PubMed Health⌕ Search

Biomedical subjects

H J Geuze

Publications and source records attributed to H J Geuze.

At least 109 records · Page 6Linked to original sources
PubMed1991

Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells.

The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogold technique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.

Adult↗
PubMed1991

Trans-Golgi reticulum.

The trans-Golgi apparatus reticulum is that portion of the Golgi apparatus located in the trans-most aspect of the stack exhibiting certain characteristic morphological and functional characteristics. The membranes of the trans-Golgi reticulum are reticular in form, thickened with plasma membrane-like characteristics and with a considerable portion of their surface covered by clathrin coats. The enzymes thiamine pyrophosphatase and sialyl- and galactosyl transferases are functional markers. Correlative studies show the trans-Golgi apparatus reticulum to be involved in glycoprotein, enzyme and receptor processing and sorting along multiple pathways. Sorting and transfer of constituents to lysosomes, to secretory granules, or to the plasma membrane emerge as dominant functions.

Animals↗
PubMed1991

Brefeldin A induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum.

The fungal drug brefeldin A (BFA) has recently been found to induce a redistribution of medial- and cis-Golgi components to the endoplasmic reticulum (ER), raising the possibility of the existence of a retrograde pathway from the Golgi complex to the ER. Here, we demonstrate a BFA-induced reversible rearrangement of the trans-Golgi membrane protein galactosyltransferase (Gal-T) to the ER in HeLa cells. With immunofluorescence microscopy we have shown that BFA first caused a rapid change of Gal-T immunolabelling from a normal Golgi complex pattern to long and slender structures emanating from the cell centre and co-localizing with tubulin. Then immunofluorescence became ER-like. This effect was not dependent on ongoing protein synthesis and was reversed to normal within 120 min after removal of the drug. Restoration of the Golgi complex after removal of brefeldin A was energy-dependent but not mediated by microtubules nor dependent on protein synthesis. BFA-induced backflow of Gal-T was inhibited by nocodazole, a microtubule-disrupting agent. Immunoelectron microscopy showed that BFA treatment resulted in the fusion of Gal-T-containing vesicles with the ER. Furthermore, sucrose gradient centrifugation showed a significant shift in density of mature Gal-T polypeptides upon BFA treatment: about 40% of the enzyme migrated from its original density (1.13 g/ml) to the density of rough ER (1.19 g/ml). Thus, BFA caused microtubule-dependent vesicular backflow from a trans-Golgi component to the ER followed by fusion of the Golgi-derived vesicles with the ER.

Antifungal Agents↗
PubMed1991

Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.

Adipose Tissue, Brown↗
PubMed1991

Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution.

Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.

Animals↗
PubMed1991

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice.

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.

Animals↗
PubMed1990

Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH.

Cytolytic T-lymphocytes (CTL) harbor cytoplasmic granules containing the lytic, pore-forming protein perforin, a family of serine proteases designated granzymes, and proteoglycans as major constituents. Growth of CTL lines in the presence of PNP-xyloside completely inhibited the glycosylation of the granule-associated chondroitin sulfate A type proteoglycans. Only short glycosaminoglycan molecules were detected. The absence of intact proteoglycans neither altered the sorting of the granule-associated proteins perforin or granzyme A nor influenced their secretion into the extracellular milieu upon T-cell receptor complex stimulation. With a weak base, the pH of the granules was determined to be acidic. At pH 5.2, granzyme A and perforin formed complexes with chondroitin sulfate A. At neutral pH, perforin and only a minor fraction of granzyme A dissociated from the proteoglycan. Upon secretion of the granule contents induced by immobilized anti-CD3 antibodies, most granzyme A molecules remained complexed with the chondroitin sulfate A glycosaminoglycans, even if synthesis of intact proteoglycans was inhibited. We suggest that granule-associated molecules complex with proteoglycans under the acidic conditions prevailing in the trans Golgi and cytolytic granules. A possible pH shift occurring during exocytosis would cause perforin, but only a minor fraction of granzyme A, to dissociate from the proteoglycans.

Animals↗
PubMed1990

No evidence for expression of the insulin-regulatable glucose transporter in endothelial cells.

A major effect of insulin is to increase glucose transport in muscle and fat. A family of genes encoding distinct mammalian glucose transporters has recently been elucidated. One of these, the insulin-regulatable glucose transporter (IRGT), is primarily expressed in muscle and fat, tissues that exhibit insulin-dependent glucose transport. Insulin promotes glucose transport in these tissues by stimulating movement of the glucose transporter from an intracellular location to the plasma membrane. Recent studies, however, suggest that an additional effect of insulin in these tissues may be the facilitation of glucose transport, presumably across capillary endothelium. This hypothesis is based on the localization of the IRGT in endothelial cells specific to muscle and adipose tissue. We report here, however, on morphological and biochemical studies using several different IRGT-specific antibodies in which we could not reproduce these results.

Adipose Tissue, Brown↗
PubMed1990

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route.

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle. In contrast, Tfr was found to internalize and recycle. The biosynthetic pathway of class II molecules differ from that of class I molecules in that it shows a delay (1-3 hr) in transport from trans-Golgi to cell surface: here it intersects the endocytic route. Immunoelectron microscopy using anti-MHC antibodies revealed the existence of vesicular structures that were intensely labeled for class II molecules. It is proposed that at this site combination of class II molecules with processed antigen could occur.

B-Lymphocytes↗
PubMed1990

A new model for lethal hit delivery by cytotoxic T lymphocytes.

Human cytotoxic T lymphocyte (CTL) granules contain an electron-dense core and small membrane vesicles. By immuno-electron microscopy, molecules relevant to CTL-target cell (TC) interactions have been identified on the membranes of the dense core and small vesicles within the granule. Moreover, perforin, the component implicated in the lethal hit, and serine esterases are localized within these granule substructures. In this article Peter Peters and colleagues argue that these observations necessitate a revision of the current model for lethal hit delivery. They suggest that the cytotoxic mediators exocytosed into the cleft between CTL and TC are not in soluble form, but rather are membrane-enveloped. The presence of the CD3-T-cell receptor (TCR) complex, CD8 and possibly other relevant molecules on these membranes may ensure unidirectional delivery of the lethal compounds to the TC.

Cytoplasmic Granules↗
PubMed1990

Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing.

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.

Animals↗
PubMed1989

Molecules relevant for T cell-target cell interaction are present in cytolytic granules of human T lymphocytes.

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

Clone Cells↗
PubMed1989

Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.

Asialoglycoprotein Receptor↗
PubMed1989

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells.

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.

Animals↗
PubMed1988

Immunoelectron microscopic localization of ubiquitin in hepatoma cells.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.

Antibody Specificity↗
PubMed1988

Recycling of 5'-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.

5'-Nucleotidase↗
PubMed1988

Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-L1 adipocytes.

A semiquantitative method using immunocytochemistry on ultrathin cryosections and the protein A-gold technique was performed to study the effect of insulin on the cellular distribution of the glucose transporters in cultured 3T3-L1 adipocytes. In basal cells a substantial portion of the label was present in a tubulovesicular structure at the trans side of the Golgi apparatus, likely to represent the trans-Golgi reticulum, and in small vesicles present in the cytoplasm. Treatment with insulin induced a rapid translocation of transporters from the tubulovesicular structure to the plasma membrane. The transporter labeling of the plasma membrane increased three-fold and that of the tubulovesicular structure decreased by half. There was no effect of insulin on the degree of label in the small cytoplasmic vesicles. Removal of insulin from stimulated cells rapidly reversed the distribution of transporters to that seen in basal cells. This study thus provides the first morphological evidence for the occurrence of transporter translocation in insulin action and identifies for the first time the intracellular location of the responsive transporters.

Adipose Tissue↗