RESUMEN
Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.
Asunto(s)
Alanina/genética , Eritromelalgia/genética , Ácido Glutámico/genética , Mutación , Canales de Sodio/genética , Trastornos Somatomorfos/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Niño , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica , Eritromelalgia/complicaciones , Ganglios Espinales/citología , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/efectos de la radiación , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Trastornos Somatomorfos/complicaciones , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. METHODS: Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. RESULTS: The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. CONCLUSIONS: These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Oligopéptidos/metabolismo , Animales , Fluorescencia , Humanos , Reproducibilidad de los ResultadosRESUMEN
Recent studies indicate that the actions of arginine vasopressin (AVP) and other agonists that stimulate electrogenic sodium transport in renal epithelial A6 cells are linked to a Ca(2+)-mobilizing signal transduction mechanism that involves generation of inositol trisphosphate. Since diacylglycerol is the other product in this pathway, studies were performed to determine the possible role of PKC in the stimulation of sodium transport. AVP induced a biphasic increase in diacylglycerol generation, characterized by an initial rapid rise and then a sustained elevation, and PKC activation, reflected by phosphorylation of a specific 80 kDa myristoylated alanine-rich PKC substrate (MARCKS). To determine the PKC isoform(s) involved in this process, immunoblot analysis was performed using antisera that recognize both classical PKC isoforms, XPKC-I and XPCK-II, cloned from Xenopus oocytes. The transcripts of both isoforms were expressed in the A6 cell. Since protein recognized by antisera was translocated from cytosol to the particulate fraction after exposure to AVP, one or both isoforms were activated in the A6 cell. Further studies showed that cyclohexyladenosine and insulin, additional agonists of sodium transport in A6 cells, also stimulated phosphorylation of MARCKS. These results argue that Ca(2+)-dependent PKC is involved in the action of AVP, and that of other agonists, which stimulate sodium transport.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Riñón/enzimología , Proteínas de la Membrana , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Vasopresinas/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Arginina Vasopresina/farmacología , Células Cultivadas , Diglicéridos/biosíntesis , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Insulina/farmacología , Isoenzimas , Riñón/citología , Riñón/efectos de los fármacos , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Fármacos Renales/farmacología , Xenopus laevisRESUMEN
MARCKS proteins are widely distributed in mammalian cells and subserve an important role as probes in the examination of signal transduction processes because they are specific endogenous phosphoreceptors for activated protein kinase C. Experiments were performed to determine whether MARCKS proteins are present in amphibia and to show their usefulness as substrates for stimulated PKC activation, using cultured renal epithelial cells (A6) derived from Xenopus laevis as an experimental model.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteína Quinasa C/metabolismo , Proteínas/aislamiento & purificación , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Immunoblotting , Riñón , Peso Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Proteínas/química , Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Vasopresinas/farmacología , Xenopus laevisRESUMEN
Previous studies showed that insulin stimulation of electrogenic Na+ transport in renal epithelial cells is mediated by a calcium-dependent signal transduction mechanism. The present study was performed to determine whether the insulin-induced increase in intracellular Ca2+ (Cai2+) was mediated by hydrolysis of phosphatidylinositol and release of inositol trisphosphate. Experiments were conducted with cultured A6 cells, derived from Xenopus Laevis, grown on permeable supports. Addition of insulin resulted in 2 to 3 fold increases in inositol trisphosphate and a 50% increase in 1,2 diacylglycerol within 10s, which corresponded to the time-course, previously reported, of insulin stimulated increases in Na+ transport and Cai2+. Further studies showed that aldosterone, previously shown to stimulate an increase in 1,4,5-inositol trisphosphate at onset of the rise in Na+ transport, also increased DAG levels during the initial phase of stimulation of Na+ transport. These studies provide the first evidence that a biological response induced by insulin is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) which results in two products, inositol trisphosphate which causes the release of Ca2+ from intracellular stores and 1,2 diacylglycerol. In addition this study provides further support for the proposal that a common signal transduction mechanism mediates electrogenic Na+ transport by multiple agonists.
Asunto(s)
Fosfatos de Inositol/metabolismo , Insulina/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sodio/metabolismo , Aldosterona/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Células Clonales , Diglicéridos/metabolismo , Transducción de Señal , Xenopus laevisRESUMEN
Vasopressin is known to activate two types of cell surface receptors; V2, coupled to adenylate cyclase, and V1, linked to a Ca(2+)-dependent transduction system. We investigated whether arginine vasopressin (AVP) stimulation of electrogenic sodium transport in A6 cells, derived from Xenopus laevis, is mediated by activation of either one or both types of AVP-specific receptors. AVP caused a rapid increase in electrogenic sodium transport, reflected by the transepithelial potential difference (VT) and equivalent short circuit current (Ieq) measurements. AVP also rapidly increased intracellular Ca2+ (Ca2+i) and total inositol trisphosphate. The increase in Ieq was dependent on the rise in (Ca2+i), because 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) dose-dependently inhibited the Ieq response. There was no evidence, however, that activation of adenylate cyclase mediated AVP-stimulated Ieq; transport was not inhibited after AVP-induced activation of adenylate cyclase was abolished by 2',5'-dideoxyadenosine or when cAMP-dependent protein kinase (PKA) activity was abolished by the specific PKA inhibitor IP20. Further studies showed that although both forskolin and 8-(4-chlorophenylthio)-cAMP stimulated Ieq, this occurred by mechanisms independent of PKA activation. These results indicate that AVP-stimulated Na+ transport is mediated by a V1 receptor and a Ca(2+)-dependent mechanism.
Asunto(s)
Arginina Vasopresina/metabolismo , Calcio/metabolismo , Receptores de Vasopresinas/metabolismo , Sistemas de Mensajero Secundario , Sodio/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Transporte Biológico , Células Clonales , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Activación Enzimática , Fosfatos de Inositol/metabolismo , Riñón/citología , Tionucleótidos/farmacología , Agua/metabolismo , Xenopus laevisRESUMEN
Studies were performed to determine the primary signal transduction mechanism that mediates adenosine stimulation of electrogenic sodium transport in renal epithelial cells. Experiments were performed on cultured amphibian A6 cells with an adenosine analogue that preferentially binds to the A1 receptor, cyclohexyladenosine (CHA). Sodium transport was assessed by the equivalent short circuit current (Ieq). CHA was found to stimulate Ieq via activation of an A1 receptor because (1) the threshold concentration was 1 nM compared to that of 10 microM for the specific A2 agonist CGS21680, (2) CHA inhibited vasopressin (AVP)-stimulated cAMP production by a pertussis toxin-sensitive mechanism, and (3) the action of CHA was inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CHA increased intracellular Ca2+ ([Ca2+]i) and stimulated phosphoinositide turnover at concentrations that increased Ieq and in a time course that paralleled the increase in Ieq. Ion transport was stimulated by a Ca(2+)-dependent mechanism because the CHA induced increase in Ieq was inhibited by chelating [Ca2+]i with 5,5'dimethyl BAPTA in a dose-dependent manner, with a Ki of approximately 10 microM. The increase in Ieq was also dose-dependently inhibited by the specific PKC inhibitors dihydroxychlorpromazine and chelerythrine, and by trifluoperazine which inhibits PKC and calmodulin. Further studies indicated that CHA-stimulated Ieq was independent of cAMP generation because CHA did not induce an increase in cAMP accumulation parallel to the increase in Ieq in a dose-response analysis, and the adenylate cyclase inhibitor 2',5' dideoxy-adenosine (DDA) did not affect the CHA-induced increase in Ieq.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina/farmacología , Calcio/fisiología , Membranas Intracelulares/metabolismo , Receptores Purinérgicos P1/fisiología , Sodio/metabolismo , Adenosina/análogos & derivados , Adenilil Ciclasas/fisiología , Animales , Transporte Biológico/efectos de los fármacos , AMP Cíclico/fisiología , Electrofisiología , Concentración Osmolar , Proteínas Quinasas/fisiología , Xenopus laevisRESUMEN
Gangliosides are implicated in cell signal transduction. Prior to investigating this phenomenon in macrophages, the in situ accessibility of gangliosides to macromolecules was assessed for peritoneal macrophages isolated from normal C3H/HeN and endotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident and thioglycolate-elicited macrophage ganglioside patterns are the same as normal strains, and no strain differences in galactose oxidase accessibility for resident or thioglycolate-elicited macrophage gangliosides were found. The only gangliosides accessible to galactose oxidase in resident macrophages are GM1a structures. In thioglycolate-elicited macrophages, an additional ganglioside is accessible. For Escherichia coli-activated macrophages, where ganglioside distribution differs between strains, a difference in galactose oxidase-accessible gangliosides also exists. Escherichia coli-activated C3H/HeN patterns show three triplets absent in C3H/HeJ patterns. There were no differences in ganglioside accessibility to Vibrio cholerae sialidase between the thioglycolate-elicited C3H/HeJ and C3H/HeN macrophages. However, despite differences in sialidase-sensitive ganglioside content between E.coli-activated macrophages of these strains, sialidase accessibility for E.coli-activated macrophages was also similar. Sialidase-susceptible GM3 was cryptic in either strain under all conditions examined. The accessibility of murine macrophage gangliosides to galactose oxidase or sialidase was independent of their sialic acid species and chain length of the ceramide fatty acid. With the exception of GM3, major murine macrophage gangliosides are accessible in situ to macromolecules, especially to exogenous pathogenic bacterial sialidase which can alter macrophage cell surface characteristics. Altered macrophage ganglioside accessibility appears sometimes as a consequence, but not a cause, of C3H/HeJ endotoxin hyporesponsiveness.
Asunto(s)
Gangliósidos/análisis , Gangliósidos/química , Macrófagos Peritoneales/química , Animales , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Galactosa Oxidasa , Gangliósidos/aislamiento & purificación , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Neuraminidasa , Especificidad de la Especie , TioglicolatosRESUMEN
WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)GM1b and (NeuAc)GM1b-GalNAc and the minor component as (NeuAc)GM1b-GalNAc-Gal. The presence of GM1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. GM1b-GalNAc and GM1b-GalNAc-Gal have been reported in T-lineage cells but not in resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoGM1 synthetic pathway. Preliminary results indicate a disialo member of this pathway, GDlc, may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.
Asunto(s)
Gangliósidos/análisis , Macrófagos/química , Animales , Secuencia de Carbohidratos , Cromatografía de Gases , Cromatografía en Capa Delgada , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Espectrometría de Masa Bombardeada por Átomos Veloces , Células Tumorales CultivadasRESUMEN
The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.
Asunto(s)
Gangliósido G(M1)/análisis , Gangliósidos/análisis , Activación de Macrófagos/fisiología , Macrófagos/química , Animales , Secuencia de Carbohidratos , Escherichia coli/inmunología , Femenino , Activación de Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Neuraminidasa , Tioglicolatos/farmacologíaRESUMEN
Bovine buttermilk contains a unique ganglioside, 9-O-acetyl-GD3. In order to isolate large quantities of this ganglioside, a simplified isolation scheme which consists of several ion-exchange and silica gel column chromatographic procedures was devised. The isolated 9-O-acetyl-GD3 was characterized on the basis of its thin-layer chromatographic behavior, its immunoreactivity with a specific monoclonal antibody, JONES, and by conversion to authentic GD3 by mild base treatment.
Asunto(s)
Gangliósidos/aislamiento & purificación , Leche/análisis , Hidróxido de Amonio , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Humanos , Concentración de Iones de Hidrógeno , Hidróxidos , Técnicas para Inmunoenzimas , Melanoma/análisisRESUMEN
We have examined the ganglioside composition of 30-day and 60-day postnatal rat oligodendroglia, adult bovine oligodendroglia, gray matter, white matter, and myelin and also the total lipid composition of the oligodendroglial preparations. The ganglioside patterns of rat and bovine oligodendroglia, as previously found for human oligodendroglia, were more complex than those of myelin. These data indicate that oligodendroglial perikarya can synthesize many brain type gangliosides, not all of which are incorporated into the compact myelin. Alternatively, the ganglioside composition of myelin may be altered in situ by the myelin-associated neuraminidase. In these two species, as in human, GM4 appears specific to oligodendroglia and myelin, while GD3 and GM3 are enriched in oligodendroglia but not myelin. In bovine oligodendrocytes GD3 is the major ganglioside. The total lipid concentration, as well as the percentage of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine, differ for 30- and 60-day-old rat oligodendroglia and may be developmentally correlated with changes in myelin composition during myelinogenesis. There are also marked differences in the lipid composition of bovine oligodendroglia compared to rat oligodendroglia, with the former having more galactolipid and less ethanolamine phosphoglycerides.
Asunto(s)
Gangliósidos/análisis , Lípidos/análisis , Neuroglía/análisis , Oligodendroglía/análisis , Animales , Química Encefálica , Bovinos , Ratas , Ratas EndogámicasRESUMEN
Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.
Asunto(s)
Encéfalo/embriología , Gangliósidos/metabolismo , N-Acetilgalactosaminiltransferasas , Animales , Encéfalo/metabolismo , Gangliósido G(M1)/metabolismo , Gangliósido G(M3)/metabolismo , Galactosiltransferasas/metabolismo , Ratas , Ratas Endogámicas , Sialiltransferasas/metabolismo , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/análisis , Globósidos/aislamiento & purificación , Glucolípidos/aislamiento & purificación , Glicoesfingolípidos/aislamiento & purificación , Feocromocitoma/análisis , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Globósidos/biosíntesis , Glucolípidos/biosíntesis , Lípidos/aislamiento & purificación , Espectrometría de Masas , Factores de Crecimiento Nervioso/farmacología , RatasRESUMEN
To understand better the molecular and cellular events associated with status epilepticus, a multifaceted analysis has begun on hippocampal tissues therapeutically removed from patients with temporal lobe epilepsy. In this first study, quantitative changes in major ganglioside species are reported, as well as the immunocytochemical localization on the ganglioside GD3 in epileptic human hippocampus. Although significant variations were found between patients, the pattern of change was consistent when compared to normal values obtained from an autopsied specimen and the literature. Total ganglioside content was reduced in epileptic hippocampi, which was attributable, in part, to pyramidal cell loss found in CA1 and CA3. In each case, the percentage of ganglioside GD3 was increased significantly, while ganglioside GD1a decreased. The former change is probably associated with reactive astrocytosis and the latter with loss of neuronal dendrites. Immunocytochemical localization revealed GD3 in the stratum radiatum and the subgranular layer of the dentate gyrus. In these areas, GD3 was present in punctate structures and astrocytes. These findings indicate that GD3 increases in selected areas of the sclerotic hippocampus and is presumably related to localized accumulation of reactive glial cells. Since gangliosides have a high affinity for calcium and localized increase in extracellular calcium could disrupt normal neuronal function, the localized increase in GD3 may not only denote reactive glial cells but may contribute directly to the altered, hyperexcitable condition of epilepsy.
Asunto(s)
Epilepsia del Lóbulo Temporal/metabolismo , Gangliósidos/metabolismo , Hipocampo/metabolismo , Cromatografía en Capa Delgada , Humanos , Técnicas para Inmunoenzimas , Lípidos de la Membrana/metabolismoRESUMEN
To examine the effect of antiglycolipid antibodies on demyelination, myelinated cultures of embryonic mouse spinal cords were treated by antigalactocerebroside (anti-GC), anti-GM1; and anti-GM4 antisera, and the lipid composition of the cultures were studied. The anti-GC antiserum-treated cultures, which exhibited severe morphologic signs of demyelination, revealed a significant reduction of cerebroside. The anti-GM4 or anti-GM1 antiserum-treated cultures, which exhibited mild degrees of demyelination, also had low contents of cerebroside. These results support our previous data showing that antiglycolipid antibodies cause demyelination in cultured mouse spinal cords, and suggest a possible role of myelin-specific glycolipids in the demyelination process.
Asunto(s)
Cerebrósidos/inmunología , Gangliósido G(M1)/inmunología , Galactosilceramidas/inmunología , Gangliósidos/inmunología , Sueros Inmunes/farmacología , Metabolismo de los Lípidos , Médula Espinal/metabolismo , Animales , Células Cultivadas , Masculino , Ratones/embriología , Vaina de Mielina/efectos de los fármacos , Conejos , Médula Espinal/citología , Médula Espinal/embriologíaRESUMEN
The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.
Asunto(s)
Gangliósidos/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Línea Celular , Gangliósido G(M3)/metabolismo , Humanos , Masculino , Melanocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We have devised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipid classes of brain tissue. We used DEAE-Sephadex column chromatography to separate the total lipid into neutral and acidic lipid fractions. The lipid fractions were then spotted on separate HPTLC plates and chromatographed in one dimension using two solvent systems. Quantitation was by in situ densitometry with absolute amounts of the lipid classes determined from co-chromatographed standards. An internal standard was also used to improve the precision. The individual lipid classes of rat whole brain, human brain gray and white matter, rat and bovine myelin, and bovine oligodendroglia were quantitated. Human brain phosphatidylethanolamine plasmalogen was also quantitated. Sensitivity was increased by using the cupric acetate charring reagent, which we found to be more sensitive than the conventional sulfuric acid-dichromate reagent. Total lipid (less than 400 micrograms) was quantitated from 5 mg of tissue wet weight. The limit of detection, on HPTLC, for the individual lipid classes was below 20 ng.
Asunto(s)
Química Encefálica , Lípidos/análisis , Animales , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada/métodos , Densitometría , Humanos , RatasAsunto(s)
Química Encefálica , Gangliósidos/análisis , N-Acetilgalactosaminiltransferasas , Enfermedad de Tay-Sachs/metabolismo , Acetilgalactosamina/metabolismo , Encéfalo/metabolismo , Gangliósido G(M2)/análisis , Gangliósido G(M3)/análisis , Galactósidos/farmacología , Galactosiltransferasas/antagonistas & inhibidores , HumanosRESUMEN
A sialyltransferase activity which catalyzes the synthesis of the tetrasialoganglioside GQ1b (N-acetylneuraminyl-N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide) from added trisialoganglioside GT1b (N-=acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) and CMP-N-acetyl[4-14C]neuraminic acid has been demonstrated using a membrane fraction of embryonic chick brain. Optimum enzymatic activity was obtained using the detergent Triton CF-54 at a pH of 6.6. Enzyme activity appeared unaffected by Ca2+, Mg2+, Mn2+, EDTA, or histone. A slight elevation in activity was seen in the presence of Hg2+. When the disialoganglioside GD1b (galactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) was used as the glycolipid substrate, approximately 15% of the radioactive label was found in GQ1b. When this GQ1b was subjected to a periodate oxidation-borohydride reduction, the distribution of radioactive label was consistent with GQ1b being the major tetrasialoganglioside product and that its synthesis could proceed via the sequence GD1b-GT1b-GQ1b.