RÉSUMÉ
BACKGROUND: Genetic factors represent a considerable part of the etiologies of intellectual disability; however, the identification of causal genetic anomaly has long been complicated by the great clinical and genetic heterogeneity of this type of disease. With advances in next-generation sequencing technologies and functional studies, the identification of genes involved in intellectual development has led to more accurate diagnostics and better understanding of the underlying biological pathways. CASE REPORT: We report on the case of two Moroccan siblings presenting mild intellectual disability with minimal dysmorphic features in which whole exome sequencing analysis revealed homozygous mutation in the METTL23 gene. Mutations in this gene have been reported to cause autosomal recessive mild intellectual disability but the association with dysmorphic features remains controversial. CONCLUSION: Hereby, we highlight the similarity of the dysmorphic traits and the characteristic facial features in patients with METTL23-related intellectual disability, suggesting the consideration of a distinct clinical entity associating mild intellectual deficiency with specific facial dysmorphy for an efficient diagnosis orientation and a better phenotype-genotype correlation in intellectual disability disorders.
Sujet(s)
Dysmorphophobies/génétique , Exome/génétique , Homozygote , Déficience intellectuelle/génétique , Methyltransferases/génétique , Mutation , Dysmorphophobies/imagerie diagnostique , Enfant , Femelle , Études d'associations génétiques , Hétérogénéité génétique , Prédisposition génétique à une maladie/génétique , Génotype , Séquençage nucléotidique à haut débit , Humains , Déficience intellectuelle/imagerie diagnostique , Déficience intellectuelle/physiopathologie , Mâle , Maroc , Pedigree ,RÉSUMÉ
BACKGROUND: Systemic lupus erythematosus is a heterogeneous chronic inflammatory autoimmune disorder characterized by an exacerbated expression of cytokines and chemokines in different tissues and organs. Renal involvement is a significant contributor to the morbidity and mortality of systemic lupus erythematosus, and its diagnosis is based on renal biopsy, an invasive procedure with a high risk of complications. Therefore, the development of alternative, non-invasive diagnostic tests for kidney disease in patients with systemic lupus erythematosus is a priority. AIM: To evaluate the plasma levels of a panel of cytokines and chemokines using multiplex xMAP technology in a cohort of Colombian patients with active and inactive systemic lupus erythematosus, and to evaluate their potential as biomarkers of renal involvement. RESULTS: Plasma from 40 systemic lupus erythematosus non-nephritis patients and 80 lupus nephritis patients with different levels of renal involvement were analyzed for 39 cytokines using Luminex xMAP technology. Lupus nephritis patients had significantly increased plasma eotaxin, TNF-α, interleukin-17-α, interleukin-10, and interleukin-15 as compared to the systemic lupus erythematosus non-nephritis group. Macrophage-derived chemokine, growth regulated oncogene alpha, and epidermal growth factor were significantly elevated in systemic lupus erythematosus non-nephritis patients when compared to lupus nephritis individuals. Plasma eotaxin levels allowed a discrimination between systemic lupus erythematosus non-nephritis and lupus nephritis patients, for which we performed a receiver operating characteristic curve to confirm. We observed a correlation of eotaxin levels with active nephritis (Systemic Lupus Erythematosus Disease Activity Index). Our data indicate that circulating cytokines and chemokines could be considered good predictors of renal involvement in individuals with systemic lupus erythematosus.
Sujet(s)
Cytokines/sang , Rein/physiopathologie , Lupus érythémateux disséminé/diagnostic , Glomérulonéphrite lupique/diagnostic , Adolescent , Adulte , Marqueurs biologiques/sang , Femelle , Humains , Lupus érythémateux disséminé/sang , Glomérulonéphrite lupique/sang , Mâle , Courbe ROC , Indice de gravité de la maladie , Jeune adulteSujet(s)
Antituberculeux/usage thérapeutique , Retard de diagnostic , Tuberculose ostéoarticulaire/diagnostic , Association thérapeutique , Hanche/imagerie diagnostique , Humains , Mâle , Adulte d'âge moyen , Tomodensitométrie , Tuberculose ostéoarticulaire/traitement médicamenteux , Tuberculose ostéoarticulaire/chirurgieRÉSUMÉ
Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-ß (TGF-ß)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1(C1039G/+)) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1(mgR/mgR), without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-ß signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-ß signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1(C1039G/+) mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.
Sujet(s)
Amino-acid oxidoreductases/métabolisme , Aorte/enzymologie , Anévrysme de l'aorte/enzymologie , Syndrome de Marfan/enzymologie , Lysyloxidase/métabolisme , Animaux , Aorte/anatomopathologie , Anévrysme de l'aorte/étiologie , Évolution de la maladie , Expression des gènes , Humains , Syndrome de Marfan/complications , Syndrome de Marfan/anatomopathologie , Souris de lignée C57BL , Souris transgéniquesRÉSUMÉ
Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads.
RÉSUMÉ
Transforming growth factor-beta (TGF-ß) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-ß in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-ß receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-ß-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1(-/-) hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-ß, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-ß-induced survival signals in Cav1(-/-) hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-ß was impaired in Cav1(-/-) hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1(-/-) hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1(+/+) cells, which was not the case in Cav1(-/-) cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-ß treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-ß-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-ß pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-ß in liver cancer cells.
Sujet(s)
Protéines ADAM/métabolisme , Cavéoline-1/métabolisme , Récepteurs ErbB/génétique , Hépatocytes/métabolisme , Activation de la transcription , Facteur de croissance transformant bêta/métabolisme , Protéines ADAM/génétique , Protéine ADAM17 , Animaux , Apoptose , Cavéoline-1/génétique , Cellules cultivées , Activation enzymatique , Récepteurs ErbB/métabolisme , Femelle , Hépatocytes/enzymologie , Mâle , Souris , Souris knockout , Phosphorylation , Espèces réactives de l'oxygène/métabolisme , Transduction du signalRÉSUMÉ
Human genome consists of 23 pairs of chromosomes, bearing our genetic information. Basically, there are two main approaches to analyse our genome: molecular genetics with direct sequencing, which detects genic mutations, and cytogenetics with the karyotype, which detects number and structural chromosomal anomalies. The main limitation of the karyotype is its level of resolution: it cannot detect abnormalities smaller than five megabases. The combined use of cytogenetics and molecular genetics has allowed the development of several new techniques that provide a comprehensive analysis of the genome with a very high level of resolution. Currently, the most efficient of those techniques is comparative genomic hybridization on microarray (array CGH), which already has diagnostic applications. However, those new methods are challenging to interpret and they raise ethical problems. Therefore they must be cautiously supervised.
Sujet(s)
Génome humain/génétique , Techniques de diagnostic moléculaire/méthodes , Maladies chromosomiques/diagnostic , Maladies chromosomiques/génétique , Hybridation génomique comparative/méthodes , Dépistage génétique/méthodes , Humains , Caryotypage/méthodes , Mutation/génétique , Tumeurs/génétique , Séquençage par oligonucléotides en batterie/méthodes , Reproductibilité des résultatsRÉSUMÉ
Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.
Sujet(s)
Astrocytes/effets des médicaments et des substances chimiques , Astrocytes/métabolisme , Éthanol/pharmacologie , Oses/métabolisme , Animaux , Astrocytes/cytologie , Transport biologique/effets des médicaments et des substances chimiques , Division cellulaire , Cellules cultivées , Électrophorèse sur gel de polyacrylamide , Transporteur de glucose de type 1 , Glycosylation/effets des médicaments et des substances chimiques , Immunohistochimie , Lectines/composition chimique , Lectines/toxicité , Mannose/composition chimique , Mannosidases/métabolisme , Glycoprotéines membranaires/analyse , Glycoprotéines membranaires/métabolisme , Masse moléculaire , Transporteurs de monosaccharides/métabolisme , Oses/pharmacocinétique , Acide N-acétyl-neuraminique/composition chimique , Rats , Facteurs tempsRÉSUMÉ
The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum-Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain beta/gamma-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.
Sujet(s)
Actines/métabolisme , Réticulum endoplasmique/métabolisme , Appareil de Golgi/métabolisme , Glycoprotéines membranaires , Shiga-toxine/métabolisme , Protéines de l'enveloppe virale/métabolisme , Cytosquelette d'actine/effets des médicaments et des substances chimiques , Cytosquelette d'actine/métabolisme , Actines/effets des médicaments et des substances chimiques , Animaux , Transport biologique/effets des médicaments et des substances chimiques , Transport biologique/physiologie , Toxines botuliniques/pharmacologie , Bréfeldine A/pharmacologie , Composés hétérocycliques bicycliques/pharmacologie , Cellules cultivées/effets des médicaments et des substances chimiques , Cellules cultivées/métabolisme , Cytochalasine D/pharmacologie , Appareil de Golgi/effets des médicaments et des substances chimiques , Cellules HeLa/effets des médicaments et des substances chimiques , Cellules HeLa/métabolisme , Humains , Membranes intracellulaires/métabolisme , Mammifères/métabolisme , Microtubules/effets des médicaments et des substances chimiques , Microtubules/métabolisme , Récepteurs peptidiques/métabolisme , Thiazoles/pharmacologie , ThiazolidinesRÉSUMÉ
Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.
Sujet(s)
Endocytose , Appareil de Golgi/métabolisme , Neurones/métabolisme , Sphingolipides/biosynthèse , Animaux , Antinéoplasiques/pharmacologie , Bréfeldine A/pharmacologie , Différenciation cellulaire , Lignée cellulaire , Membrane cellulaire/métabolisme , Cellules cultivées , Chromatographie sur couche mince , Cytoplasme/métabolisme , Endosomes/métabolisme , Glucosylcéramides/biosynthèse , Hippocampe/cytologie , Hippocampe/métabolisme , Lysosomes/métabolisme , Microscopie de contraste de phase , Nocodazole/pharmacologie , Inhibiteurs de la synthèse protéique/pharmacologie , Rats , Sphingomyéline/métabolisme , Température , Facteurs temps , Transferrine/métabolismeRÉSUMÉ
It has been shown previously that the morphology and subcellular positioning of the Golgi complex is controlled by actin microfilaments. To further characterize the association between actin microfilaments and the Golgi complex, we have used the Clostridium botulinum toxins C2 and C3, which specifically inhibit actin polymerization and cause depolymerization of F-actin in intact cells by the ADP ribosylation of G-actin monomers and the Rho small GTP-binding protein, respectively. Normal rat kidney cells treated with C2 showed that disruption of the actin and the collapse of the Golgi complex occurred concomitantly. However, when cells were treated with C3, the actin disassembly was observed without any change in the organization of the Golgi complex. The absence of the involvement of Rho was further confirmed by the treatment with lysophosphatidic acid or microinjection with the constitutively activated form of RhoA, both of which induced the stress fiber formation without affecting the Golgi complex. Immunogold electron microscopy in normal rat kidney cells revealed that beta- and gamma-actin isoforms were found in Golgi-associated COPI-coated buds and vesicles. Taken together, the results suggest that the Rho signaling pathway does not directly regulate Golgi-associated actin microfilaments, and that beta- and gamma-actins might be involved in the formation and/or transport of Golgi-derived vesicular or tubular intermediates.
Sujet(s)
Actines/métabolisme , Complexe I de protéines de revêtement/métabolisme , Appareil de Golgi/métabolisme , ADP ribose transferases/pharmacologie , Adénosine diphosphate ribose/métabolisme , Animaux , Toxines botuliniques/pharmacologie , Cellules cultivées , Technique d'immunofluorescence , Appareil de Golgi/effets des médicaments et des substances chimiques , Microinjections , Microscopie immunoélectronique , Rats , Transduction du signal , Protéines G rho/métabolismeRÉSUMÉ
We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.
Sujet(s)
Adénosine diphosphate ribose/métabolisme , Bréfeldine A/antagonistes et inhibiteurs , Protéines de transport/métabolisme , Antienzymes/pharmacologie , Appareil de Golgi/effets des médicaments et des substances chimiques , Membranes intracellulaires/effets des médicaments et des substances chimiques , Morpholines/pharmacologie , Facteurs de transcription , ADP/métabolisme , Animaux , Protéine du coatomère/métabolisme , Cytosol/métabolisme , Appareil de Golgi/métabolisme , Membranes intracellulaires/métabolisme , RatsRÉSUMÉ
In this report we have studied the morphological changes of the Golgi complex (GC) that specifically accompany F-actin reorganizations. In starved rat RBL-2H3 tumor mast cells, the GC, that was visualized at immunofluorescence level with antibodies raised against the Golgi-resident proteins giantin, mannosidase II, or TGN-38, showed a compacted morphology with a supranuclear positioning. Concomitant to membrane ruffle formation induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA), and stress fiber formation induced by lysophosphatidic acid (LPA), specific GC morphological changes were observed. When cells were stimulated with EGF or PMA, the compacted GC morphology was transformed into a reticular network that was extended towards the cell periphery. When cells were incubated with LPA, the GC acquired a characteristic ring-shaped morphology. Brefeldin A (BFA) did not affect the PMA- or LPA-induced membrane ruffling and stress fiber formation, respectively, indicating that actin rearrangements occurred independent of the presence of the GC. Upon BFA removal, the presence of PMA or LPA during the recovery process induced the GC to acquire the morphological appearance described above for each agent. Moreover, the PMA- but not the LPA-induced GC rearrangements were sensitive to the actin perturbing agents cytochalasin D and jasplakinolide. When cells were preincubated with the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin or LY294002, the PMA-induced GC morphological changes were inhibited but not membrane ruffles. Finally, the PMA-induced increase in the post-Golgi transport of glycosaminoglycans to the cell surface was not altered by cytochalasin D or jasplakinolide. Altogether, these data suggest that: (1) the shape of the GC is influenced by the 3D arrangement of actin microfilaments; (2) PI3K regulates the association of the GC with actin microfilaments; and (3) actin microfilaments are not essential for the post-Golgi transport to the plasma membrane.
Sujet(s)
Actines/métabolisme , Depsipeptides , Appareil de Golgi/ultrastructure , Androstadiènes/pharmacologie , Animaux , Transport biologique , Bréfeldine A/pharmacologie , Membrane cellulaire/métabolisme , 4H-1-Benzopyran-4-ones/pharmacologie , Cytochalasine D/pharmacologie , Cytosquelette , Antienzymes/pharmacologie , Facteur de croissance épidermique/pharmacologie , Glycosaminoglycanes/métabolisme , Appareil de Golgi/effets des médicaments et des substances chimiques , Lysophospholipides/pharmacologie , Morpholines/pharmacologie , Inhibiteurs de la synthèse d'acide nucléique/pharmacologie , Peptides cycliques/pharmacologie , Inhibiteurs de la synthèse protéique/pharmacologie , Rats , 12-Myristate-13-acétate de phorbol/pharmacologie , Cellules cancéreuses en culture , WortmannineRÉSUMÉ
In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375-P) and high metastatic (A375-MM) potential. Immunofluorescence analysis showed that in A375-P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375-MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375-P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375-P, but not in A375-MM cells. In addition, the F-actin content in A375-P cells was significantly lower than in A375-MM cells. These morphological differences in A375-P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans-Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well-structured morphofunctional organization of the Golgi complex and actin cytoskeleton.
Sujet(s)
Cytosquelette/anatomopathologie , Appareil de Golgi/anatomopathologie , Mélanome/métabolisme , Mélanome/anatomopathologie , Tumeurs cutanées/métabolisme , Tumeurs cutanées/anatomopathologie , Transport biologique , Humains , Invasion tumorale , Cellules cancéreuses en cultureRÉSUMÉ
A monoclonal antibody (mAb; A10) raised against murine Ehrlich tumor cell surface carbohydrates was tested for reactivity with human normal and malignant tissues. A10 reacted strongly, with a high proportion of adenocarcinomas arising from colon and other tissues but not with breast carcinomas or other malignant tumors. Normal tissues were virtually A10 unreactive, except for the duct cells from breast and pancreas and some bronchial mucosae. Ultrastructural studies showed mAb A10 immunolabeling of both microvilli and mucin droplets in colon cancer cells but not in normal absorptive or globet cells. A10 reacted strongly with mucin-enriched fractions from colon cancer tissues and HT-29 xenografts but not from normal colon tissues. A10 epitope was carried on MUC1 derived from colon adenocarcinomas and probably on other mucin species, although not on MUC2 molecules. A10 epitope was resistant to exoglycosidases and periodate oxidation but sensitive to the Smith's degradation and beta-elimination, suggesting the involvement of O-linked carbohydrates in nonterminal reducing positions. A mucin-type glycosidic linkage was supported because of the lack of A10 reactivity with HT-29 cells grown with phenyl-N-acetyl-alpha-D-galactosaminide. Deglycosylation studies with trifluoromethanesulfonic acid pointed to the involvement of core mucin glycans in the A10 epitope. This epitope was resistant to protease, O- and N-glycanase treatments carried out on trifluoromethanesulfonic acid-deglycosylated mucins. Inhibition studies with core 1, core 2, core 3, and core 6 suggested the latter [GlcNAcbeta(1-6)GalNAc] as being involved in A10 epitope. Taken together, the present results point to A10 defining a core 6-related epitope on core mucin glycans expressed by colon cancer MUC1 not previously associated with human cancer.
Sujet(s)
Adénocarcinome/anatomopathologie , Carcinome d'Ehrlich/immunologie , Tumeurs du côlon/anatomopathologie , Polypes coliques/anatomopathologie , Muqueuse intestinale/anatomopathologie , Mucine-1/analyse , Mucine-1/immunologie , Adénocarcinome/composition chimique , Adénocarcinome/immunologie , Animaux , Anticorps monoclonaux , Spécificité des anticorps , Séquence glucidique , Côlon/cytologie , Côlon/anatomopathologie , Tumeurs du côlon/composition chimique , Tumeurs du côlon/immunologie , Polypes coliques/immunologie , Test ELISA , Épitopes/analyse , Humains , Muqueuse intestinale/cytologie , Souris , Microscopie immunoélectronique , Données de séquences moléculaires , Oligosaccharides/composition chimique , Oligosaccharides/immunologie , Valeurs de référence , Cellules cancéreuses en cultureRÉSUMÉ
Aberrant glycosylation of proteins and lipids is a common feature of many tumor cell types, and is often accompanied by alterations in membrane traffic and an anomalous localization of Golgi-resident proteins and glycans. These observations suggest that the Golgi complex is a key organelle for at least some of the functional changes associated with malignant transformation. To gain insight into this possibility, we have analyzed changes in the structure and function of the Golgi complex induced by the conditional expression of the transforming N-Ras(K61) mutant in the NRK cell line. A remarkable and specific effect associated with this N-Ras-induced transformation was a conspicuous rearrangement of the Golgi complex into a collapsed morphology. Ultrastructural and stereological analyses demonstrated that the Golgi complex was extensively fragmented. The collapse of the Golgi complex was also accompanied by a disruption of the actin cytoskeleton. Functionally, N-Ras-transformed KT8 cells showed an increase in the constitutive protein transport from the trans-Golgi network to the cell surface, and did not induce the appearance of aberrant cell surface glycans. The Golgi complex collapse, the actin disassembly, and the increased constitutive secretion were all partially inhibited by the phospholipase A2 inhibitor 4-bromophenylacyl bromide. The results thus suggest the involvement of the actin cytoskeleton in the shape of the Golgi complex, and intracellular phospholipase A2 in its architecture and secretory function.
Sujet(s)
Gènes ras/génétique , Appareil de Golgi/génétique , Appareil de Golgi/métabolisme , Protéines/métabolisme , Acétophénones/pharmacologie , Actines/effets des médicaments et des substances chimiques , Actines/métabolisme , Animaux , Lignée de cellules transformées , Dexaméthasone/pharmacologie , Antienzymes/pharmacologie , Glycosylation , Appareil de Golgi/ultrastructure , Liquide intracellulaire/enzymologie , Liquide intracellulaire/métabolisme , Protéines membranaires/métabolisme , Microscopie électronique , Phospholipases A/antagonistes et inhibiteurs , Phospholipases A/métabolisme , Phospholipases A2 , Rats , Transduction du signal/génétique , TransfectionRÉSUMÉ
The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the centrosome region. Golgi complex alteration affected all Golgi subcompartments as revealed by double fluorescence staining with antibodies to the cis/middle Mannosidase II and the trans-Golgi network TGN38 proteins or vital staining with the lipid derivate C6-NBD-ceramide. The ultrastructural and stereological analysis showed that the Golgi cisternae remained attached in a stacked conformation, but they were swollen and contained electron-dense intra-cisternal bodies. The Golgi complex cluster remained linked to microtubules since it was fragmented and dispersed after treatment with nocodazole. Moreover, the reassembly of Golgi fragments after the disruption of the microtubuli with nocodazole does not utilize the actin microfilaments. The actin microfilament requirement for the disassembly and reassembly of the Golgi complex and for the ER-Golgi vesicular transport were also studied. The results show that actin microfilaments are not needed for either the retrograde fusion of the Golgi complex with the endoplasmic reticulum promoted by brefeldin A or the anterograde reassembly after the removal of the drug, or the ER-Golgi transport of VSV-G glycoprotein. However, actin microfilaments are directly involved in the subcellular localization and the morphology of the Golgi complex.
Sujet(s)
Cytosquelette d'actine/physiologie , Actines/physiologie , Appareil de Golgi/physiologie , Cytosquelette d'actine/effets des médicaments et des substances chimiques , Cytosquelette d'actine/ultrastructure , Animaux , Cellules cultivées , Cytosquelette/effets des médicaments et des substances chimiques , Appareil de Golgi/ultrastructure , Rein/cytologie , Microtubules , RatsRÉSUMÉ
In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.
Sujet(s)
Calcium/métabolisme , Cyclopentanes/antagonistes et inhibiteurs , Réticulum endoplasmique/effets des médicaments et des substances chimiques , Appareil de Golgi/effets des médicaments et des substances chimiques , Morpholines/pharmacologie , Sphingolipides/métabolisme , Animaux , Antibactériens/antagonistes et inhibiteurs , Antibactériens/pharmacologie , Transport biologique actif/effets des médicaments et des substances chimiques , Bréfeldine A , Lignée cellulaire , Céramides/métabolisme , Cyclopentanes/pharmacologie , Réticulum endoplasmique/métabolisme , Glucosylcéramides/métabolisme , Appareil de Golgi/métabolisme , Cellules HT29 , Homéostasie/effets des médicaments et des substances chimiques , Humains , Membranes intracellulaires/effets des médicaments et des substances chimiques , Membranes intracellulaires/métabolisme , Métabolisme lipidique , Macrolides , Propionates/pharmacologie , Quinoléines/pharmacologie , Rats , Sphingomyéline/biosynthèseRÉSUMÉ
Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.