neurotóxicas/etiologia , Propofol/farmacologia
2.
Mol Genet Metab Rep ; 1: 431-442, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25405082

RESUMO

Smith-Lemli-Opitz syndrome (SLOS) is a congenital, autosomal recessive metabolic and developmental disorder caused by mutations in the enzyme which catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. Herein we show that dermal fibroblasts obtained from SLOS children display increased basal levels of LC3B-II, the hallmark protein signifying increased autophagy. The elevated LC3B-II is accompanied by increased beclin-1 and cellular autophagosome content. We also show that the LC3B-II concentration in SLOS cells is directly proportional to the cellular concentration of 7DHC, suggesting that the increased autophagy is caused by 7DHC accumulation secondary to defective DHCR7. Further, the increased basal LC3B-II levels were decreased significantly by pretreating the cells with antioxidants implicating a role for oxidative stress in elevating autophagy in SLOS cells. Considering the possible source of oxidative stress, we examined mitochondrial function in the SLOS cells using JC-1 assay and found significant mitochondrial dysfunction compared to mitochondria in control cells. In addition, the levels of PINK1 which targets dysfunctional mitochondria for removal by the autophagic pathway are elevated in SLOS cells, consistent with mitochondrial dysfunction as a stimulant of mitophagy in SLOS. This suggests the increase in autophagic activity may be protective, i.e., to remove dysfunctional mitochondria. Taken together, these studies are consistent with a role for mitochondrial dysfunction leading to increased autophagy in SLOS pathophysiology.

3.
Mol Genet Metab ; 104(3): 346-55, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21724437

RESUMO

The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BK(Ca) K(+) channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BK(Ca) protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.


Assuntos
Cavéolas/metabolismo , Desidrocolesteróis/metabolismo , Fibroblastos/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Desidrocolesteróis/química , Genótipo , Humanos , Immunoblotting , Membranas Artificiais , Microscopia Eletrônica , Estrutura Molecular , Pele/citologia , Esteróis/metabolismo , Difração de Raios X
4.
Reprod Sci ; 16(10): 1001-4, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19602724

RESUMO

Studies were undertaken to evaluate the effect of Botulinum neurotoxin type-A (BoNTA) preparation on oxytocin-induced contractions of pregnant human myometrium in vitro. Human myometrial tissue was exposed to increasing concentrations (1-50 000 U/mL) of BoNT/A. Isometric contractions were measured using a force displacement transducer. The cumulative effect of BoNT/A on myometrial activity (time to half relaxation [TTR50], frequency, and amplitude) was evaluated. The frequency of myometrial contractions was depressed by 40% from baseline (P < .05) and relaxation time was increased by 30% (P < .05) from baseline within a narrow range of concentrations. There was no significant difference in amplitude. The observed effects were rapidly reversed after complete wash out of the tissue. BoNT/A or its analogues with more specific tissue affinity may be of value as future agents for prevention of unwanted uterine contractile activity associated with preterm labor and fetal surgery.


Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Contração Uterina/efeitos dos fármacos , Contração Uterina/fisiologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Miométrio/efeitos dos fármacos , Miométrio/fisiologia , Gravidez
5.
Artigo em Inglês | MEDLINE | ID: mdl-12136186

RESUMO

YAC clones were modified via homologous recombination by transfecting yeast cells containing YAC with plasmid PRAN4 DNAs. The integration of neo gene into YAC DNA was identified using Southern blotting and PCR methods. A modified YAC that carried 330 kb human DNA was introduced into L929 cells through PEG mediated spheroplast fusion, and G418 resistant colonies were obtained.

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