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1.
Biochemistry (Mosc) ; 80(1): 120-5, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25754047

RESUMO

The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.


Assuntos
Adenosina Desaminase/análise , Células Parietais Gástricas/química , Receptor A2B de Adenosina/análise , Animais , Western Blotting , Citometria de Fluxo , Microscopia Confocal , Células Parietais Gástricas/enzimologia , Coelhos
2.
Anal Bioanal Chem ; 401(1): 29-51, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21308368

RESUMO

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.


Assuntos
Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biologia Computacional/métodos , Biologia Computacional/tendências , Humanos , Lipídeos/química , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/tendências
3.
J Physiol Biochem ; 66(1): 73-83, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20414760

RESUMO

Although the human homologue of SND p102, p100 coactivator, was initially described as a nuclear protein, the p100 coactivator protein family members have non-nuclear localization in mammalian cells with active lipid handling, storage, and secretion. However, their role in lipid homeostasis remains unresolved. Here, we investigate the distribution of the rat homologue SND p102 (also called SND1) and its association with newly formed lipid droplets in the liver parenchyma and cultured hepatocytes. Sucrose gradient fractionation showed that SND p102 cofractionated with endoplasmic reticulum and Golgi markers. Such cofractionation was not altered in regenerating steatotic rat liver. However, SND p102 was also detected in lipid droplets from regenerating liver, showing a specific directionalization to the least dense ones. Confocal microscopy of cultured hepatocytes confirmed the findings of gradient fractionation. In addition, p100 coactivator was consistently encountered in microsomes and lipid droplets in control and oleate-treated HepG2 cells. The total amount of SND p102 in hepatocytes was similar in both conditions, suggesting a specific translocation of the protein. Our findings indicate that SND p102 and the human p100 coactivator have a ubiquitous cytoplasmic distribution in hepatocytes and that steatogenic conditions promote the targeting of SND p102 from other cell compartments to specific low density lipid droplets.


Assuntos
Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Animais , Endonucleases , Retículo Endoplasmático/metabolismo , Feminino , Imunofluorescência , Complexo de Golgi/metabolismo , Humanos , Ratos , Ratos Sprague-Dawley
4.
J. physiol. biochem ; 66(1): 73-83, mar. 2010.
Artigo em Inglês | IBECS | ID: ibc-122852

RESUMO

No disponible


Although the human homologue of SND p102, p100 coactivator, was initially described as a nuclear protein, the p100 coactivator protein family members have non-nuclear localization in mammalian cells with active lipid handling, storage, and secretion. However, their role in lipid homeostasis remains unresolved. Here, we investigate the distribution of the rat homologue SND p102 (also called SND1) and its association with newly formed lipid droplets in the liver parenchyma and cultured hepatocytes. Sucrose gradient fractionation showed that SND p102 cofractionated with endoplasmic reticulum and Golgi markers. Such cofractionation was not altered in regenerating steatotic rat liver. However, SND p102 was also detected in lipid droplets from regenerating liver, showing a specific directionalization to the least dense ones. Confocal microscopy of cultured hepatocytes confirmed the findings of gradient fractionation. In addition, p100 coactivator was consistently encountered in microsomes and lipid droplets in control and oleate-treated HepG2 cells. The total amount of SND p102 in hepatocytes was similar in both conditions, suggesting a specific translocation of the protein. Our findings indicate that SND p102 and the human p100 coactivator have a ubiquitous cytoplasmic distribution in hepatocytes and that steatogenic conditions promote the targeting of SND p102 from other cell compartments to specific low density lipid droplets (AU)


Assuntos
Animais , Ratos , Fígado Gorduroso/fisiopatologia , Proteína p120 Ativadora de GTPase/farmacocinética , Hepatócitos/ultraestrutura , Modelos Animais de Doenças
5.
Clín. investig. arterioscler. (Ed. impr.) ; 18(1): 9-17, ene.-feb. 2006. ilus
Artigo em Es | IBECS | ID: ibc-042451

RESUMO

Introducción. Tras su administración sistémica, los adenovirus recombinantes se concentran en el hígado, lo que les validaría para la transferencia génica a este órgano, pero existen efectos colaterales. En este trabajo se ha analizado el impacto de la infección sobre la cantidad y composición de las lipoproteínas de muy baja densidad (VLDL) secretadas en hepatocitos de rata en cultivo. Métodos. Se generaron 2 vectores adenovirales, con ADNc en dirección sentido o antisentido, ambos con capacidad infectiva pero no de propagación. Se infectaron cultivos de hepatocitos de rata con una dosis no citotóxica y se analizaron las VLDL secretadas durante los 3 períodos de 24 h posteriores a la infección y el contenido en lípidos del citosol. Resultados. La infección causó un moderado descenso en la secreción de apo B48 y de éster de colesterol durante las primeras 24 h, pero posteriormente se redujo drásticamente la secreción de partículas con apo B48 y apo B100 y su contenido en lípidos. Además, modificó sus proporciones, aumentando el porcentaje de colesterol libre a costa del esterificado, que llegó a ser indetectable. La menor secreción de VLDL no conllevó una acumulación de lípido intracelular e, incluso, descendió la masa citosólica de éster de colesterol. Conclusión. La infección con adenovirus reduce la secreción hepatocitaria de VLDL y su porcentaje de colesterol esterificado y, a diferencia de otros virus, estas acciones no parecen ir acompañadas de esteatosis hepatocelular (AU)


Introduction. After systemic administration, recombinant adenoviruses are rapidly concentrated in the liver, making them good candidates for gene transfer to this organ. However, there are collateral effects. In this study, we analyzed the effect of adenoviral infection on the number and composition of very low-density lipoproteins (VLDL) secreted by cultured rat hepatocytes. Methods. Two recombinant adenoviruses were developed, each containing cDNA in sense or antisense orientation, and with infective but not propagating ability. Cultured rat hepatocytes were infected with a subcytotoxic adenoviral dose. Secretion of VLDL particles during 3 consecutive 24 h-periods after infection and cytosolic lipid content were characterized. Results. During the first 24 h period, apo B48 and cholesteryl ester secretion moderately decreased. However, in longer incubations, infection dramatically reduced both apo B48 and apo B100 secretion, as well as VLDL lipid content. Lipid proportion was also modified: free cholesterol increased while cholesteryl ester decreased to undetectable levels. Decreased VLDL secretion was not associated with intracellular lipid accumulation; indeed, cholesteryl ester mass was even lower than in noninfected cells. Conclusion. Adenovirus infection causes hepatocytes to secrete less VLDL and to reduce their cholesteryl ester percentage. In contrast with the effects of other viruses, these effects are not accompanied by hepatocellular steatosis (AU)


Assuntos
Ratos , Animais , Hepatócitos/virologia , Adenoviridae/patogenicidade , Lipoproteínas VLDL , Infecções por Adenoviridae/imunologia , Ésteres do Colesterol/análise , Hepatócitos , Lipídeos/imunologia
6.
Hepatology ; 33(3): 662-7, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11230747

RESUMO

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.


Assuntos
Ésteres do Colesterol/metabolismo , Retículo Endoplasmático/enzimologia , Hepatócitos/enzimologia , Hidrolases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Técnicas de Cultura , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Microscopia Imunoeletrônica , Ratos , Frações Subcelulares/enzimologia , Distribuição Tecidual
7.
J Physiol Biochem ; 57(4): 291-301, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12005032

RESUMO

Radiolabeling of low density lipoprotein (LDL) apoB100 with 125I, an oxidative process, is commonly used in lipoprotein investigation. Since 1) LDL is unstable and oxidation-prone, 2) the modification of apoB100 by oxidation increases the negative charge of particles and leads to the uptake of modified LDL through the scavenger receptor pathway, and 3) oxidized LDL is cytotoxic, it is relevant to investigate whether the oxidative stability of LDL is influenced by its labeling with 125I. The aim of this study was to investigate and compare lipid and protein oxidation markers in human LDL after labeling with 125I by two widely adopted methods that use ICl or the chloramide 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril as the oxidizing agent. Native LDL served as a common control and sham-iodinated LDL as a handling control for each procedure. The resistance against copper-induced oxidation of 125I-LDL labeled with ICl was similar to that of controls with regard to the lag time and maximal amount of conjugated diene formed, as there were levels of initial conjugated diene, alpha-tocopherol, and tryptophan. However, radioiodination with the chloramide accelerated the onset of the rapid phase of LDL oxidation due to a drastic depletion of alpha-tocopherol and increased conjugated diene content. Measurements of copper-induced LDL oxidizability showed enhanced indices of lipid oxidation. The lag time and the time to maximal diene production were 65% and 30% shorter than controls. This was accompanied by a 50% reduced tryptophan fluorescence. The anionic surface charge of the LDL particle increased moderately with both labeling procedures. The results indicate that labeling of LDL with 125I may oxidize lipids and apoB100 to a variable extent, depending on the nature of the iodinating agent. This is why assessment of the oxidizability properties of 125I-labeled LDL is recommended for reliable biological studies.


Assuntos
Radioisótopos do Iodo/metabolismo , Marcação por Isótopo/métodos , Lipoproteínas LDL/metabolismo , Ureia/análogos & derivados , Adulto , Antioxidantes/metabolismo , Cloretos/metabolismo , Cobre/metabolismo , Humanos , Indicadores e Reagentes/metabolismo , Iodetos/metabolismo , Lipoproteínas LDL/efeitos da radiação , Oxirredução , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Triptofano/metabolismo , Ureia/metabolismo , alfa-Tocoferol/metabolismo
8.
Lipids ; 34(9): 907-13, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10574654

RESUMO

The periportal (PP) and perivenous (PV) zones of the liver acinus differ in enzyme complements and capacities for cholesterol and bile acid synthesis and other metabolic processes. The aim of this investigation was to determine the acinar distribution of the catalytic activity of the enzymes governing the formation and hydrolysis of cholesteryl esters using PP and PV hepatocytes from normal or cholestyramine-fed rats. The hepatocyte subpopulations were isolated by centrifugal elutriation, characterized according to the distribution pattern of a number of cell parameters and marker enzymes, and assayed for acyl-CoA:cholesterol acyltransferase (ACAT) and lysosomal, cytosolic and microsomal cholesteryl ester hydrolase (CEH). In normally fed rats, no zonation was found in the activity of lysosomal CEH and ACAT, and the activity of both cytosolic and microsomal CEH zonated toward the PV zone of the acinus. Concentrations of free and esterified cholesterol in homogenates, cytosol, and microsomes of PP and PV cells were, however, similar. Cholestyramine raised significantly the PV/PP ratio of ACAT because of an exclusive PP reduction of activity and abolished the heterogeneity in microsomal CEH because of a greater inhibitory PV response, whereas the PV dominance of cytosolic CEH and the homogeneous distribution of lysosomal CEH were unaffected. These results demonstrated homogeneity within the liver acinus for the enzymatic degradation of endocyted lipoprotein-derived cholesteryl esters, a structural zonation of the cytosolic CEH and a dynamic zonation of ACAT and the microsomal CEH, with a PV dominance of the enzymatic capacity for the degradation of stored cholesteryl esters in normal livers.


Assuntos
Ésteres do Colesterol/metabolismo , Fígado/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Resina de Colestiramina/administração & dosagem , Citosol/enzimologia , Esterificação , Feminino , Veias Hepáticas , Hidrólise , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Lisossomos/enzimologia , Microssomos Hepáticos/enzimologia , Veia Porta , Ratos , Ratos Sprague-Dawley , Esterol Esterase/metabolismo , Esterol O-Aciltransferase/metabolismo
9.
J Lipid Res ; 40(4): 715-25, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10191296

RESUMO

This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.


Assuntos
Microssomos Hepáticos/enzimologia , Esterol Esterase/isolamento & purificação , Esterol Esterase/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Western Blotting , Fracionamento Celular , Ésteres do Colesterol/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Cinética , Fluoreto de Fenilmetilsulfonil/farmacologia , Ratos , Ratos Sprague-Dawley , Coloração pela Prata , Esterol Esterase/química , Especificidade por Substrato , Reagentes de Sulfidrila/farmacologia , Ácido Taurocólico/farmacologia
10.
Gen Physiol Biophys ; 17(1): 79-94, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9675558

RESUMO

The binding and internalization of chylomicron remnants in rat hepatocytes originating from the periportal and perivenous zones was compared. The hepatocyte sub-populations were separated by centrifugal elutriation and incubated with 125I-labelled chylomicron remnants at 37 degrees C (to measure binding and internalization) or 4 degrees C (to measure initial binding). Periportal and perivenous cells bound and internalised similar amounts of remnants up to a concentration of about 25 micrograms remnant protein per assay, but at higher concentrations the periportal cells were able to internalise significantly more remnants. When excess unlabelled low density lipoprotein was added to the incubations, little effect on the kinetics of either binding or internalization of the remnants was observed. Lactoferrin, an inhibitor of uptake via the remnant receptor, also did not affect the initial binding of the remnants to either cell type, but decreased internalization to similar extents in both sub-populations. These results suggest that periportal hepatocytes have a greater capacity for the uptake of chylomicron remnants than perivenous cells, and that the remnant receptor plays a more important role than the low density lipoprotein receptor in both sub-populations. This acinar heterogeneity parallels that reported previously for cholesterol de novo synthesis, bile formation, lipid content and hepatic lipase secretion.


Assuntos
Quilomícrons/metabolismo , Fígado/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Humanos , Radioisótopos do Iodo , Cinética , Lipoproteínas LDL/sangue , Fígado/citologia , Linfa , Masculino , Ratos , Ratos Sprague-Dawley
12.
Lipids ; 31(3): 323-9, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8900462

RESUMO

The utility of 2-hydroxypropyl-beta-cyclodextrin for increasing the sensitivity of assays for the microsomal acylCoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-beta-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-beta-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.


Assuntos
Ciclodextrinas/farmacologia , Compostos de Organossilício , Esterol Esterase/metabolismo , Esterol O-Aciltransferase/metabolismo , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Amidas/farmacologia , Animais , Fracionamento Celular , Colesterol/farmacologia , Citoplasma/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hidroxicolesteróis/farmacologia , Cinética , Lisossomos/enzimologia , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Ratos , Fluoreto de Sódio/farmacologia , Solubilidade , Esterol O-Aciltransferase/antagonistas & inibidores
13.
Exp Clin Endocrinol Diabetes ; 104(2): 137-44, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8740937

RESUMO

We have used primary monolayer cultures of hepatocytes from rats fed standard and cholestyramine-diet to study the effects of 17 beta-estradiol and progesterone on the activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) and bile acid synthesis. Cholesterol 7 alpha-hydroxylase activity in hepatocytes freshly isolated from rats fed either diet mentioned above declined gradually during attachment and the first day of culture. Exposure of cell monolayers to 1 or 10 microM estradiol or progesterone resulted in rapid and transient increases in cholesterol 7 alpha-hydroxylase activity, the maximal stimulation of enzyme activity being observed after a 6 h culture period. Bile acid synthesis in standard cells was markedly activated by both hormones, but in cholestyramine cells only the effect caused by 10 microM progesterone was significant. The cellular content of total bile acids was not significantly altered by the presence of the hormones, except by 10 microM progesterone, which provoked an initial cellular depletion of bile acids that was rapidly restored. Bile acid output was enhanced by treating primary cultures with 10 microM estradiol or progesterone, but whereas the increases caused by progesterone were marked and sustained, those caused by estradiol were minor and transient. We conclude that progesterone and 17 beta-estradiol, in this order of potency, enhance short-term bile acid synthesis in rat hepatocyte monolayers.


Assuntos
Anticolesterolemiantes/farmacologia , Ácidos e Sais Biliares/biossíntese , Resina de Colestiramina/farmacologia , Estradiol/farmacologia , Fígado/metabolismo , Progesterona/farmacologia , Animais , Células Cultivadas , Colesterol 7-alfa-Hidroxilase/metabolismo , Feminino , Cinética , Fígado/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
14.
Biochim Biophys Acta ; 1258(2): 90-4, 1995 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-7548188

RESUMO

Okadaic acid, a potent and specific inhibitor of protein phosphatases 1 (IC50 10-20 nM) and 2A (IC50 0.05-2 nM) caused early and sustained inhibitions of microsomal cholesterol ester hydrolase activity in hepatocyte suspensions. The changes in the kinetic properties of the esterase and its response to exogenous alkaline phosphatase and cyclic AMP-dependent protein kinase after cell exposure to 1 microM or 1 nM okadaic acid differed markedly among themselves, which suggests the involvement of both protein phosphatases 1 and 2A in the regulation of the microsomal hydrolysis of cholesterol esters. Furthermore, the inhibitory effect of okadaic acid is likely to be independent of the dibutyryl-cyclic AMP promoted cell events leading to stimulation of esterase activity.


Assuntos
Inibidores Enzimáticos/farmacologia , Éteres Cíclicos/farmacologia , Microssomos Hepáticos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Esterol Esterase/antagonistas & inibidores , Animais , AMP Cíclico/metabolismo , Feminino , Cinética , Ácido Okadáico , Fosforilação , Ratos , Ratos Sprague-Dawley , Esterol Esterase/metabolismo
15.
Steroids ; 59(9): 528-35, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7846735

RESUMO

The regulation of cholesterol 7 alpha-hydroxylase activity by estradiol and progesterone was investigated in liver microsomes isolated from rats fed standard diet, either ad libitum or fasted for 24 h, and diet containing the bile acid sequesterant cholestyramine. Differential effects were observed when the direct action of estradiol and progesterone on microsome preparations was examined. Cholesterol 7 alpha-hydroxylase activity was inhibited by progesterone in a dose-dependent way to almost complete abolition; similar patterns of declines were found in the three feeding groups under study. In contrast, the addition of 5 microM estradiol induced small and selective 7 alpha-hydroxylase increases in fasting and cholestryamine-fed animals, then activity declined to control values and consistent decreases were found from 20 microM. The administration of estradiol (50 micrograms) or progesterone (100 micrograms) for 21 days resulted in depressed cholesterol 7 alpha-hydroxylase activity in rats with high bile acid synthesis basal rate due to cholestyramine feeding. In rats receiving a standard diet, either ad libitum or after 24 h fasting, the hormonal effects did not reach significance. Declines in the content of free cholesterol were provoked by progesterone, not by estradiol, in liver microsomes prepared from all feeding groups. No changes in cholesterol 7 alpha-hydroxylase activity and microsomal free cholesterol were observed after administration of the sex hormones for 3 days. Rapid and transient inhibitions in 7 alpha-hydroxylase activity were found after the single injection of progesterone to fed animals. Estradiol, on the contrary, was unable to alter rapidly the hepatic 7 alpha-hydroxylase capacity.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Colesterol 7-alfa-Hidroxilase/efeitos dos fármacos , Resina de Colestiramina/administração & dosagem , Dieta , Estradiol/farmacologia , Progesterona/farmacologia , Inanição/enzimologia , Animais , Ácidos e Sais Biliares/biossíntese , Colesterol/metabolismo , Feminino , Homeostase/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Ratos , Ratos Sprague-Dawley
16.
FEBS Lett ; 282(2): 300-4, 1991 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-1903716

RESUMO

When pigments of the non-N2-fixing cyanobacterium Phormidium laminosum were carefully extracted and analyzed in a completely O2-free atmosphere, by either high performance liquid chromatography (HPLC) or thin layer chromatography (TLC), the presence of only two carotenoids (namely, beta-carotene and nostoxanthin) was detected. However, exposure of pigments to an air atmosphere during their manipulation led to the rapid appearance in the organic extracts of at least three additional carotenoids (identified as caloxanthin, zeaxanthin and beta-cryptoxanthin). This fact could explain the presence in cyanobacteria of such hydroxylated derivatives of beta-carotene widely reported in the literature. Nitrogen starvation also resulted in an important decrease on the relative beta-carotene/nostoxanthin content of cells, suggesting that this nutritional condition affects thylakoid membranes more drastically than cytoplasmic membranes.


Assuntos
Carotenoides/química , Cianobactérias/análise , Nitrogênio/metabolismo , Carotenoides/análise , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Cianobactérias/metabolismo , Nitrogênio/química , Fixação de Nitrogênio , Oxirredução , Pigmentos Biológicos/análise , Pigmentos Biológicos/metabolismo
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