RESUMO
The lethal effect of 7 beta-hydrocholesterol (7 beta-OHC) on spontaneously transformed cell lines, derived from neonatal rat astrocyte primary cultures and the extent of 7 beta-OHC esterification by naturally occurring fatty acids on C-3 -OH (metabolite) was investigated. The extent of cellular death and metabolite biosynthesis matched with the 7 beta-OHC concentrations. Incubation of the cells with 10 microM 7 beta-OHC in the presence of either lipoproteins depleted fetal calf serum or with increasing serum concentrations revealed proportionality between the degree of cellular cytotoxicity and metabolite levels. The use of tetracaine or progesterone as acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors indicated that ACAT was involved in metabolite production; the inhibition of metabolite biosynthesis slowed down 7 beta-OHC lethal effect. Incubation of the cells with 1 mM db-cAMP, prior 7 beta-OHC treatment, enhanced both metabolite production and cellular death. These findings support the view that the metabolite is directly implicated in the cytotoxic action.
Assuntos
Astrócitos/metabolismo , Ésteres do Colesterol/metabolismo , Hidroxicolesteróis/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Ésteres do Colesterol/toxicidade , Hidroxicolesteróis/toxicidade , Progesterona/farmacologia , Ratos , Esterol O-Aciltransferase/antagonistas & inibidores , Tetracaína/farmacologiaRESUMO
The lethal effect of 7 beta-hydroxycholesterol (7 beta-OHC) on neonatal rat astrocyte primary cultures and spontaneously transformed cell lines derived from them was investigated. Confluent astrocyte primary cultures were not affected by 30 microM 7 beta-OHC over a period of 72 h. In contrast, spontaneously transformed cells were killed by 20 microM 7 beta-OHC within the first 48 h. Further studies indicated that the cell lines metabolized 7 beta-OHC to a product the polarity of which was less than that of 7 beta-OHC. The metabolite was identified as 7 beta-OHC esterified on C-3 by naturally occurring fatty acids. Incubation of the cell lines with 0.5 microM metabolite markedly affected the cells within 24 h. These observations suggest that the 7 beta-OHC metabolite is implicated in the mechanism of action of 7 beta-OHC cytotoxicity on spontaneously transformed cells.
Assuntos
Astrócitos/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/citologia , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Colesterol Oxidase/metabolismo , Esterificação , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hidroxicolesteróis/metabolismo , RatosRESUMO
The correlation between the lethal effect of 7 beta-hydroxycholesterol (7 beta-OH-CH) on spontaneously transformed cell lines derived from rat astrocyte primary cultures (normal cells) and de novo cholesterogenesis was investigated. Both 7 beta-OH-CH and 7-keto-CH were not cytotoxic on normal cells but 7 beta-OH-CH affected markedly the viability of the transformed cells. The use of [14C]acetate or [14C]mevalonate indicated that 7-keto-CH inhibits de novo cholesterogenesis upstream of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in both cell types whereas 7 beta-OH-CH also inhibits downstream of HMGR. The accumulation of two radiolabelled products X1 and X2 between mevalonate and CH was found in unsaponifiable neutral lipids extracted from 7 beta-OH-CH treated transformed cells. HPLC and GC-MS revealed that X1 and X2 are not lanosterol and 24,25-epoxylanosterol, respectively. Incubation of the transformed cells with X1 and X2 did not affect their viability. Our data demonstrate that, under our experimental conditions, 7 beta-OH-CH cytotoxicity is not linked to the inhibition of de novo cholesterogenesis in cultured glial transformed cells.
Assuntos
Astrócitos/efeitos dos fármacos , Colesterol/biossíntese , Hidroxicolesteróis/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Acetatos/metabolismo , Animais , Astrócitos/enzimologia , Linhagem Celular Transformada , Ácido Mevalônico/metabolismo , RatosRESUMO
Incubation of spontaneous transformed cells derived from astrocyte primary cultures with 30 microM 7 beta-hydroxycholesterol (7 beta-OH-CH) which is lethal to the cells or with 150 microM isoproterenol reduces the intracellular level of cAMP (4- and 2-fold respectively). Treatment of the cultures with 0.5 mM dibutyryl (db)-cAMP and 7 beta-OH-CH increases 3-fold the intracellular level of cAMP and both, db-cAMP and isoproterenol, raise the lethal effect of 7 beta-OH-CH and its esterification on C-3-OH by naturally occurring fatty acids (metabolite). Kinetic studies of net steryl-3-esters hydrolysis revealed that db-cAMP and isoproterenol lower that of cholesteryl-3-esters (2-fold) whereas the opposite is found for the metabolite. These data demonstrate that (i) high cAMP intracellular levels modulate differently the net hydrolysis of cholesteryl-3-esters and metabolite, (ii) isoproterenol acts otherwise than cAMP on 7 beta-OH-CH esterification, (iii) the cytotoxicity of 7 beta-OH-CH is linked to its own esterification. The accumulation of metabolite subsequent to db-cAMP or isoproterenol treatment as a result of acyl-CoA:cholesterol acyl transferase activation is discussed.
Assuntos
Bucladesina/farmacologia , Hidroxicolesteróis/farmacologia , Isoproterenol/farmacologia , Animais , Astrócitos/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Ésteres do Colesterol/metabolismo , AMP Cíclico/análise , Hidroxicolesteróis/metabolismo , RatosRESUMO
PURPOSE: To compare the accuracy of left atrial enlargement (LAE) diagnosis made by electrocardiographic criteria with those obtained using M-mode echocardiography. METHODS: We studied 273 patients age 17 to 87 (mean 49) years, 115 men, white 95.5%, black 3.5% mulattos 1%, with or without heart disease of different etiologies. The ECG criteria studied were: a) P terminal force in V1 > or = 0.04 mmx s; b) Pt force in V1 duration > 0.04s; c) Pt force in V1 depth > or = 1 mm; d) P wave notching in D2 with interpeak distance > or = 40ms; f) presence of atrial fibrillation. The gold-standard for LAE was left atrial dimension > 40 mm obtained by echocardiography. RESULTS: The percentage of correct diagnosis were: atrial fibrillation (88%), Morris index (75%), PtfV1 negativity > or = 1 mm (74%), notched P wave in D2 with interpeak distance > or = 0.04 s (70%), PtfV1 with duration > 0.04 s (64%) and P wave duration in D2 > 0.11s (46%). CONCLUSION: Conventional ECG has limited value for detecting LAE. A higher correlation was found between atrial fibrillation and changes in P wave in V1 and the echocardiographic LAE.
Assuntos
Cardiomegalia/diagnóstico , Ecocardiografia , Eletrocardiografia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cardiomegalia/diagnóstico por imagem , Feminino , Átrios do Coração/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
A molecular beam technique has been used to study the dynamics and mass accommodation of HCl molecules in collision with sulfuric acid-water surfaces. The experiments were performed by directing a nearly mono-energetic beam of HCl molecules onto a continuously renewed liquid film of 54-76 wt% sulfuric acid at temperatures between 213 K and 243 K. Deuterated sulfuric acid was used to separate sticking but non-reactive collisions from those that involved penetration through the phase boundary followed by dissociation and recombination with D+. The results indicate that the mass accommodation of HCl on sulfuric acid-water surfaces decreases sharply with increasing acidity over the concentration range 54-76 wt%. Using the capillary wave theory of mass accommodation this effect is explained by a change of the surface dynamics. Regarding the temperature dependence it is found that the mass accommodation of HCl increases with increasing temperature and is limited by the bulk phase viscosity and driven by the restoring forces of the surface tension. These findings imply that under atmospheric conditions the uptake of HCl from the gas phase depends crucially on the bulk phase parameters of the sulfuric acid aerosol.
Assuntos
Aerossóis/química , Atmosfera/química , Ácido Clorídrico/química , Modelos Químicos , Material Particulado/química , Ácidos Sulfúricos/química , Gases/química , Cinética , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The rate and thermodynamics of the adsorption of acetone on ice surfaces have been studied in the temperature range T = 190-220 K using a coated-wall flow tube reactor (CWFT) coupled with QMS detection. Ice films of 75 +/- 25 microm thickness were prepared by coating the reactor using a calibrated flow of water vapor. The rate coefficients for adsorption and desorption as well as adsorption isotherms have been derived from temporal profiles of the gas phase concentration at the exit of the flow reactor together with a kinetic model that has recently been developed in our group to simulate reversible adsorption in CWFTs (Behr, P.; Terziyski, A.; Zellner, R. Z. Phys. Chem. 2004, 218, 1307-1327). It is found that acetone adsorption is entirely reversible; the adsorption capacity, however, depends on temperature and decreases with the age of the ice film. The aging effect is most pronounced at low acetone gas-phase concentrations (< or = 2.0 x 10(11) molecules/cm(3)) and at low temperatures. Under these conditions, acetone is initially adsorbed with a high rate and high surface coverage that, upon aging, both become lower. This effect is explained by the existence of initially two adsorption sites (1) and (2), which differ in nature and number density and for which the relative fractions change with time. Using two-site dynamic modeling, the rate coefficients for adsorption (k(ads)) and desorption (k(des)) as well as the Langmuir constant (K(L)) and the maximum number of adsorption sites (c(s,max)), as obtained for the adsorption of acetone on sites of types (1) and (2) in the respective temperature range, are k(ads)(1) = 3.8 x 10(-14) T(0.5) cm(3) s(-1), k(des)(1) = 4.0 x 10(11) exp(-5773/T) s(-1), K(L) (1) = 6.3 x 10(-25) exp(5893/T) cm(3), c(s,max)(1) < or = 10(14) cm(-2) and k(ads)(2) = 2.9 x 10(-15) T(0.5) cm(3) s(-1), k(des)(2) = 1.5 x 10(7) exp(-3488/T) s(-1), K(L)(2) = 5.0 x 10(-22) exp(3849/T) cm(3), c(s,max)(2) = 6.0 x 10(14) cm(-2), respectively. On the basis of these results, the adsorption of acetone on aged ice occurs exclusively on sites of type (2). Among the possible explanations for the time-dependent two-site adsorption behavior, i.e., crystallographic differences, molecular or engraved microstructures, or a mixture of the two, we tentatively accept the former, i.e., that the two adsorption sites correspond to cubic (1, I(c)) and hexagonal (2, I(h)) sites. The temporal change of I(c) to I(h) and, hence, the time constants of aging are consistent with independent information in the literature on these phase changes.
RESUMO
Studies of the oxidation of ethanol, serine and pyruvate by non proliferating suspensions of P. fluorescens show that the rate of oxidation of these substrates is appreciably increased by the addition of NADH. The observed effects are interpreted on the basis of apparent oxidation quotients of NADH which exceed the theoretical values.
Assuntos
Etanol/metabolismo , NAD/metabolismo , Pseudomonas fluorescens/metabolismo , Piruvatos/metabolismo , Serina/metabolismo , Oxirredução , Consumo de Oxigênio , Pseudomonas fluorescens/crescimento & desenvolvimentoRESUMO
Simultaneous addition of succinate (or fumarate) and NADH to a non-proliferating suspension of P. fluorescens results in an enhancement of the oxydation of the endogenous substrates. This conclusion is in accord with the determination of the respiratory and the oxidation quotients of the analysed exogenous substrates.
Assuntos
NAD/farmacologia , Pseudomonas fluorescens/efeitos dos fármacos , Succinatos/farmacologia , Dióxido de Carbono/metabolismo , Ciclo do Ácido Cítrico , Fumaratos/farmacologia , Ácidos Cetoglutáricos/farmacologia , Malatos/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Pseudomonas fluorescens/metabolismoRESUMO
The cytotoxicity of 7 beta-hydroxycholesterol (7 beta-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 microM 7 beta-OHC over a period of 72 h whereas 30 microM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7 beta-OHC added to the cultures at concentrations of 20 microM or 30 microM. Cellular fractionation after incubation with 20 microM or 30 microM 7 beta-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7 beta-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 microM 7 beta-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 microM 7 beta-OHC treatment. Fluorescence anisotropy measurements indicated that 20 microM 7 beta-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 microM 7 beta-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 microM 7 beta-OHC. These findings demonstrate that 7 beta-OHC is lethal to highly proliferating cultured glial cells. The high accumulation of 7 beta-OHC in the plasma membrane and its decrease in fluidity, by themselves, do not seem to be involved in the processes leading to cellular death. However, increase of plasma membrane fragility associated with the decrease of CH/PL, which occurs exclusively in plasma membranes isolated from 7 beta-OHC treated transformed cells together with high 7 beta-OHC uptake, are probably implicated in 7 beta-OHC cytotoxicity. The possibility of an additional action mechanism is discussed.
Assuntos
Astrócitos/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Lipídeos de Membrana/análise , Proteínas de Membrana/análise , Animais , Astrócitos/química , Astrócitos/metabolismo , Linhagem Celular Transformada , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hidroxicolesteróis/metabolismo , RatosRESUMO
We have reported previously that oxysterols inhibit astrogliosis and intracranial glioblastoma growth. To elucidate the mechanism of action of these molecules in vivo, we have investigated their effect on the cholesterol biosynthesis in the injured brain. In a bilateral lesion model, injection of liposomes containing 7 beta-hydroxy-cholesterol decreased [3H]acetate incorporation into neutral lipids and cholesterol by 30% and 40%, respectively. Structural analogues were tested using a unilateral lesion model. The injury did not significantly affect cholesterogenesis; injection of 7 beta-hydroxycholesterol or 7 beta-hydroxycholesteryl-3-oleate reduced acetate incorporation into cholesterol by 47% and 43%, respectively. Both 7-ketocholesteryl-3-oleate and 7 alpha-hydroxycholesteryl-3-oleate inhibited cholesterogenesis by 32%. As cholesterol and by-products of the cholesterol pathway play a key role in cell division, we have assessed the effect of oxysterols on reactive astrocyte proliferation. The incorporation of bromodeoxyuridine showed that up to 46% of astrocytes were proliferating 24 h after the injury. Injection of 12 nmol of 7 beta-hydroxycholesterol or 7 beta-hydroxycholesteryl-3-oleate reduced the labelling index to 26%, whereas the labelling index in the 7-keto-cholesteryl-3-oleate-treated cortex was 37%. These findings demonstrate that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis in brain and show a correlation between cholesterogenesis and reactive astrocyte proliferation.