RESUMO
1,3,8-Trihydroxy-6-methylanthaquinone (emodin) is recognized as an antiproliferative compound. In the present study, however, we show that emodin has both toxic and survival effects in glioma cells and that the survival effects involve Mdr1a. Emodin inhibited the proliferation and induced apoptosis of C6 cells in a 12-h treatment, but C6 cells survived a 72-h drug treatment, indicating resistance to emodin. Emodin-induced apoptosis was reduced by inhibition of the expression and activation of apoptosis-associated proteins including p53, Bax, Bcl-2, Fas, and caspase-3. C6 cells could express antioxidant proteins (superoxide dismutase and catalase) to decrease reactive oxygen species-induced cytotoxicity of emodin and overexpress multidrug resistance genes (Mdr1a, MRP2, MRP3, and MRP6) to decrease the intracellular accumulation of emodin. Electrophoretic mobility shift analysis showed that emodin decreased nuclear factor kappaB (NF-kappaB) expression in 24 h of treatment, but in 48 h, emodin increased NF-kappaB activity. A confocal microscope showed that emodin induced NF-kappaB translocation from cytoplasm to nuclei. C6 cells would activate the mitogen-activated protein kinase survival pathway and express the DNA repair gene (MGMT) and associated proteins (PARP and XRCC1) to recover the cell activity. C6 cells also expressed GRP78 to decrease emodin-induced endoplasmic reticulum (ER) stress that would cause apoptosis in C6 cells, and GRP78 inhibited the expression of GADD153 to enhance the expression of Bcl-2 that could balance the ER- and mitochondria-induced apoptosis of C6 cells.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos , Emodina/farmacologia , NF-kappa B/fisiologia , Substâncias Protetoras , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dano ao DNA , Fragmentação do DNA/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Fluoresceínas , Corantes Fluorescentes , Microscopia de Fluorescência , RNA Interferente Pequeno/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
Nondeletion forms of hereditary persistence of fetal hemoglobin may result from regulatory disorders of globin gene expression. The defects in two such conditions were localized by demonstrating a tight genetic linkage between the disorders and polymorphic restriction endonuclease sites within the beta-like globin gene complex. In one instance, the defect probably occurred outside the region of DNA between the epsilon- and beta-globin genes.
Assuntos
Hemoglobina Fetal/genética , Talassemia/genética , Deleção Cromossômica , Enzimas de Restrição do DNA , Feminino , Ligação Genética , Globinas/genética , Humanos , Masculino , Linhagem , Polimorfismo GenéticoRESUMO
Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from the whole bone marrow of beta-thalassemia heterozygotes by a density gradient centrifugation procedure. In experiments using total cell "globin," a gradient of alpha/beta chain ratios was observed, increasing with erythroid cell maturation from unity in the basophilic cells up to 2.0 in reticulocytes. Gel filtration of the lysates from these marrow fractions revealed the presence of free alpha chains even in the most immature cells, the amount of which increased with erythroid cell age; the total alpha/beta ratio derived from gel filtration experiments showed a gradient similar to that observed in the total globin experiments. However, the alpha/beta ratio of the hemoglobin fraction obtained by gel filtration remained constant throughout maturation at an average of 0.65. This latter finding is incompatible with balanced synthesis at any stage of red cell development and excludes the possibility that total beta chain production is higher in the early cells than in the later cells or that alpha chain production in the early cells is reduced to the level of beta chain synthesis. Furthermore, in a Hb S/beta-thalassemia marrow examined, the beta A/beta S ratio remained constant throughout maturation while the alpha/non-alpha ratio showed an increase like that observed in the simple beta-thalassemia heterozygotes. This argues strongly against increased synthesis from either the thalassemic or nonthalassemic beta chain gene being responsible for the balanced synthesis in the immature cells. These findings lead us to suggest that, in beta-thalassemia heterozygotes, a large alpha chain pool is present throughout erythroid cell maturation and that the observed increase in alpha/beta ratios is a function of the ability of those cells to degrade the excess alpha chains.
Assuntos
Eritrócitos/metabolismo , Globinas/biossíntese , Heterozigoto , Talassemia/metabolismo , Adulto , Animais , Basófilos/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea , Bovinos , Separação Celular , Centrifugação com Gradiente de Concentração , Cromatografia , Cromatografia em Gel , Contagem de Eritrócitos , Eritrócitos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/biossíntese , Reticulócitos/metabolismo , Análise Espectral , Talassemia/sangue , Talassemia/genéticaRESUMO
Hemoglobin E interacts with beta-thalassemia to produce a disorder of variable severity that is the most common form of symptomatic thalassemia in Southeast Asia. The beta E-globin gene acts as a mild thalassemia gene; there are low levels of beta E-messenger RNA (mRNA) in reticulocytes, and preliminary evidence had suggested that this might be due to instability of the beta E-mRNA. Analysis of beta E-mRNA levels in the nuclei and cytoplasm of bone marrow erythroblasts compared with reticulocytes has shown higher levels of beta E-mRNA in the former, providing direct evidence that this is the case.
Assuntos
Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Hemoglobina E/genética , Hemoglobinas Anormais/genética , RNA Mensageiro/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea , Diferenciação Celular , Eritrócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , HumanosRESUMO
The elongated alpha-globin chains of hemoglobin Constant Spring (alpha cs chain of HbCS ) are produced in low amounts such that the alpha cs-gene acts as a form of alpha-thalassemia; yet in the homozygous state the pathophysiological effects of this mutant are more severe than in the corresponding conditions that result from alpha-globin gene deletions. In studies designed to examine this discrepancy, we have demonstrated that a significant proportion of red cells produced in an HbCS homozygote has a much reduced red cell life span. Contrary to previous reports, we have been able to demonstrate the expected deficit in alpha-chain production in this condition and have shown that both the cessation of globin chain synthesis in vitro and the destruction of the excess beta-chains occur unusually rapidly. Comparison with various deletion forms of alpha-thalassemia suggests that, in terms of intracellular globin chain precipitates and free beta-chain pool, homozygous HbCS red cells more closely resemble those of HbH disease, with three of the four alpha-genes inactivated, than they do the more comparable alpha-thalassemia carriers with only two genes deleted.
Assuntos
Eritrócitos/fisiologia , Hemoglobinas Anormais/metabolismo , Homozigoto , Adulto , Medula Óssea/ultraestrutura , Sobrevivência Celular , Envelhecimento Eritrocítico , Globinas/genética , Hemoglobinas/análise , Humanos , Cinética , Masculino , Microscopia Eletrônica , Talassemia/sangueRESUMO
The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.
Assuntos
Globinas/genética , Família Multigênica , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , DNA , Desoxirribonuclease I/metabolismo , Resistência a Medicamentos/genética , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Neomicina/farmacologia , Mapeamento por Restrição , Transformação GenéticaRESUMO
The beta-globin gene complex is regulated by an upstream locus control region (LCR) which is responsible for high-level, position-independent, erythroid-cell-specific expression of the genes in the cluster. Its role in the developmental regulation of beta-like globin gene transcription remains to be established. We have examined the effect of a single LCR element, hypersensitive site 2 (HS2), on the developmental regulation of the human fetal gamma and adult beta genes in transgenic mice. In mice bearing HS2A gamma beta and HS2G gamma A gamma-117 delta beta human globin gene constructs, switching from gamma- to beta-gene expression begins at about day 13.5 of gestation and is largely completed shortly after birth. The larger construct also demonstrates a switch in G gamma- to A gamma-gene expression during the gamma-to-beta switch similar to that observed during normal human development. We conclude that HS2 alone is sufficient for developmental regulation of the human beta-globin genes.
Assuntos
Regulação da Expressão Gênica , Genes Reguladores , Globinas/genética , Família Multigênica , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Envelhecimento , Animais , Cruzamentos Genéticos , DNA/genética , DNA/isolamento & purificação , Feminino , Idade Gestacional , Hemoglobinas/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por RestriçãoRESUMO
Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.
Assuntos
Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Troca/genética , Globinas/genética , Animais , Sangue , Embrião de Mamíferos/citologia , Eritroblastos/efeitos da radiação , Sangue Fetal/citologia , Raios gama , Globinas/biossíntese , Humanos , Células Híbridas , Cariotipagem , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Família Multigênica/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Baço/citologia , Baço/embriologiaRESUMO
Amyloid beta-protein (Abeta) increases apolipoprotein E (apoE) levels in astrocytes which could alter lipid trafficking. The mechanism for the Abeta-induced increase in apoE levels is not well understood. It is well established that stimulation of beta-adrenergic receptors (betaARs) increases cAMP levels. Elevation of cAMP levels increases apoE abundance. The current study determined if Abeta(1-42) stimulation of cAMP and apoE levels could be inhibited by betaAR antagonists in astrocytes. We demonstrate that Abeta(1-42) but not the reverse protein Abeta(42-1) or Abeta(1-40) stimulated cAMP formation and this stimulation was inhibited by selective betaAR antagonists in mouse primary cortical astrocytes. Abeta(1-42) significantly increased apoE levels which were significantly inhibited by the betaAR selective antagonists with the greatest inhibition observed with the beta(2) antagonist. Separate lines of evidence have suggested that agonist-induced stimulation of betaARs and increases in apoE abundance may serve a neuroprotective role in astrocytes. Our results indicate a potential interaction between betaARs and apoE which may contribute to reducing Abeta(1-42) neurotoxicity.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Peptídeos beta-Amiloides/farmacologia , Apolipoproteínas E/metabolismo , Astrócitos/efeitos dos fármacos , AMP Cíclico/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Western Blotting/métodos , Células Cultivadas , Interações Medicamentosas , Expressão Gênica/efeitos dos fármacos , Técnicas Imunoenzimáticas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
This communication deals with a longitudinal evaluation of C-reactive protein (CRP) analysis during a one-year period using a single lot of liquid control sera (3 levels) (BIOREF-CRP levels 1, 2 and 3) in different laboratories. A total of 652 sets of data were returned from 20 participating laboratories using 13 different reagent-measuring device combinations. The use of the control materials was defined in a standard operating procedure. Data was returned to the organizers on a monthly basis and questions could be asked or problems presented during the evaluation period. Although the performance of different reagents varied, the control materials were shown to be stable over the whole of the evaluation period when stored at 4-7 degrees C in a refrigerator/cold room. Typical problems were encountered, examples of which are presented here in graphical and tabular form.
Assuntos
Proteína C-Reativa/análise , Humanos , Indicadores e Reagentes , Estudos Longitudinais , Nefelometria e Turbidimetria/métodos , Reprodutibilidade dos TestesRESUMO
The interaction of Abeta with synaptosomal plasma membranes decreases membrane fluidity. Using model membrane/liposome systems the interaction of Abeta with specific lipids (e.g. phospholipids, gangliosides, cholesterol) has been defined. The formation of the beta-sheet structure of Abeta when undergoing peptide aggregation is important for Abeta's membrane perturbing properties. This effect can be correlated with the peptide length of Abeta, the longer Abeta1-42 having the greatest effect on membrane fluidity and on neurotoxicity.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Membrana Celular/ultraestrutura , Membranas Intracelulares/ultraestrutura , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Colesterol/metabolismo , Humanos , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Camundongos , Conformação Proteica , Ratos , Sinaptossomos/metabolismoRESUMO
Accurate, economical methods for haemoglobin determination by laboratories in countries with limited resources are not available. This report provides the results of an international collaborative study evaluating the alkaline haematin detergent (AHD575) method as a reference method for laboratory services with limited resources. The study included 6 laboratories; 3 in East Mediterranean countries, 1 in East Africa and 3 in Europe. The (AHD575) method was evaluated against the HiCN method, with blood samples drawn from healthy and sick subjects. The results indicate that the AHD575 method is suitable for measuring haemoglobin in laboratories at all levels.
Assuntos
Hemina/normas , Hemoglobinometria/métodos , Hemoglobinas , África Oriental , Calibragem , Estudos de Casos e Controles , Países em Desenvolvimento , Europa (Continente) , Hemoglobinometria/economia , Hemoglobinometria/normas , Hemoglobinas/análise , Hemoglobinas/metabolismo , Hemoglobinas/normas , Hemólise , Humanos , Região do Mediterrâneo , Fotometria , Padrões de Referência , Fatores de TempoRESUMO
Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.
Assuntos
Encéfalo/citologia , Etanol/farmacologia , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Polarização de Fluorescência , Masculino , Matemática , Camundongos , Camundongos Endogâmicos C57BL , Sinapses/citologiaRESUMO
Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.
Assuntos
Alcoolismo/fisiopatologia , Membrana Celular/fisiologia , Colesterol/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Compartimento Celular , Membrana Celular/ultraestrutura , Ergosterol/análogos & derivados , Ergosterol/metabolismo , Bicamadas Lipídicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipídeos/metabolismo , Membranas Sinápticas/metabolismoRESUMO
Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.
Assuntos
Diterpenos/farmacologia , Dolicóis/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Membranas Sinápticas/fisiologia , Animais , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Difenilexatrieno/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Polarização de Fluorescência , Camundongos , Ácidos Esteáricos , Sinaptossomos , TemperaturaRESUMO
Liver fatty acid binding protein, L-FABP, is an abundant protein that binds fatty acids in vitro. The effects of L-FABP on plasma membrane lipid composition, distribution, and physical structure were determined in intact L-cell fibroblasts transfected with cDNA encoding L-FABP. L-FABP expression altered plasma membrane phospholipids by decreasing both phosphatidylethanolamine and esterified oleic acid content, and increasing sphingomyelin. L-FABP also binds sterols and stimulates sterol uptake and esterification. The fluorescent sterol dehydroergosterol was used to examine sterol distribution in the transfected cell plasma membrane. Dehydroergosterol codistributed equally with the cholesterol in both the bulk membrane and the individual bilayer leaflets. The sterol/phospholipid ratio was decreased in the inner leaflet due to sterol depletion. Concomitantly, intermembrane sterol transfer from the rapidly exchangeable lateral sterol domains as measured by exchange of dehydroergosterol, was reduced. The fluidity of the plasma membrane was measured with the fluorescent molecule diphenylhexatriene by multifrequency (1-250 MHz) phase and modulation fluorometry. Both the bulk plasma membrane and the plasma membrane outer leaflet lipids were fluidized in transfected cells. These alterations of plasma membrane structure and composition are consistent with a role for L-FABP in regulating intracellular sterol and fatty acid distribution and thereby membrane lipid domain structure.
Assuntos
Proteínas de Transporte/metabolismo , Fígado/metabolismo , Lipídeos de Membrana/química , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Fosfolipídeos/química , Animais , Difenilexatrieno , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Células L/metabolismo , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Camundongos , Estrutura Molecular , Ácido Oleico , Ácidos Oleicos/análise , Fosfatidiletanolaminas/análise , Fosfolipídeos/metabolismo , TransfecçãoRESUMO
The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.
Assuntos
Colesterol/química , Ergosterol/análogos & derivados , Membrana Eritrocítica/química , Colesterol/análogos & derivados , Difenilexatrieno/química , Ergosterol/química , Eritrócitos/química , Fluorescência , Humanos , Cinética , Bicamadas Lipídicas/química , Lipossomos/química , Fosfatidiletanolaminas/química , Fosfolipases A/metabolismo , Ácido Trinitrobenzenossulfônico/farmacologiaRESUMO
Sterol carrier protein-2 (SCP-2) is an intracellular lipid carrier protein that binds cholesterol, phospholipids, fatty acids and other ligands. It has been reported that expression of SCP-2 was increased in brain nerve endings or synaptosomes of chronic ethanol-treated mice and it was shown that cholesterol homeostasis was altered in brain membranes of chronic ethanol-treated animals. Ethanol may interfere with the capacity of SCP-2 to bind cholesterol as well as other lipids. This hypothesis was tested using recombinant SCP-2 and fluorescent-labeled cholesterol, phosphatidylcholine (PC), and stearic acid. The association constants (Ka) of the ligand-SCP-2 complex were in the following order: NBD-cholesterol>NBD-PC>NBD-stearic acid. Ethanol, beginning at a concentration of 25 mM, significantly reduced the affinity of NBD-cholesterol and NBD-PC for SCP-2. Effects of ethanol on the Ka of NBD-stearic acid was significant only at the highest concentration that was examined (200 mM). Ethanol significantly increased the Bmax of NBD-cholesterol for SCP-2 but did not have a significant effect on the Bmax of NBD-PC. Similar results were found for effects of ethanol on the Kas and Bmaxs using pyrene-labeled cholesterol and PC. In conclusion, ethanol beginning at a physiological concentration of 25 mM inhibited binding of cholesterol and PC to SCP-2. However, effects of ethanol on lipid binding to SCP-2 were dependent on the type of lipid. Ethanol in vivo may interfere with lipid binding to SCP-2 and disrupt lipid trafficking within cells.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Etanol/farmacologia , Proteínas de Plantas , 4-Cloro-7-nitrobenzofurazano , Ligação Competitiva , Proteínas de Transporte/antagonistas & inibidores , Interações Medicamentosas , Fosfatidilcolinas/farmacologia , Pirenos , Espectrometria de Fluorescência , Ácidos Esteáricos/farmacologiaRESUMO
The metabolic fate of silicone gel leaked into the body from an implant is unknown. In this study, serum from 72 women with silicone gel breast implants and 55 control women was blindly assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental silicon. Samples were processed using materials free of silicon. The mean silicon level in controls was 0.13 +/- 0.07 mg/l (range 0.06-0.35 mg/l), while in implant patients, the mean was significantly higher at 0.28 +/- 0.22 mg/l (range 0.06-0.87 mg/l) (P < 0.01, Student's t-test with correction for unequal variances). Using the mean of the control group + 2 SD as a cutoff for normal range (0.27 mg/l), 25/72 (34.7%) implant patients exceeded this value, compared with 2/55 (3.6%) controls. There was no significant correlation between past rupture of one or both implants, current rupture at the time of the blood draw or the number of years with implants and silicon levels. The results suggest that elevations of serum silicon are seen in many women with silicone gel breast implants. The kinetics of this elevation and the actual chemical species of the measured silicon remain to be determined.
Assuntos
Implantes de Mama , Silício/sangue , Silicones/farmacocinética , Adulto , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Apolipoprotein E (apoE) 4 and aging are risk factors for Alzheimer's disease (AD). Mice expressing human apoE4 and aged wild-type mice show a similarity in the transbilayer distribution of cholesterol in synaptic plasma membranes (SPMs) but differ markedly compared with apoE3 mice and young mice. The largest changes in cholesterol distribution were observed in the SPM exofacial leaflet where there was a doubling of cholesterol. Lipid rafts are thought to be associated with the exofacial leaflet, and we proposed that lipid raft protein and lipid composition would be associated with apoE genotype and age. Lipid rafts were isolated from synaptosomes of different age groups (2, 12, 24 months) of mice expressing human apoE3 and apoE4. Lipid raft markers, alkaline phosphatase (ALP), flotillin-1, cholesterol and sphingomyelin (SM) were examined. Lipid rafts of young apoE4 mice were more similar to older mice as compared with young apoE3 mice in reductions in alkaline phosphatase activity and flotillin-1 abundance. Lipid raft cholesterol and sphingomyelin levels were not significantly different between the young apoE3 and apoE4 mice but cholesterol levels of lipid rafts did increase with age in both genotypes. Results of the present study demonstrate that the two risk factors for Alzheimer's disease, apoE4 genotype and increasing age have similar effects on brain lipid raft protein markers and these findings support the notion that the transbilayer distribution of cholesterol is associated with lipid raft function.