GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.     

Signalling molecules and the regulation of intracellular transport

Sixty-seven insulinomas were investigated by immunohistochemistry using site-directed antibodies against insulin, proinsulin, chromogranin A, HISL-19, and four proteins directly or indirectly involved in the proteolytic processing of proinsulin: the prohormone convertases PC2 and PC3, carboxypeptidase H (CPH) and 7B2. Results were expressed in a six-grade score according to the frequency of immunoreactive tumour cells. Insulin was expressed by all tumours, appearing in either a diffuse or a polarized pattern and being detected in more than 30% of tumour cells in all cases but three. Proinsulin was also expressed in all tumours, with more than 50% of tumour cells immunoreactive in all cases but 5. It was consistently localized in the Golgi apparatus. In about half the cases, moreover, it also showed diffuse cytoplasmic staining, usually with a very sparse distribution. Trabecular and solid insulinomas did not present specific, homogeneous patterns of insulin immunostaining. However, insulin immunoreactivity was much more abundant in trabecular than in solid neoplasms, being present in virtually all tumour cells (score 6) in 50% and 8% of cases, respectively. Virtually all insulinomas expressed PC2, PC3, CPH and 7B2, usually in 30-100% of tumour cells, with a frequency significantly related to that of insulin. However, detection of PC2 and 7B2 was slightly less frequent than that of PC3 and CPH. In consecutive sections these proteins were found to be mostly co-localized with insulin and chromogranin A but not with proinsulin. They were heavily expressed in all 10 tumours with more than 10% of cells showing cytoplasmic proinsulin immunoreactivity, indicating that the leakage of proinsulin from the Golgi compartment is not associated with faulty expression of converting enzymes and possibly reflects a saturated processing capacity. HISL-19 immunoreactivity was found in both Golgi apparatus and insulin stores, indicating that the relevant antigen is different from all other proteins investigated. These results do not support a defect in expression or localization of proinsulin-processing enzymes in most insulinomas.     

Altered cell signaling and mononuclear phagocyte deactivation during intracellular infection

Given the critical antimicrobial properties of mononuclear phagocytes, an important concern in cell biology and immunology has been to understand how intracellular microbes are able to establish states of chronic infection within these cells. Recent studies indicate that mononuclear phagocytes become functionally deactivated during intracellular infection. Here, Neil Reiner considers the experimental evidence to indicate that this is a frequent event that may be accounted for by induced defects in the signaling pathways required to bring cells to an activated state.     

Pathways downstream of Shc and Grb2 are required for cell transformation by the tpr-Met oncoprotein

The Tpr-Met oncoprotein, which is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement, consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. We have previously demonstrated that a single tyrosine residue in the carboxyl terminus, Tyr489, is highly phosphorylated and is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and for the association of Tpr-Met with the Grb2 adaptor protein and phosphatidylinositol 3'-kinase. We show here that Tyr489 is also required for association of Tpr-Met with phospholipase Cgamma and the tyrosine phosphatase, SHPTP2/Syp. To distinguish which of these substrates are required for cell transformation by the Tpr-Met oncoprotein, we generated a novel Tpr-Met mutant that selectively fails to associate with the Grb2 adaptor protein. Utilizing this mutant, together with additional Tpr-Met mutants containing Tyr to Phe substitutions, we have demonstrated that transformation of Fr3T3 fibroblasts by the Tpr-Met oncoprotein is dependent upon pathways downstream of Shc and Grb2 and that pathways downstream of phosphatidylinositol 3'-kinase, phospholipase Cgamma, and SHPTP2/Syp are insufficient for transformation.     

Identification of a core functional and structural domain of the v-Ski oncoprotein responsible for both transformation and myogenesis

The v-ski oncogene promotes cellular transformation and myogenic differentiation. In quail embryo fibroblasts the two properties are displayed simultaneously and terminal muscle differentiation occurs only among cells already transformed by v-ski. To understand how the two phenotypes are derived from a single gene, we have undertaken to identify functionally important regions in v-ski and to test whether these regions can promote one phenotype without the other. We have generated both random and targeted mutations in v-ski and evaluated the effects of these mutations on expression, intracellular location, transformation, and myogenesis. Among a total of 26 mutants analysed, we have not found complete separation of the myogenic and transforming properties. Mutations in the region of v-Ski encoded by exon 1 of c-ski frequently abolish both its transformation and muscle differentiation activities, whereas mutations outside of this region are always tolerated. When expressed in cells from a minigene containing only the exon 1 sequence, the protein displays the transforming and myogenic activities similar to v-Ski. These results argue that the amino acid sequence encoded by exon 1 contains the core functional domain of the oncoprotein. To determine whether this functional domain has a structural counterpart, we have fragmented the v-Ski protein by limited proteolysis and found a single proteolytically stable domain spanning the entire exon 1-encoded region. Physical studies of the polypeptide encoded by exon 1 confirms that it folds into a compact, globular protein. The finding that both the transforming and myogenic properties of v-Ski are inseparable by mutation and are contained in a single domain suggests that they are derived from the same function.     

Glutathione peroxidase degrades intracellular hydrogen peroxide and thereby inhibits intracellular protein iodination in thyroid epithelium

Protein iodination in the thyroid is largely confined to the surface of the epithelium. Intracellular iodine binding is insignificant. We have tested our hypothesis that the key mechanism in the control of intracellular iodination is the control of the intracellular availability of H2O2. The sites of iodination were identified by locating bound radioiodine in electron microscopic autoradiographs, produced from porcine thyroid epithelium grown on filter in Transwell bicameral culture chambers. Autoradiographs obtained after standard incubations with 125I for 15 min to 3 h were all characterized by concentrations of autoradiographic grains along the external surface of the plasma membrane and very few grains over the cytoplasm. The presence of 10 microM H2O2 in the incubation medium resulted in a drastically changed labeling pattern now showing a dissemination of grains over the entire cytoplasm. Epithelia with elevated GSH peroxidase activity produced autoradiographs showing the same restriction of grains to the cell surface as controls; this pattern was the same in the absence and presence of H2O2 (up to 10 microM). Cultures with subnormal GSH peroxidase activity presented cytoplasmic labeling both in the absence and presence of H2O2. In conclusion, iodine binding in filter-cultured thyroid epithelium under normal conditions is an extracellular process located at the cell surface. When H2O2 is available intracellularly, iodination takes place in the cytoplasm, evidently catalyzed by intracellular thyroperoxidase. Normally, this iodination is prevented by cytosolic GSH peroxidase that effectively degrades H2O2 and thus controls intracellular iodination. The observations should be applicable to the thyroid in vivo.     

Ketamine inhibits the proinflammatory cytokine-induced reduction of cardiac intracellular cAMP accumulation

The proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), are increased in heart failure and sepsis, clinical conditions for which the IV anesthetic ketamine is useful. The proinflammatory cytokines cause beta-adrenergic receptor (betaAR) hypofunction secondary to reduced function of the enzyme adenylylcyclase (AC). In this study, we evaluated the effect of ketamine alone, TNF-alpha and IFN-gamma, and ketamine plus TNF-alpha and IFN-gamma, on isoproterenol (ISO, a betaAR agonist) and forskolin (FSK, an activator of AC)-induced intracellular accumulation of cAMP. An in vitro culture of a rat heart cell line (H9c2) was labeled with [3H]adenine to produce [3H]ATP, and we measured the intracellular accumulation of [3H]cAMP after stimulation with ISO or FSK to convert the [3H]ATP to [3H]cAMP. Pretreatment with either cytokine alone did not significantly affect ISO or FSK-induced intracellular cAMP accumulation, whereas the combination of TNF-alpha and IFN-gamma caused a significant (P < 0.05 compared with untreated cells) reduction. Pretreatment with ketamine caused a significant (P < 0.05 compared with untreated cells) increase in ISO or FSK-induced cAMP accumulation. Pretreatment of the H9c2 cells with ketamine, plus the combination of TNF-alpha and IFN-gamma, inhibited the reduction of ISO or FSK-induced intracellular cAMP accumulation caused by the proinflammatory cytokines alone. These results demonstrate that the combination of the proinflammatory cytokines TNF-alpha and IFN-gamma reduce poststimulation (ISO or FSK) intracellular cAMP accumulation. This action of the proinflammatory cytokines is consistent with the observation of betaAR hyporesponsiveness to betaAR agonist therapy in sepsis and heart failure. Ketamine augments the poststimulation cAMP accumulation in H9c2 cells while inhibiting the cytokine-induced reduction of cAMP accumulation. This may partly explain the improvement in cardiac function after ketamine use in clinical conditions known to have increased systemic levels of proinflammatory cytokines, such as sepsis and heart failure. IMPLICATIONS: Tumor necrosis factor-alpha and interferon-gamma reduced poststimulation intracellular cAMP levels, whereas ketamine inhibits this action of the proinflammatory cytokines. Because cAMP is the second messenger for the beta-adrenergic receptor, this may be a mechanism for improved blood pressure and cardiac output in sepsis and heart failure after ketamine use.     

Na, K-ATPase--its structure, function and intracellular transport

Na, K-ATPase is an integral plasma membrane protein and plays essential roles such as maintaining sodium and potassium ion gradients across the plasma membrane. The enzyme consists of the alpha and the beta subunits with the stoichiometry of one to one. Three alpha subunit and two beta subunit isoforms have been detected in animal cells with the tissue-specific expression of both subunits. Recent advances in molecular biological studies on the Na, K-ATPase enable us to understand the structure-function relationships and mechanisms of intracellular transport of the enzyme. In this article we review the findings deduced from these studies, especially on the assembly and transport to the plasma membrane of the alpha and beta subunits.     

Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glycosylation in diabetes

Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonezymatic glycosylation of basic fibroblast growth factor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3-phosphate (G3P) reduced high affinity heparin-binding activity of recombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosylation in GM7373 endothelial cells, we first demonstrated that GLUT-1 transporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, and the intracellular glucose concentration increased sixfold at 24 h and fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation end-products (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 nM glucose, demonstrating that all the mitogenic activity was due to bFGF. Immunoblotting and ELISA showed that 30 mM glucose did not decrease detectable bFGF protein, suggesting that the marked decrease in bFGF mitogenic activity resulted from posttranslational modification of bFGF induced by elevated glucose concentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. These data are consistent with the hypothesis that nonenzymatic glycosylation of intracellular protein alters vascular cell function.     

Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.     

Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.     

Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis promastigotes

Pentamidine-resistant clones of Leishmania donovani and L. amazonensis promastigotes were developed by increase of the drug pressure in the culture medium and characterized. The resistant clones could grow in 40 and 20 microM pentamidine as determined for L. donovani and L. amazonensis, respectively, with 50% inhibitory concentrations (IC50 values) being 140 and 60 microM, which were 18 and 75 times higher than those recorded for the parental clones, respectively. Biochemical analysis of the clones showed that the acquired pentamidine resistance was specific (no cross-resistance to unrelated drugs and no reversibility with verapamil) and stable in vitro and in vivo. Pentamidine resistance is related to decreased drug uptake and highly increased efflux in both clones of Leishmania spp., accompanied by an alteration in polyamine carriers. Furthermore, a modification of the uptake of pyrimidine nucleosides and several amino acids by these resistant clones indicates alterations in the surface membrane.     

Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines

The expression of the tumor suppressor/oncoprotein p53 has been investigated in normal human placental villous trophoblast, in vitro propagated invasive extravillous trophoblast, SV40 tumor antigen (Tag)-immortalized extravillous trophoblast, human cytomegalovirus (hCMV)-infected syncytiotrophoblast and malignant trophoblast (choriocarcinoma) cell lines (JAR, JEG-3 and BeWo) using quantitative enzyme-linked immunosorbent assay (ELISA) and Western immunoblot methods using monoclonal antibodies specific for wild-type and mutant p53. The normal villous and extravillous trophoblast cells expressed low levels of the wild-type p53 protein, whereas normal terminally differentiated multinucleated syncytiotrophoblast cells, as well as hCMV-infected syncytiotrophoblast, showed a higher expression of the wild-type p53 protein. SV40 Tag-immortalized invasive trophoblast cells also showed a high expression of the wild-type p53 protein which remained complexed with the Tag protein. All the choriocarcinoma cell lines over expressed the mutant form of the p53 protein. The increased expression of p53 protein in the SV40 Tag-immortalized invasive trophoblast and choriocarcinoma cells paralleled with increased expression of the mouse double minute 2 (mdm2) oncogenic protein. Transforming growth factor (TGF)-beta inhibited proliferation of normal extravillous trophoblast cells. The antiproliferative effects of TGF-beta were reduced in SV40 Tag-immortalized cells and non-detectable in choriocarcinoma cell lines JAR, BeWo and JEG-3. The inactivation of p53 owing to complexing with Tag in the immortalized premalignant trophoblast and p53 mutation in the malignant trophoblast may be responsible for their aberrant proliferation and refractoriness to antiproliferative effects of TGF-beta observed in these cells as compared to the normal trophoblast. These results may suggest the role of p53 protein in trophoblast differentiation, transformation and tumorigenesis.     

Altered intracellular processing and enhanced secretion of procathepsin D in a highly deviated rat hepatoma

Using immunomagnetic cell separation and fluorescent in situ hybridization (FISH), we studied nine patients who had chronic granulocytic leukemia (CGL) for the proportion of interphase nuclei with Mbcr/abl fusion in a direct preparation of the bone marrow and also in the mononuclear cell (MNC), neutrophil, and B- and T-cell fractions of the peripheral blood. In five untreated patients, conventional cytogenetics revealed 97% to 100% Philadelphia chromosome (Ph)+ metaphases. In three of these five patients, FISH studies on bone marrow direct preparations and peripheral blood MNCs indicated that an Mbcr/abl fusion occurred in 62% to 69% of the cells. We observed 69% to 88% of nuclei with Mbcr/abl fusion within the neutrophil fractions. In contrast, the values were 12% to 39% within the T-cell fractions in the four patients we studied. B-cell fractions were studied in three patients, and only one had an abnormal value (58%). In the four patients receiving alpha-interferon therapy, the degree of conventional cytogenetic remission correlated best with the degree of FISH remission observed in the peripheral blood neutrophil fraction. Our results are in agreement with earlier studies in that both B and T lymphocytes may be involved with the clonal process in CGL. The FISH-based detection of Mbcr/abl fusion in the peripheral blood neutrophil compartment provided the best estimate for the proportion of Ph metaphases determined by conventional cytogenetics.     

Acute stretching of peripheral nerves inhibits retrograde axonal transport

The conjugation of horseradish peroxidase with wheat germ agglutinin was used to identify the effect on retrograde axonal transport of stretching the rat sciatic nerve indirectly by 10% and 20% femoral lengthening with a unilateral external fixator. To investigate the relationship between retrograde axonal transport and blood flow in the stretched nerve, nerve blood flow in the sciatic nerve was measured by a hydrogen washout technique. At 11% strain (20% femoral lengthening), the numbers of horseradish peroxidase-labelled motor neuron cells and nerve blood flow had decreased by 43% and 50%, respectively. Histological examination demonstrated ischaemic changes, but not mechanical damage. However, at 6% strain (10% femoral lengthening) there were no significant abnormalities. These findings suggest that the inhibition of retrograde axonal transport can be induced by acute stretching of the peripheral nerve and that circulatory disturbance is the main cause of the inhibition of retrograde axonal transport at the low strain.     

Synthesis and intracellular transport of aminoglycerophospholipids in permeabilized cells of the yeast, Saccharomyces cerevisiae

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.