RESUMEN
In the present report, the action of leukemia inhibitory factor (LIF) on testicular steroid hormone formation was studied. For this purpose, the direct effects of LIF were evaluated on basal and human (h)CG-stimulated testosterone synthesis by cultured, purified Leydig cells isolated from porcine testes. LIF reduced (more than 60%) hCG-stimulated testosterone synthesis. This inhibitory effect was exerted in a dose- and time-dependent manner. The maximal and half-maximal effects were obtained with, respectively, 10 ng/ml (0.5 nM ) and 2.5 ng/ml (0.125 nM ) of LIF after a 48-h treatment of the Leydig cells. Such an effect of the cytokine was not a cytotoxic effect, because it was reversible and Leydig cells recovered most of their steroidogenic activity after the removal of LIF. Considering the sites of action of LIF in inhibiting gonadotropin-stimulated testosterone formation, it was shown that LIF significantly (P < 0.002) reduced, in a comparable range (about 60% decrease), testosterone synthesis stimulated with LH/hCG or with pharmacological agents that enhance cAMP levels (cholera toxin, forskolin, and PG E2), and testosterone synthesis stimulated with 8-bromo-cAMP. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step (or steps) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 microg/ml, 2 h), a cholesterol substrate derivative that does not need an assisted process to be delivered to the inner mitochondrial membrane, reversed most of the inhibitory effect of LIF on the steroid hormone formation. Such results indicate that LIF acts by reducing cholesterol substrate availability in the mitochondria. Consequently, LIF action was tested on steroidogenic acute regulatory protein and PBR (peripheral benzodiazepine receptor) shown to be potentially involved in such a cholesterol transfer. LIF reduced, in a dose- and time-dependent manner, LH/hCG-induced steroidogenic acute regulatory protein messenger RNA levels. The maximal inhibitory effect was obtained with 6.6 ng/ml of LIF after 48 h of treatment. In contrast, LIF had no effect on PBR messenger RNA expression or PBR binding. This inhibitory effect of LIF on Leydig cell steroidogenesis is probably exerted via an auto/paracrine action of the cytokine. Indeed, by immunohistochemistry, LIF and LIF receptor proteins were identified in Leydig and Sertoli cells but not in other testicular cell types, except for LIF receptor in spermatogonia. Furthermore, the presence of LIF and its receptor in Leydig cells at the neonatal and adult periods suggests that the inhibitory effect of LIF on androgen formation reported here probably occurs in both the fetal and the adult Leydig cell populations during testicular development.
Asunto(s)
Gonadotropina Coriónica/farmacología , Inhibidores de Crecimiento/farmacología , Interleucina-6 , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Linfocinas/farmacología , Testosterona/biosíntesis , Androstenodiona/metabolismo , Animales , Células Cultivadas , Toxina del Cólera/farmacología , Colesterol/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Deshidroepiandrosterona/metabolismo , Dinoprostona/farmacología , Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/genética , Hidroxicolesteroles/metabolismo , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Células Intersticiales del Testículo/ultraestructura , Hormona Luteinizante/farmacología , Linfocinas/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfoproteínas/genética , Pregnenolona/metabolismo , ARN Mensajero/análisis , Receptores de Citocinas/genética , Receptores OSM-LIF , Porcinos , Testículo/crecimiento & desarrolloRESUMEN
Porcine Leydig cells in primary cultures are resistant to tumor necrosis factor alpha (TNFalpha) cytotoxicity. Here we report that these cells can be rendered sensitive to TNFalpha killing by treatment with the translational inhibitor cycloheximide, suggesting the existence of proteins that can suppress the death stimulus induced by the cytokine. In search of these cytoprotective proteins, we focused on the constituents of the mitochondrial permeability transition pore (PT pore), whose opening has been shown to play a critical role in the TNFalpha-mediated death pathway. We found that TNFalpha up-regulated mRNA and protein expression of the mitochondrial peripheral benzodiazepine receptor (PBR), an outer membrane-derived constituent of the pore. A strong correlation was established between the resistance of the cells to TNFalpha killing and the density of PBR-binding sites. Concomitantly, TNFalpha down-regulated Bcl-2 mRNA and protein expression. As Bcl-2 has been shown to be an endogenous inhibitor of the PT pore, we hypothesize that the TNFalpha-induced up-regulation of PBR expression may compensate for the decrease in Bcl-2 levels to prevent the opening of the PT pore.
Asunto(s)
Células Intersticiales del Testículo/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Receptores de GABA-A/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antifúngicos/farmacología , Antineoplásicos/farmacología , Sitios de Unión , Supervivencia Celular/fisiología , Cicloheximida/farmacología , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Isoquinolinas/farmacología , Células Intersticiales del Testículo/metabolismo , Masculino , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Receptores de GABA-A/genética , Porcinos , Testículo/citología , Testículo/efectos de los fármacos , Tritio , Regulación hacia ArribaRESUMEN
The aim of the present study was to identify the sites of the inhibitory action of TNFalpha (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFalpha was shown to inhibit testosterone secretion when testicular cells were stimulated with hCG but not when incubated with 22R-hydroxycholesterol (a cholesterol substrate derivative that readily passes through cell and mitochondrial membranes). Such an observation suggested that the cytokine may affect cholesterol transport and/or availability to cytochrome P450scc in the mitochondria. Specifically, we report here that TNFalpha reduced in a dose- and time-dependent manner hCG-induced StAR (steroidogenic acute regulatory protein) levels. The maximal and half-maximal effects were obtained with 20 ng/ml (1.2 nM) and 1.6 ng/ml (0.09 nM) of TNFalpha, respectively. Maximal inhibitory effects of TNFalpha on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of TNFalpha receptors P55 in terms of protein (identified through cross-linking experiments) and messenger RNA (identified through RT-PCR analysis) suggested that the effects of the cytokine are directly exerted on the testicular steroidogenic cell type.
Asunto(s)
Células Intersticiales del Testículo/metabolismo , Fosfoproteínas/metabolismo , Testosterona/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Humanos , Hormona Luteinizante/farmacología , Masculino , Fosfoproteínas/genética , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Porcinos , Testosterona/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The rate-limiting and hormonally regulated step in steroid hormone biosynthesis is the delivery of cholesterol from the outer to the inner mitochondrial membrane where cytochrome P450scc resides. Although the exact mechanism of intramitochondrial cholesterol translocation remains unknown, the formation of contact sites between outer and inner mitochondrial membranes appears as a necessary component for cholesterol transfer. Several pieces of evidence suggest that local formation of intermembrane contact is a consequence of a non-bilayer arrangement of polymorphic lipids which are enriched in the junctions. As a step toward clarifying mitochondrial contact sites formation and thus cholesterol translocation in steroidogenic cells, we have undertaken studies to identify the factors which might result in non-bilayer structure to be adopted by mitochondrial phospholipids on stimulation of MA-10 Leydig cells. Our results demonstrate that an increase in the unsaturation of the cardiolipin acyl groups on hormonal stimulation might favor the formation of non-bilayer adhesion points.
Asunto(s)
Cardiolipinas/efectos de los fármacos , Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Membranas Intracelulares/metabolismo , Células Intersticiales del Testículo/metabolismo , Mitocondrias/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cardiolipinas/metabolismo , Ácidos Grasos Insaturados/análisis , Células Intersticiales del Testículo/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Fosfolípidos/metabolismo , Células Tumorales CultivadasRESUMEN
The distribution of sialic acid-containing glycoproteins was investigated in highly purified mitochondrial membranes using labeled Sambucus nigra agglutinin as a detection system. Two sialylated glycoproteins were shown to be true components of the mitochondrial outer membrane. Relative to monoamine oxidase activity, these glycoproteins were found to be preferentially located in the "free" outer membrane fraction. As sialic acid is thought to be involved in molecular recognition, a role for these glycoproteins in mediating the interactions between mitochondria and other sub-cellular organelles is considered.
Asunto(s)
Membranas Intracelulares/química , Mitocondrias/química , Sialoglicoproteínas/análisis , Animales , Femenino , Ratones , Peso Molecular , OrgánulosRESUMEN
The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidylethanolamine N-methyltransferase) and a mitochondrial one (phosphatidylserine decarboxylase) occurs in reconstituted cell-free systems. Subfractionation of crude mitochondria after swelling and separating on a sucrose density gradient resulted in the isolation of two contact site-enriched fractions from total outer membranes and inner membranes, respectively. Estimation of marker enzyme activities shows a high recovery of glucose-6-phosphate phosphatase (a marker for the endoplasmic reticulum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosynthetic pathway was absent from purified contact site-enriched fractions correlative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restoration of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. These functional interactions between the endoplasmic reticulum and mitochondria are confirmed at the ultrastructural level using either chemical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maintenance of a specific lipid composition and distribution within membranes.
Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Microsomas Hepáticos/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosfolípidos/biosíntesis , Partículas Submitocóndricas/metabolismo , Animales , Sitios de Unión , Biomarcadores/análisis , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Carboxiliasas/metabolismo , Fraccionamiento Celular , Sistema Libre de Células , Centrifugación por Gradiente de Densidad , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares/ultraestructura , Hígado/ultraestructura , Metiltransferasas/metabolismo , Ratones , Microscopía Electrónica , Microsomas Hepáticos/ultraestructura , Mitocondrias Hepáticas/ultraestructura , Dilatación Mitocondrial , Fosfatidiletanolamina N-Metiltransferasa , Partículas Submitocóndricas/ultraestructuraRESUMEN
We previously demonstrated that the translocation of phospholipids between the mitochondrion and the endoplasmic reticulum occurs via highly specialized membrane microdomains of both organelles that are in situ closely associated. As understanding of the interactions between both organelles requires characterization of the translocation sites organization, we first analysed the amino acid compositions of these sites. Using principal component analysis, we have shown that the translocation sites exhibit characteristic patterns when compared with the membranes from which they are derived. The results are discussed in terms of both functional and structural microcompartmentation within the membranes of mitochondria and endoplasmic reticulum.
Asunto(s)
Aminoácidos/análisis , Compartimento Celular , Retículo Endoplásmico/química , Membranas Intracelulares/química , Mitocondrias Hepáticas/química , Análisis de Varianza , Animales , Ratones , Análisis Multivariante , Fracciones Subcelulares/químicaRESUMEN
Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.
Asunto(s)
Centrifugación por Gradiente de Densidad/métodos , Proteínas de la Membrana/aislamiento & purificación , Mitocondrias/química , Placenta/química , Porinas , Fraccionamiento Celular/métodos , Estudios de Evaluación como Asunto , Femenino , Humanos , Proteínas de la Membrana/química , Microscopía Electrónica , Mitocondrias/ultraestructura , Peso Molecular , Placenta/ultraestructura , Povidona , Embarazo , Dióxido de Silicio , Canales Aniónicos Dependientes del VoltajeRESUMEN
Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.
Asunto(s)
Inmunohistoquímica , Manosiltransferasas/aislamiento & purificación , Microscopía Electrónica , Mitocondrias Hepáticas/enzimología , Animales , Western Blotting , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Inmunoglobulina G , Membranas Intracelulares/enzimología , Manosiltransferasas/análisis , Manosiltransferasas/antagonistas & inhibidores , Ratones , Peso MolecularRESUMEN
Previous studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was undertaken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p-CMB, allowed us to discriminate between a galactoside alpha(2-3) sialyltransferase and a galactoside alpha(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events.
Asunto(s)
Membranas Intracelulares/enzimología , Mitocondrias Hepáticas/enzimología , Ácidos Siálicos/metabolismo , Sialiltransferasas/metabolismo , Animales , Asialoglicoproteínas/metabolismo , Fraccionamiento Celular , Activación Enzimática/efectos de los fármacos , Fetuínas , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/ultraestructura , Ratones , Ratones Endogámicos , Mitocondrias Hepáticas/ultraestructura , Ácido N-Acetilneuramínico , Ovinos , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacología , Transferrina/análogos & derivados , Transferrina/metabolismo , alfa-Fetoproteínas/metabolismo , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.
Asunto(s)
Glucosa/metabolismo , Microsomas/metabolismo , Mitocondrias Musculares/metabolismo , Músculos/metabolismo , Adolescente , Adulto , Dolicoles/metabolismo , Femenino , Galactosa/metabolismo , Glucosiltransferasas/metabolismo , Humanos , Masculino , Membranas/metabolismo , Microsomas/enzimología , Persona de Mediana Edad , Mitocondrias Musculares/enzimología , Proteínas Musculares/metabolismo , Músculos/enzimología , Músculos/ultraestructura , Enfermedades Musculares/metabolismo , alfa-Fetoproteínas/metabolismoRESUMEN
Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.
Asunto(s)
Glucosiltransferasas/metabolismo , Mitocondrias Hepáticas/enzimología , Aminoácidos/análisis , Animales , Membranas Intracelulares/enzimología , Ratones , TripsinaRESUMEN
Previous studies have shown the existence of an autonomous mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase, located in mitochondrial outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.
Asunto(s)
Guanosina Difosfato Manosa/análisis , Hexosiltransferasas/análisis , Membranas Intracelulares/enzimología , Manosiltransferasas/análisis , Mitocondrias Hepáticas/enzimología , Azúcares de Nucleósido Difosfato/análisis , Animales , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Activación Enzimática/efectos de los fármacos , Membranas Intracelulares/ultraestructura , Manosiltransferasas/metabolismo , Ratones , Ratones Endogámicos , Mitocondrias Hepáticas/ultraestructura , Concentración Osmolar , Tripsina/farmacologíaRESUMEN
We report 185 children with clinical manifestations of various conditions classically described as giant hamartoma, angiodysplasia, congenital hypertrophy, congenital trophoedema, localised gigantism (e.g. macrodactyly), etc. It is proposed to group all these conditions into a single entity: congenital soft-tissue dysplasia (CSTD). According to recent advances in fundamental embryology and cell biology, CSTD appears to be a consequence of embryonal or fetal cell biosynthetic dysregulation. The concept of the CSTD entity leads to a common protocol for clinical investigation and a common therapeutic plan, with special reference to the stability and the benign nature of the condition. Treatment should be confined to improving function rather than attempting to correct cosmetic deficits.
Asunto(s)
Neoplasias de los Tejidos Blandos/congénito , Adolescente , Niño , Preescolar , Anomalías Congénitas/patología , Displasia Ectodérmica/complicaciones , Displasia Ectodérmica/patología , Femenino , Hamartoma/patología , Humanos , Hipertrofia , Lactante , Recién Nacido , Masculino , Neoplasias Primarias Múltiples/patología , Neoplasias Primarias Múltiples/terapia , Neoplasias de Tejido Vascular/complicaciones , Neoplasias de Tejido Vascular/congénito , Neoplasias de Tejido Vascular/patología , Neoplasias de Tejido Vascular/terapia , Neurofibromatosis 1/patología , Neoplasias de los Tejidos Blandos/complicaciones , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/terapiaRESUMEN
1. Mitochondria outer membranes prepared from mouse livers were purified on a discontinuous sucrose gradient. Control in electron microscopy and marker enzymes assays confirmed purity and homogeneity of this fraction. 2. Purified mitochondria outer membranes exhibited significant UDP-galactose: glycoprotein galactosyltransferase activities when incubated with endogenous or exogenous glycoprotein acceptors in presence of detergent (Nonidet P40). 3. Some properties of two distinct mitochondrial galactosyltransferases, acting respectively on ovomucoid and ovine asialo-mucin were investigated. 4. Transfer of galactose on ovomucoid was maximal for a pH of 7.6 at 33 degrees C whereas asialo-mucin galactosyltransferase exhibited an optimum pH of 5.6 for an optimal temperature of 46 degrees C. 5. These two distinct membrane-bound enzymes were both inhibited by diacylglycerophospholipids whereas lysophospholipids modulated both enzymes in a different way: at 5 mM lysophosphatidylcholine, asialo-mucin galactosyltransferase was slightly stimulated while ovomucoid galactosyltransferase was markedly activated. 6. The most important activating effect on ovomucoid galactosyltransferase was obtained with a phospholipid containing a long aliphatic side chain linked by an ester bond in sn-1 of glycerol, an hydroxyl group or hydrogen atoms in sn-2 and a phosphorylcholine head group in sn-3.
Asunto(s)
Galactosiltransferasas/metabolismo , Membranas Intracelulares/enzimología , Mitocondrias Hepáticas/enzimología , Animales , Glicoproteínas/biosíntesis , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Ratones , Microscopía Electrónica , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructuraRESUMEN
Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.
Asunto(s)
Fosfatos de Dolicol/biosíntesis , Galactosa/biosíntesis , Galactosiltransferasas/metabolismo , Mitocondrias/enzimología , Fosfatos de Poliisoprenilo/biosíntesis , Animales , Ratones , Uridina Difosfato Galactosa/metabolismoRESUMEN
The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated by choline-containing lipids such as phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl phosphate-mannose. The partially purified mannosyltransferase exhibits a Km of 1.33 microM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient centrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.
Asunto(s)
Hexosiltransferasas/aislamiento & purificación , Manosiltransferasas/aislamiento & purificación , Mitocondrias Hepáticas/enzimología , Animales , Centrifugación por Gradiente de Densidad , Cromatografía DEAE-Celulosa , Cromatografía por Intercambio Iónico , Detergentes , Fosfatos de Dolicol/farmacología , Liposomas/aislamiento & purificación , Ratones , Fosfolípidos/farmacología , SolubilidadRESUMEN
Seven cases of papillary meningioma are reported. The patients, 3 females and 4 males, were aged between 21 and 69 years. Five tumors were supratentorial, 1 was located in the left temporal bone, and 1 in the thoracic spinal canal. Five patients had local recurrences and died within 1.4 to 9 years of the original operation. In Case 2, one small pulmonary metastatic nodule was found at autopsy. Microscopically, these meningiomas showed foci of necrosis, numerous mitotic figures and local invasiveness. Psammoma bodies were occasional or absent. Forty-six papillary meningiomas have been identified in the literature. Certain histologic features (necrosis, high mitotic index, rich peripapillary reticulin network) and evolutive events (high rate of local recurrence, development of distant metastases) suggest that this aggressive variant of meningioma could form a histologic link between syncytial, fibroblastic, and hemangiopericytic meningiomas.
Asunto(s)
Meningioma/patología , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Meningioma/epidemiología , Meningioma/terapia , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Factores SexualesAsunto(s)
Anomalías Múltiples/patología , Ectodermo/patología , Mesodermo/patología , Anomalías Múltiples/terapia , Niño , Preescolar , Femenino , Gigantismo/patología , Hamartoma/patología , Humanos , Lactante , Recién Nacido , Síndrome de Klippel-Trenaunay-Weber/patología , Linfedema/congénito , Masculino , Neurofibromatosis 1/patología , Neoplasias de los Tejidos Blandos/patologíaRESUMEN
Difficulties encountered during surgery on maxillary angiodysplasias were studied in relation to case-reports of patients treated in the stomatology and maxillofacial surgery unit directed by professor Vaillant, Hôpital Salpêtrière, Paris. Findings allowed certain conclusions to be drawn with respect to diagnostic and therapeutic features and to compare these with those reported in the international literature.