RESUMEN
Rabbit antibody produced in response to the purified mitogenic glycoprotein lectin from Wistaria floribunda seeds (WFM) contains anti-carbohydrate antibody. This antibody, which represents 25% of the total antibody precipitated by the homologous antigen cross-reacts with the glycoprotein hemagglutinating lectins from Sophora japonica (SJL), W. floribunda (WFA) and the glycoprotein bromelain, but not the protein lectin from Maclura pomifera seeds. The cross-reactive reaction is totally abolished by the presence of glycopeptides obtained from SJL. Utilization of a fluorometric binding assay employing fluorescein derivatized glycopeptides from SJL, bromelain, fetuin and ovalbumin, it was found that the total anti-carbohydrate antibody population best reacts with the following carbohydrate structure: MAN alpha 1 leads to 6 MAN alpha 1 leads to 6 MAN beta 1 leads to 4 GLCNAC beta 1 leads to 4 GLCNAC beta 1 leads to Asn. Substitution of the beta-mannosyl moiety at position 3 results in structures not capable of binding to the anti-carbohydrate antibody. This antibody appears to distinguish between those glycan moieties of glycoproteins commonly found in animals from those lacking 3-O-substitution of the beta-mannosyl residue as found in some plant glycoproteins.
Asunto(s)
Carbohidratos/inmunología , Glicoproteínas/inmunología , Lectinas/inmunología , Animales , Formación de Anticuerpos , Reacciones Cruzadas , Glicopéptidos/inmunología , Sueros Inmunes/inmunología , Inmunodifusión , Lectinas de Plantas , Conejos , SemillasRESUMEN
Since hapten inhibition of precipitation is relatively time consuming, we have developed a hapten inhibition of adsorption assay to explore the sugar binding specificity of the Sophora japonica lectin. Adsorbents were prepared with Sephadex and p-aminophenyl beta-D-galactopyranoside using the CNBr procedure. The ability of simple saccharides to inhibit the binding of lectin to the adsorbent was performed employing a fixed amount of adsorption with respect to the logarithm of micromoles of inhibitor yielded sigmoid shaped curves. The quantities of various saccharides required to cause 50% inhibition of the lectin--adsorbent interaction relative to D-galactose were within+/-5% of the relative inhibiting potencies of the sugars in
Asunto(s)
Adsorción , Especificidad de Anticuerpos , Lectinas , Sistema del Grupo Sanguíneo ABO , Compuestos de Anilina , Carbohidratos , Bromuro de Cianógeno , Dextranos , Galactosa , Glicósidos , Haptenos , Concentración de Iones de HidrógenoRESUMEN
The preparation of chlorophyllin copper complex (CCC), shown to be a tumor promoter in an animal model (Nelson, R.L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res., 12, 737-740), also inhibits the activities of direct- and indirect-acting mutagens in the Salmonella assay and exhibits cytostatic and cytocidal effects toward myeloma cells. Data from elemental analyses, spectrophotometry and reversed-phase high-performance liquid chromatography indicate that CCC preparations generally used in antimutagenic/anticarcinogenic experiments are variable, complex mixtures of structurally distinct porphyrins lacking copper in some instances. This variability of the composition may be a cause for the differences reported for the tumor promotion activity of CCC.
Asunto(s)
Antimutagênicos/farmacología , Antineoplásicos/farmacología , Clorofilidas/química , Clorofilidas/farmacología , Animales , Antineoplásicos/química , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cobre/análisis , Relación Dosis-Respuesta a Droga , Ratones , Plasmacitoma/patología , Salmonella typhimurium/efectos de los fármacos , Espectrofotometría , Células Tumorales CultivadasRESUMEN
The acetoxymethyl ester of chlorin e6 (CAME) was initially designed to be a hydrophobic photosensitizing agent that would be recognized by an endocytic pathway and initially accumulated in lysosomes. This was expected to lead to hydrolysis of the ester groups, followed by redistribution of the free chlorin to other subcellular sites. In this study, we examined the patterns of localization of CAME and of subsequent photodamage in murine leukemia L1210 cells. The drug was initially localized at intracellular sites, yielding a pattern similar to that obtained with a fluorescent probe for acidic intracellular vesicles and endosomes. A brief (30 min) incubation with 10 microM CAME followed by irradiation led to mitochondrial photodamage and apoptotic cell death. At a higher drug level, or with a longer incubation time, we observed additional photodamage to the plasma membrane and to lysosomes. The higher photodynamic therapy dose led to inhibition of apoptosis, with cell death likely occurring via a necrotic process. Distribution of CAME among the components of human plasma was to albumin > high-density lipoprotein > low-density lipoprotein. These results have implications concerning the likely mechanism of CAME accumulation and subcellular distribution.
Asunto(s)
Fotoquimioterapia/efectos adversos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Animales , Compartimento Celular , Humanos , Leucemia L1210/patología , Ratones , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/farmacocinéticaRESUMEN
Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.
Asunto(s)
Clorofila/análogos & derivados , Grupo Citocromo c/efectos de la radiación , Clorofila/farmacología , Grupo Citocromo c/metabolismo , Técnicas In Vitro , Oxidación-Reducción , Fotoquímica , Fotoquimioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Superóxidos/metabolismoRESUMEN
We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Clorofila/análogos & derivados , Fármacos Fotosensibilizantes/química , Porfirinas/química , Carcinoma/enzimología , Clorofila/química , Clorofila/metabolismo , Clorofilidas , Esterasas/metabolismo , Esterificación , Ésteres/química , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Solubilidad , Espectrometría de Fluorescencia , Espectrofotometría , Neoplasias de la Vejiga Urinaria/enzimologíaRESUMEN
A new, 'diffuse, multiple banding', electrophoretic variant of arylsulfatase A protein was found in two patients with major depression. Protein analyses showed that this variant and the normal enzyme differed in amino acid sequence and/or post-translational modifications unrelated to phosphate groups and oligomannose glycans. Analysis of the arylsulfatase A genes from a subject with the new variant identified three mutations; one gene had the two mutations associated with arylsulfatase A pseudodeficiency, and the other had a G to T transversion which changes a tryptophan to cysteine in the protein. These mutations result in an arylsulfatase A protein heteromer with diffuse electrophoretic banding. The possible association of these mutations with major depression is discussed.
Asunto(s)
Cerebrósido Sulfatasa/genética , Trastorno Depresivo/genética , Isoenzimas/genética , Adulto , Anciano , Electroforesis de las Proteínas Sanguíneas , Cerebrósido Sulfatasa/deficiencia , Análisis Mutacional de ADN , Trastorno Depresivo/diagnóstico , Trastorno Depresivo/enzimología , Femenino , Variación Genética , Humanos , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/enzimología , Leucodistrofia Metacromática/genética , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Lead exposure causes cognitive and behavioral deficits in some children. We have proposed that the effects of single nucleotide polymorphisms (SNP) of the human pseudodeficient arylsulfatase A (ARSA) gene that result in reduced levels of the enzyme, and lead concentrations that decrease ARSA activity, culminate in cellular enzymic activity that is below a critical threshold required for the normal nervous system function. Human fibroblasts grown in the presence of lead acetate exhibit a 65% decrease in ARSA protein, resulting in a significant decrease in the ability to catabolize sulfatide in cells from individuals with the SNP(s) of pseudodeficient ARSA, but not those from subjects with the normal gene (Poretz et al., Neurotoxicology 21 (2000) 379). The present study examines the potential of lead to affect the biosynthesis, trafficking and turnover of ARSA in human fibroblasts. Fibroblasts, grown in 20 microM lead, displayed a 44--58% increase in the rate of proliferation. Lead caused a decrease of approximately 33% in the accumulation of newly synthesized intracellular ARSA. This difference was not due to increased rates of intracellular degradation of ARSA or decreased levels of ARSA mRNA. Lead, however, caused the newly synthesized enzyme to be trafficked through the secretion pathway, resulting in decreased amounts of the enzyme in intracellular compartments. Though lead exposure results in increased cellular proliferation, it appears to cause decreased intracellular steady-state levels of ARSA by affecting the sorting cues and/or mechanisms directing the enzyme to lysosomes.
Asunto(s)
Cerebrósido Sulfatasa/metabolismo , Fibroblastos/efectos de los fármacos , Plomo/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Cerebrósido Sulfatasa/biosíntesis , Cerebrósido Sulfatasa/genética , Fibroblastos/enzimología , Homocigoto , Humanos , Lisosomas/enzimología , Biosíntesis de Proteínas , ARN Mensajero/biosíntesisRESUMEN
Lead is a neurotoxicant that can cause myelin deficits. Galactolipids are expressed during differentiation of oligodendrocyte lineage cells and accumulate in myelin. To examine the impact of lead on oligodendroglial differentiation, galactolipid metabolism in cultured oligodendrocyte lineage cells exposed to the metal was studied. Oligodendrocyte progenitor cells obtained from newborn rat pups were exposed to 1 microM lead acetate for 24 h prior to maintenance of the cells in medium containing the metal salt for 0, 2, or 6 days of differentiation. Lead caused approximately 50% reduction in levels of the galactolipid biosynthetic transferases, UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:galactocerebroside sulfotransferase, as compared to sodium-treated controls, in cultures of oligodendrocyte lineage cells following 2 days of differentiation. The activities of the galactolipid catabolic hydrolases, galactocerebroside-beta-galactosidase and arylsulfatase A, were reduced by 20%. Following 6 days of differentiation, lead-exposed cells exhibited levels of all the enzymes, except for arylsulfatase A, similar to those of the control cells. These results are consistent with the lead-induced delay of oligodendrocyte differentiation, as evidenced by the emergence of stage-specific immunochemical markers and the observed change in the developmental activity profile of 2',3'-cyclic nucleotide 3'-phosphohydrolase. The activity of arylsulfatase A in lead-treated 6-day oligodendrocytes was significantly less than that found in control cultures. This effect is consistent with the lead-induced reduction of arylsulfatase A in human fibroblasts caused by mis-sorting the newly-synthesized enzyme. The perturbation of galactolipid metabolism by lead during developmental maturation of oligodendrocytes may represent a contributing mechanism for lead-induced neurotoxicity.
Asunto(s)
Glucolípidos/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/enzimología , Compuestos Organometálicos/toxicidad , Animales , Animales Recién Nacidos , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Células Cultivadas , Cerebrósido Sulfatasa/metabolismo , Galactolípidos , Galactosilceramidasa/metabolismo , Glucolípidos/antagonistas & inhibidores , Oligodendroglía/citología , Prosencéfalo/citología , Prosencéfalo/efectos de los fármacos , Prosencéfalo/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
Lead exposure causes cognitive and behavioral deficits in some affected children. We propose that a contributing mechanism for the neurological damage is that lead induces critically low levels of arylsulfatase A (ASA) at sensitive stages of nervous system development. It is hypothesized that the combined effects of a single nucleotide polymorphism (SNP) in human ASA which results in reduced levels of the enzyme, and lead concentrations which decrease ASA activity culminate in cellular enzymic activity that is below a critical threshold required for the maintenance of normal nervous system function. Human fibroblasts grown in the presence of 20 microM lead acetate exhibit a more than 60% decrease of cellular ASA enzyme protein. Lead treatment of cells from individuals with the SNP(s) of pseudodeficient ASA, but not those from subjects with the normal gene, results in a significant decrease in ability of the cells to desulfate sulfatide, the substrate of ASA. The decrease in the degree of sulfatide catabolism is consistent with possible enhanced lead-induced neurobehavioral effects in individuals homozygous for the pseudodeficiency polymorphism(s) of ASA.
Asunto(s)
Cerebrósido Sulfatasa/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Compuestos Organometálicos/farmacología , Sulfoglicoesfingolípidos/metabolismo , Células Cultivadas , Cerebrósido Sulfatasa/genética , Cerebrósido Sulfatasa/metabolismo , Fibroblastos/metabolismo , Genotipo , HumanosRESUMEN
An increasing number of plant glycoproteins have been shown to possess a characteristic N-glycan component containing a beta-(1----2)-linked D-xylose unit on the core beta-D-mannose unit, and an alpha-(1----3)-linked L-fucose unit on the asparagine-linked 2-acetamido-2-deoxy-D-glucose unit. Wistaria floribunda seeds have two distinct lectins; the erythroagglutinin, WFA, and the lymphocyte mitogen, WFM. Earlier studies indicated that both lectins belong to such a class of glycoproteins. We now report the complete structural analysis of Pronase glycopeptides derived from WFA. On the basis of chemical treatment of the glycopeptides, carbohydrate composition and methylation analysis of fluorescein-labeled glycopeptides, and their susceptibility to specific exoglycosidases, the structure of the WFA glycan was found to be, alpha-D-Manp-(1----6)-[beta-D-Xylp-(1----2)]- [alpha-D-Manp(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc-[ alpha-L- Fucp-(1----3)]-beta-D-Glcp-NAc-(1----N). Quantitative studies on the interaction of the original fluorescein-labeled glycopeptide and its specific degradation products with a rabbit anti-glycan antibody, developed against WFM, showed that the (1----3)-linked alpha-L-fucose unit is essential for interaction. Loss of the terminal alpha-D-mannosyl groups resulted in decreased, though detectable binding.
Asunto(s)
Lectinas/química , Lectinas de Plantas , Animales , Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Fucosa/química , Fucosa/inmunología , Glicopéptidos/química , Glicopéptidos/inmunología , Glicopéptidos/aislamiento & purificación , Inmunoquímica , Lectinas/inmunología , Datos de Secuencia Molecular , Estructura Molecular , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Polisacáridos/química , Polisacáridos/inmunología , Conejos , Receptores N-AcetilglucosaminaRESUMEN
The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein). The results demonstrate that two-dimensional PAGE is applicable to the separation and characterization of complex mixtures of FGPs. The procedure is rapid, sensitive, and convenient for glycopeptide mapping, and for the purification and structural characterization of glycans. Furthermore, the FGPs can be characterized with affinity matrices, such as lectins, and by methylation analysis.
Asunto(s)
Fluoresceínas , Glicopéptidos/análisis , Ovalbúmina/química , Polisacáridos/química , Secuencia de Carbohidratos , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , PronasaRESUMEN
Pheophorbide a is a photocytotoxic agent. To develop a tissue-specific, intracellularly targeted photoactive system, pheophorbide a was incorporated into immunoliposomes coated with a monoclonal antibody (T-43) directed against the T-24 bladder tumor cell line. The efficacy of this system was studied in vitro using the human bladder tumor cell line MGH-U1. Uptake and localization were determined by the fluorescence of the immunoliposome markers within biochemically resolved subcellular components. The results demonstrate localization of the immunoliposome markers within the lysosomes of the tumor cells. Specific monoclonal antibody enhancement of the immunoliposomes uptake by MGH-U1 cells was demonstrated by the use of soluble T-43 monoclonal antibody as a competitive inhibitor. Pheophorbide-a-loaded immunoliposomes were shown to be photocytotoxic towards MGH-U1 cells at concentrations equivalent to photosensitizer at 500 ng ml-1. Treated cells, when protected from light, showed no cytotoxicity. These results demonstrate that uptake of pheophorbide-a-containing immunoliposomes by target cells and subsequent delivery to the lysosomes cause photoactivated killing of tumor cells. The utilization of immunoliposomes for intracellular lysosomal targeting of photoactive drugs to tumor cells constitutes a potentially valuable approach to photodynamic therapeutics.
Asunto(s)
Clorofila/análogos & derivados , Lisosomas/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/toxicidad , Anticuerpos Monoclonales , Carcinoma de Células Transicionales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Clorofila/toxicidad , Oscuridad , Portadores de Fármacos , Humanos , Luz , Liposomas , Células Tumorales Cultivadas , Neoplasias de la Vejiga UrinariaAsunto(s)
Encéfalo/enzimología , Cerebrósido Sulfatasa/metabolismo , Intoxicación del Sistema Nervioso por Plomo en la Infancia/enzimología , Células-Madre Neurales/enzimología , Neurogénesis , Oligodendroglía/enzimología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/fisiopatología , Cerebrósido Sulfatasa/genética , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Intoxicación del Sistema Nervioso por Plomo en la Infancia/genética , Intoxicación del Sistema Nervioso por Plomo en la Infancia/patología , Intoxicación del Sistema Nervioso por Plomo en la Infancia/fisiopatología , Intoxicación del Sistema Nervioso por Plomo en la Infancia/psicología , Aprendizaje , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/patología , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Fenotipo , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Transducción de SeñalAsunto(s)
Glicósidos , Lectinas , Unión Proteica , Derivados del Benceno , Fenómenos Químicos , Química Física , Concanavalina A , Cristalización , Glucosa , Lectinas de Plantas , Plantas ComestiblesRESUMEN
Maclura promifera seeds contain a protein which agglutinates human erythrocytes at concentrations as low as 4 ng/mL. This property is related to its ability to bind with high specificity various alpha-D-galactopyranosides. The agglutinin, which was pruified by affinity adsorption, exhibits one band on immunoelectrophoresis and displays one peak during ultracentrifugation, isoelectric focusing, and gel permeation chromatography. The active protein has a molecular weight of 40 000-43 000 and contains two dissimilar polypeptide chains of 12 000 and 10 000, respectively.
Asunto(s)
Aglutininas , Hemaglutininas , Lectinas , Sistema del Grupo Sanguíneo ABO , Aglutininas/aislamiento & purificación , Aminoácidos/análisis , Carbohidratos , Eritrocitos/inmunología , Glicósido Hidrolasas/aislamiento & purificación , Hemaglutininas/aislamiento & purificación , Humanos , Lectinas/aislamiento & purificación , Peso Molecular , Lectinas de Plantas , Semillas/enzimologíaRESUMEN
Morphological studies have indicated divergent pathways for the endocytosis of epidermal growth factor (EGF) and transferrin (Tf). In order to obtain biochemical evidence for the pathways associated with the endocytosis of EGF and Tf, a series of Percoll density gradients were employed to separate individual cellular components. Subcellular fractionation of murine fibroblasts exposed to a 2-min pulse of either 125I-Tf or 125I-EGF results in the detection of a total of six cellular compartments related to the internalization process of these ligands. The results of kinetic analysis of the entry of EGF into five membranous fractions is consistent with a model in which ligand is transferred sequentially from the plasma membrane through three distinct prelysosomal environments prior to reaching secondary lysosomes. Each prelysosomal compartment exhibits distinct density and temporal properties in a Percoll density gradient and may represent preexisting endocytic vesicles and/or specific domains of a continuous tubular structure, vesicularized during the process of cell disruption. In addition, the observed differential migration on Percoll density gradients of Tf and EGF containing compartments indicates that the majority of cell bound Tf segregates from EGF and enters a compartment lacking EGF within 5 min of internalization.
Asunto(s)
Compartimento Celular , Endocitosis/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Transferrina/metabolismo , Animales , Fraccionamiento Celular , Femenino , Fibroblastos/citología , Cinética , Ratones , EmbarazoRESUMEN
Fundamental questions remain unanswered regarding the effect of the acceptor polypeptide structure on the fine structure of the N-linked glycan of glycoproteins and conversely, the effect of the glycan structure of IgG on the function and structure of the protein. The construction of myeloma hybrids capable of secreting multiple IgG which differ with regard to the fine structure of their N-linked oligosaccharides would be a valuable model for studying these questions. P3X63Ag8 analogous glycan of the IgG2b secreted by Sp2/HLBu. Fusion hybrids of these cells secrete parental IgG1, and to a lesser degree IgG2b, as well as a heterodimeric IgG containing both the gamma 1 and gamma 2b chains. The oligosaccharide of each chain is identical in structure to the appropriate parental IgG. Such cells allow for the analysis of acceptor properties that influence glycan fine structure, as well as the role of glycan structure on the stability of the IgG.
Asunto(s)
Galactósidos/química , Inmunoglobulinas/química , Mieloma Múltiple/inmunología , Proteínas de Mieloma/química , Oligosacáridos/química , Animales , Ascitis , Fusión Celular , Cromatografía de Afinidad , Células Clonales/química , Hibridomas , Concentración de Iones de Hidrógeno , Inmunoglobulinas/aislamiento & purificación , Ratones , Proteínas de Mieloma/aislamiento & purificación , Oligosacáridos/aislamiento & purificación , Proteína Estafilocócica A , Células Tumorales CultivadasRESUMEN
Lead poisoning is known to cause myelin defects. Galactolipids are the major lipid components of myelin and myelin-competent oligodendrocytes. The present study examines the cellular activity of enzymes involved in the galactolipid pathway, tissue concentrations of galactolipids, and the cellular activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in rat pups exposed to lead in utero and subsequently through maternal milk from exposed mothers and in drinking water following weaning. Pups from control and lead-treated groups (500 or 2000 ppm lead in the drinking water) were euthanized by decapitation on postnatal day 7, 14, 21, 35, or 56. Lead decreased levels of galactolipids and the oligodendrocyte marker CNPase in the brain to a similar degree. The ratios of galactocerebrosides/sulfatides and nonhydroxy fatty acid/hydroxy fatty acid forms of the galactolipids were not altered by lead treatment. In contrast, the activities of the galactolipid metabolic enzymes were reduced to a degree significantly greater than that of CNPase or galactolipids. These results are consistent with previously obtained data indicating that in vitro cultured oligodendroglial progenitor cells are a target for Pb toxicity. Chronic Pb exposure may impact on brain development by impairing timely myelin production due to perturbation of the early developmental commitment of oligodendroglial progenitors. It is further suggested that perturbation of the galactolipid pathway during the developmental maturation of oligodendrocytes may represent a contributing mechanism for Pb-induced neurotoxicity.