RESUMEN
Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.
Asunto(s)
Núcleo Celular/metabolismo , Lectinas/química , Lectinas/metabolismo , Aglutininas/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Cromatografía de Afinidad/métodos , Citoplasma/metabolismo , Galanthus , Glicosilación , Humanos , Lectinas de Plantas , Polisacáridos/química , Polisacáridos/metabolismo , Especificidad por Sustrato , TritioRESUMEN
Since nuclear lectins were first characterized several years ago, six lectins have been isolated. Furthermore, the existence of nuclear glycoproteins containing N-linked complex-oligosaccharide chains or O-linked GlcNAc residues was evidenced. These latter are abundant in the nucleus and are well-studied so far. The presence of both glycoprotein and lectin in the cell nucleus led us to postulate that these two proteins could interact and play a role in some nuclear activities such as the modulation of transcription and/or nuclear cytoplasmic exchanges or by the disruption of protein-protein interactions. In such context, the recent data concerning the GlcNAc-binding activity of CBP70 argued this postulate. However, to study the possible role of a glycoprotein-lectin complex, it was critical to isolate the two partners. Because CBP70 was also a cytoplasmic protein, the lectin was isolated in both cytoplasmic and nuclear compartments in order to investigate the putative ligand in the two cellular compartments. The results obtained with cross-linking experiments on isolated and membranedepleted nuclei incubated with the CBP70 bearing an iodinatable, cleavable, photoreactive cross-linking agent (sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate) and immunoprecipitation experiments with polyclonal antibodies raised against CBP70, revealed that both nuclear and cytoplasmic CBP70 have the same 82 kDa nuclear ligand which is absent in the cytoplasmic fraction. In addition, this ligand is glycosylated, containing GlcNAc residues, and, therefore, the complex between CBP70 and the 82 kDa polypeptide could be due to a glycoprotein-lectin interaction. These results raised the possibility that nuclear glycoprotein-lectin interaction could be involved in nuclear activities.
Asunto(s)
Lectinas/metabolismo , Extractos Celulares , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados , Citoplasma/metabolismo , Células HL-60 , Humanos , Ligandos , Pruebas de Precipitina , Unión Proteica , Aglutininas del Germen de Trigo/metabolismoRESUMEN
The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 and p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3.
Asunto(s)
Lectinas/metabolismo , Macrófagos/química , Diferenciación Celular , Núcleo Celular/química , Supervivencia Celular , Citoplasma/química , Citometría de Flujo , Células HL-60 , Humanos , Macrófagos/citología , Microscopía Fluorescente , Peso Molecular , Células Tumorales CultivadasRESUMEN
Using neoglycoproteins, lectins that recognize different sugars, including N-acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N-acetylglucosamine-binding proteins from HL60 cell nuclei: i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and ii) a 70 kDa polypeptide with an isoelectric point of 7.8. This latter protein corresponds to the glucose-binding protein (CBP70) previously isolated, based on the following similarities: i) they have the same molecular mass, ii) they have the same isoelectric point, iii) they are recognized by antibodies raised against CBP70, and iv) both are lectins from the C group of Drickamer's classification. CBP70 appeared to recognize glucose and N-acetylglucosamine; however, its affinity for N-acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N-acetylglucosamine-binding proteins and their potential ligands, such as O-N-acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein-lectin interactions.
Asunto(s)
Acetilglucosamina/química , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas Nucleares/aislamiento & purificación , Acetilglucosamina/metabolismo , Sitios de Unión , Línea Celular , Núcleo Celular/química , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Humanos , Lectinas/clasificación , Lectinas/metabolismo , Leucemia Mieloide/patología , Proteínas Nucleares/metabolismo , Células Tumorales CultivadasRESUMEN
We have previously reported that two carbohydrate-binding proteins (CBP35 and CBP70) can, under appropriate conditions of affinity chromatography, be isolated from HL60 cell nuclear extracts as a complex. Moreover, we have demonstrated that, during affinity chromatography, the CBP70-CBP35 association can be modified by the binding of lactose to CBP35. To determine whether the CBP70-CBP35 association could be disrupted in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, incubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that was specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immunofluorescent staining, immunoblotting experiments, and quantitative flow-cytofluorometric analysis show that (i) CBP70 and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuclei, and (iii) glucose failed to have the same effect as lactose. These results strongly suggest that, in membrane-depleted nuclei, CBP35 and CBP70 interactions can be altered by a conformational change of CBP35 induced by the binding of lactose to its carbohydrate-recognition domain.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Lactosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos de Diferenciación/aislamiento & purificación , Unión Competitiva , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Cromatografía de Afinidad , ADN de Neoplasias/análisis , Electroforesis en Gel de Poliacrilamida , Galectina 3 , Humanos , Immunoblotting , Inmunohistoquímica , Leucemia Promielocítica Aguda , Peso Molecular , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Proteínas Nucleares/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Zajdela hepatoma cells are poorly-adherent cells derived from an undifferentiated tumour and transplanted into rat. We compared the biosynthesis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte fibronectin receptor has been isolated. It is composed of two subunits: alpha 5 (molecular mass 155 kDa) and beta 1 (molecular mass 115 kDa). However, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we observed that the alpha 5-subunit was synthesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta 1-subunit was 130 kDa in Zajdela hepatoma cells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta 1-precursor to the 130-kDa mature form was abnormally prolonged in Zajdela hepatoma cells since the latter was not detected until 24 h, while the transition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta 1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized in normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta 1-subunit with peptide N-glycosidase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SDS/PAGE, while the molecular mass of the abnormal mature Zajdela hepatoma cell beta 1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, 'ascitic growth' induced other post-translational modifications in the Zajdela hepatoma cell beta 1-subunit. Furthermore, both the abnormal mature 130-kDa and precursor 100-kDa beta 1-subunits were detected on the surface of Zajdela hepatoma cells, associated with the alpha 5-subunit. The relationship between these structural alterations in the fibronectin receptor and the impaired Zajdela hepatoma cell binding to soluble fibronectin or to a coated fibronectin matrix that was observed in this study is discussed.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Receptores de Fibronectina/fisiología , Amidohidrolasas/metabolismo , Animales , Adhesión Celular , Fibronectinas/metabolismo , Glicosilación , Hexosaminidasas/metabolismo , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Receptores de Fibronectina/biosíntesis , Receptores de Fibronectina/químicaRESUMEN
Nuclear proteins were extracted in 2 M NaCl from membrane-depleted nuclei isolated from HL60 cells. Extracted proteins were submitted to affinity chromatography columns containing immobilized glucose, galactose or lactose. The polypeptides present in the different eluted fractions were resolved by SDS-PAGE and were either silver stained or analysed by immunoblotting with monoclonal or polyclonal antibodies, respectively, raised against the glucose-binding protein CBP67 and the galactose-binding proteins CBP35 and L14. The results presented here show that HL60 cell nuclei contain CBP35 and a glucose-binding lectin of 70 kDa (CBP70). These data account for the previously reported binding of neoglycoproteins containing glucosyl and galactosyl residues to HL60 cell nuclei. Furthermore, the present study provides the new information that CBP35 can associate with CBP70 by interactions dependent on the binding of CBP35 to lactose, and the results of some affinity chromatography experiments strongly suggest that CBP35 and CBP70 associate by protein-protein interactions. The potential function of this lactose-mediated interaction is discussed with respect to data recently reported by others showing that CBP35 is involved in in vitro mRNA splicing and that lactose inhibits the processing of the pre-RNA substrate.
Asunto(s)
Proteínas Portadoras/metabolismo , Lactosa/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular , Antígenos de Diferenciación/aislamiento & purificación , Antígenos de Diferenciación/metabolismo , Proteínas Portadoras/aislamiento & purificación , Núcleo Celular/química , Cromatografía de Afinidad , Galectina 3 , Glucosa/metabolismo , Humanos , Peso Molecular , Proteínas Nucleares/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.
Asunto(s)
5'-Nucleotidasa/metabolismo , Glicoproteínas/metabolismo , Músculos/embriología , Músculos/enzimología , 5'-Nucleotidasa/análisis , Animales , Anticuerpos Monoclonales , Membrana Basal/metabolismo , Embrión de Pollo , Pollos , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Fibronectinas/análisis , Técnica del Anticuerpo Fluorescente , Glicoproteínas/análisis , Integrinas/análisis , Laminina/análisis , Microscopía Electrónica , Peso Molecular , Músculos/citología , Miosinas/análisis , Nucleotidasas/análisis , Nucleotidasas/metabolismo , Sarcolema/enzimología , Sarcolema/ultraestructuraRESUMEN
Previous studies have shown that 5'-nucleotidase, an ectoenzyme from chicken gizzard, interacts specifically with laminin and fibronectin, two glycoproteins of the extracellular matrix. Recently, we demonstrated that 5'-nucleotidase was involved in the spreading of chick embryo fibroblast on laminin. In the present communication, we report that a monoclonal antibody (CG37) raised-directed against 5'-nucleotidase inhibited the spreading of chick embryo myoblasts on laminin after their initial attachment to the substrate. Furthermore, monoclonal antibody CG37 specifically eluted 5'-nucleotidase from immobilized laminin and thus enabled its isolation from other myoblast laminin-binding proteins.
Asunto(s)
5'-Nucleotidasa/fisiología , Músculos/citología , 5'-Nucleotidasa/aislamiento & purificación , Animales , Anticuerpos Monoclonales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Cromatografía de Afinidad , Laminina , Músculos/embriologíaRESUMEN
Eight d (8d) and 16d (16d) chick embryo fibroblasts (CEF) exhibited marked differences in their adhesive capacity on plastic support, but not on fibronectin substratum. This suggests differences in fibronectin (FN) expression and/or FN receptor expression. Both 8d and 16d CEF expressed an identical number of membrane receptors for FN with similar affinity. In contrast, the newly synthesized FN appeared de novo in 30 min in 8d CEF versus 60 min in 16d CEF. This difference is not due to a modification of the polypeptide chain biosynthetic rate. The FN synthesized in 8d CEF became insensitive to endo beta-N-acetyl-glucosaminidase H (endo H) treatment after 20 min, whereas it remained sensitive to endo H until 60 min in 16d CEF. Post-translational modifications of N-linked mannose-rich chains to complex type chain may account for the difference in the expression of cell surface FN and thus for the difference in cell adhesion capacity to plastic.
Asunto(s)
Fibroblastos/citología , Fibronectinas/biosíntesis , Animales , Sitios de Unión , Adhesión Celular , Membrana Celular/metabolismo , Embrión de Pollo , Hexosaminidasas/farmacología , Cinética , Manosa/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Procesamiento Proteico-Postraduccional , Receptores de Fibronectina , Receptores Inmunológicos/metabolismo , Factores de TiempoRESUMEN
Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.
Asunto(s)
Fibroblastos/citología , Laminina , Receptores de Concanavalina A/fisiología , Animales , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Embrión de Pollo , Concanavalina A/farmacología , Fibroblastos/efectos de los fármacos , Glicoproteínas/fisiología , Metilmanósidos/farmacología , Receptores de Concanavalina A/farmacologíaRESUMEN
The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.
Asunto(s)
Concanavalina A/farmacología , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Laminina/metabolismo , Animales , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Peso MolecularRESUMEN
Polyclonal and monoclonal antibodies raised against chicken gizzard 5'-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5'-nucleotidase. However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard. In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5'-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.
Asunto(s)
Adhesión Celular , Laminina/metabolismo , Nucleotidasas/metabolismo , 5'-Nucleotidasa , Animales , Anticuerpos/inmunología , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Células Cultivadas , Pollos , Matriz Extracelular/metabolismo , Fibroblastos , Fibronectinas/metabolismo , Molleja de las Aves/enzimología , Inmunoglobulina G/inmunología , Cinética , Nucleotidasas/inmunologíaRESUMEN
Eight day (8-d CEF) and 16 day old chick embryo fibroblasts (16-d CEF) obtained after a mild trypsin treatment (50 micrograms/ml in Ca2+ and Mg2+-free PBS, plus 10 mM EDTA) for 10 min at 37 degrees C present the same number of fibronectin (FN) binding sites at their surface (approximately 550,000 sites per cell) with a Kd approximately equal to 1.40 microM in both cases. Furthermore, FN interacted with high molecular weight plasma membrane proteins (150,000 and 125,000) insensitive to trypsin treatment. Both 8-d and 16-d CEF adhered and spread to the same extent on a fibronectin coated substratum (80% of the CEF adhered in 60 min). In contrast, 8-d and 16-d CEF behaved differently towards laminin (LM). 8-d CEF exhibited approximately 5500 binding sites per cell with a Kd of 1.5 nM (Codogno P., Doyennette, M.-A. and Aubery M., 1987, Experimental Cell Research, 169, 478-489.) and were highly sensitive to trypsin treatment, whereas 16-d CEF do not express cell surface binding sites for laminin. Differences were also observed in the adhesive capacities of 8-d and 16-d CEF on LM substrata: 8-d CEF adhered and spread on LM in a very specific manner (60% of the cells adhere in 60 min) and 16-d CEF did not adhere to LM even after long periods of incubation exceeding 360 min.
Asunto(s)
Fibroblastos/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Embrión de Pollo , Unión Proteica , Factores de TiempoRESUMEN
Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.
Asunto(s)
Adhesión Celular , Fibronectinas , Laminina , Animales , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Embrión de Pollo , Fibroblastos/citología , Glicoproteínas/análisis , Radioisótopos de Yodo , Proteínas de la Membrana/análisisRESUMEN
After the destruction of the noradrenergic ascending pathways, the localization of frontal cortical fields receiving fibers from the dopaminergic mesocortical system has been studied in rats, using a glyoxylic-paraformaldehyde method. The dopaminergic innervation was distributed in two main areas. The area of highest density was a medial field which spread in the medial cortex anterior and dorsal to the genu of the corpus callosum. It did not reach the shoulder region except in the foremost part of the frontal lobe where dopaminergic fibers were scattered in the whole cortex, including the molecular layer. A deep sulcal field was situated between the dorsal bank of the rhinal sulcus and the lateral cortex above it. In addition, a moderately dense band of dopaminergic fibers was observed between the corpus callosum and the anterior commissura, beside the accumbens nucleus. Similar data were obtained with dopamine uptake experiments on reserpine-treated but otherwise normal animals. The frontal areas receiving dopaminergic innervation coincide strikingly with the 'prefrontal cortex' as defined by neuroanatomical studies, which is assumed to be more or less equivalent to the prefrontal cortex of primates and derives direct projections from the amygdala. The functional implications of these findings are discussed.
Asunto(s)
Dopamina/metabolismo , Lóbulo Frontal/metabolismo , Animales , Lóbulo Frontal/efectos de los fármacos , Histocitoquímica , Hidroxidopaminas , Masculino , Nialamida/farmacología , Norepinefrina/metabolismo , Pargilina , Ratas , Reserpina/farmacologíaRESUMEN
Catecholamine axons have been visualized in human cerebral cortex obtained during routine neurosurgical operations. The fluorescence histochemical method of Lindvall et al. was used, slightly modified (calcium-deprived buffer, glyoxylic acid fixation followed by formaldehyde vapours exposition). The frontal cortex was more richely provided with catecholamine terminals than the parietal cortex. Two general types of axon morphology are evident. The most frequent is thin and sinous, sometimes forming clews, or loose basket-like arrangement around presumed nerve cells. The other one is moniliform and demonstrates spherical evenly-spaced varicosities. They look like, respectively, the well characterized dopaminergic and noradrenergic axons of the rat cerebral cortex. In two cases of Alzheimer's disease, noradrenergic-like fibers were missing and voluminous green-fluorescent varicosities, sometimes in obvious connection with typical axons, were observed in the proximity of senile plaques.