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P A Gleeson

Publications and source records attributed to P A Gleeson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule. A new model for parietal cell transformation.

The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic 'tubulovesicles' as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.

Animals↗
PubMed1995

A novel method for isolating mononuclear cells from the stomachs of mice with experimental autoimmune gastritis.

Autoimmune gastritis induced in BALB/c mice by neonatal thymectomy is a CD4+ T cell-mediated disease. The disease is characterised by mononuclear cell infiltrates in the gastric mucosa, loss of gastric parietal and chief cells and autoantibodies to the gastric H/K ATPase. Here we describe a simple non-enzymatic method for isolating cellular infiltrates from stomachs of gastric mice by injection of medium directly into stomach walls, causing swelling and rupture. Using this method, large numbers of viable lymphocytes were released from stomachs for analysis by flow cytometry. An 8.3 fold increase in the total number of lymphocytes from diseased stomachs compared to normal controls was observed. Total cell numbers of CD4+ and B cells were increased 4.8 fold and 39.5 fold respectively, in diseased stomachs compared with controls. No change was observed in the CD8+ T cell population. This method will allow detailed quantitative analysis of cellular infiltrates during the development of the gastric lesion and enrichment of pathogenic T cells for analysis and cloning. This procedure may have general application for the isolation of cellular infiltrates from lesion sites of other organs.

Animals↗
PubMed1994

Medial-Golgi retention of N-acetylglucosaminyltransferase I. Contribution from all domains of the enzyme.

We have previously shown that the transmembrane domain and flanking residues of beta-1,2-N-acetylglucosaminyltransferase I (GnTI) can localize a hybrid molecule to medial-Golgi cisternae (Burke, J., Pettitt, J. M., Schachter, H., Sarkar, M., and Gleeson, P.A. (1992) J. Biol. Chem. 267, 24433-24440). Here, we have further examined the contribution of the cytoplasmic tail, transmembrane domain, and the catalytically active, luminal domain of GnTI in medial-Golgi localization, by analyzing the localization of hybrid molecules stably expressed in murine cells. In contrast to wild-type GnTI, which was efficiently localized to the medial-Golgi and not detected at the cell surface, hybrid molecules containing any two of the three domains of GnTI were localized to the medial-Golgi and were also present at low levels at the cell surface. Hybrid molecules containing only the transmembrane domain or the luminal domain of GnTI showed partial Golgi retention together with an increased level of cell surface expression compared with molecules containing two GnTI domains. The cytoplasmic tail independently was unable to retain reporter sequences to the Golgi but increased the ability of constructs containing either the luminal or transmembrane domain of GnTI to localize to the Golgi apparatus. Therefore, all three domains of GnTI contribute significantly to medial-Golgi localization. Furthermore, GnTI hybrid molecules showing increased cell surface expression were more readily extracted in a low salt buffer, suggesting that Golgi localized GnTI differs in physicochemical properties from cell surface GnTI. Based on an aggregation model of localization, we propose that Golgi retention of these hybrid molecules is mediated by the interaction of their GnTI domains with the corresponding domains of endogenous glycosyltransferase aggregates within the Golgi membranes of the transfected cell.

Amino Acid Sequence↗
PubMed1994

Targeting of proteins to the Golgi apparatus.

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.

Biological Transport↗
PubMed1994

Heterogeneity of human anti-Golgi auto-antibodies: reactivity with components from 35 to 260 kDa.

The Golgi auto-antigens recognized by five human autoimmune sera were characterized with anti-Golgi auto-antibodies. The five sera showed strong anti-Golgi reactivity, together with weak anti-nuclear reactivity, as assessed by indirect immunofluorescence using the human HEp2 epithelial cell line. The Golgi auto-antigens recognized by the autoimmune sera were identified by immunoprecipitation and immunoblotting of post-nuclear supernatants. These sera reacted with at least 14 components of molecular mass (M(r)) 35-260 kDa; three components were detected only by immunoprecipitation, six components detected only by immunoblotting and five components appear to be detected by both techniques. We have previously shown that a patient with Sjögren's syndrome has auto-antibodies specific for a 230 kDa Golgi auto-antigens; a bacterial fusion protein containing auto-epitope(s) of this Golgi auto-antigen is not recognized by the other four autoimmune sera. Taken together, this study shows that the five anti-Golgi autoimmune sera recognize distinct sets of auto-antigens which include both conformational and/or sequential epitopes.

Autoantibodies↗
PubMed1994

Post-translational modifications distinguish cell surface from Golgi-retained beta 1,4 galactosyltransferase molecules. Golgi localization involves active retention.

beta 1,4 Galactosyltransferase (GalT) is a membrane-bound enzyme localized predominantly to the trans-Golgi cisternae. Our previous studies have shown that the transmembrane domain of bovine GalT plays a critical role in Golgi localization (Teasdale, R.D., D'Agostaro, G. and Gleeson, P.A., J. Biol. Chem., 267, 4084-4096, 1992). Here we have compared the localization and post-translational modifications of full-length bovine GalT with a GalT/hybrid molecule where the transmembrane domain of GalT was replaced with that of the transferrin receptor. GalT/hybrid molecules were expressed on the surface of transfected cells; however, differences were observed in the distribution of the hybrid molecules between transfected COS and murine L cells. In transfected COS cells, the GalT/hybrid protein was expressed efficiently at the cell surface, with little Golgi-localized material, whereas in stable murine L cells, which expressed lower levels of the construct, hybrid molecules were detected both at the cell surface and within the Golgi apparatus. Expression of the GalT constructs in either COS or L cells produced two glycoprotein products which differed in molecular mass by 7 kDa. The difference in size between the two products is due to post-translational modifications which are inhibited by brefeldin A and are therefore likely to occur in the trans-Golgi network (TGN). Very little of the high-molecular-weight species was detected for full-length GalT, whereas it was a major product for the GalT/hybrid protein. Only the higher molecular weight species was expressed at the cell surface. Thus, this additional 7 kDa post-translational modification distinguishes molecules retained within the Golgi apparatus (lower M(r) species) from those transported through the TGN to the cell surface. These studies indicate that (i) the level of expression influences the intracellular distribution of GalT/hybrid molecules and (ii) the localization of full-length GalT involves active retention within the Golgi stack, and not retrieval from later compartments. After treatment of membrane preparations from stable L cell clones with a heterobifunctional cross-linking agent, full-length bovine GalT molecules were found almost exclusively as high-molecular-weight aggregates, suggesting that GalT exists as an oligomer or aggregate. This ability to oligomerize may be a requirement for Golgi retention.

Animals↗
PubMed1993

An autoimmune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus.

Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.

Animals↗
PubMed1993

Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.

This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

Amino Acid Sequence↗
PubMed1993

Gastric parietal cell development: expression of the H+/K+ ATPase subunits coincides with the biogenesis of the secretory membranes.

The early development of the parietal cell in the embryonic murine gastric mucosa was investigated with particular attention paid to the biogenesis of the secretory membranes and the localization of the gastric H+/K+ ATPase alpha and beta subunits. Gastric glands were recognized in the day 18 foetus. However, at this stage in development no parietal cells could be distinguished ultrastructurally in the glands, and no immunoreactivity was detected with monoclonal antibodies to either the alpha or beta subunits of the gastric H+/K+ ATPase. In the 19 day embryo, parietal cells were recognizable morphologically by the presence of slender microvilli on the apical (lumenal) surface and differentiating intracellular canaliculi in the apical cytoplasm. Both subunits of the proton pump were found to be specifically associated with the apical and canalicular membranes and with the membranes of relatively large vesicles distributed in the subapical cytoplasm and the cytoplasm surrounding the canaliculi. In the parietal cells of the day 1 neonate, the intracellular canaliculi had extended basally to form the extensive compartments typical of parietal cells in the adult animal. Again, profiles of vesicles showing H+/K+ ATPase immunoreactivity were present in the pericanalicular cytoplasm. These results indicate that the intracellular canaliculi are formed by expansion of the apical surface and suggest that the delivery of newly synthesized gastric H+/K+ ATPase alpha and beta subunits to the apical plasma membrane is mediated by typical Golgi transport vesicles. The large immunoreactive vesicles that occur in the apical and pericanalicular cytoplasm of the developing cells may represent artefacts generated by fixation-induced fragmentation of the differentiating canalicular membrane system during preparation.

Animals↗
PubMed1993

Alpha and beta subunits of the gastric H+/K(+)-ATPase are concordantly targeted by parietal cell autoantibodies associated with autoimmune gastritis.

We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen, subsequently identified as the beta subunit of the gastric H+/K(+)-ATPase (EC 3.6.1.3) (proton pump) whereas Karlsson et al showed that these autoantibodies primarily target the 95 kDa alpha subunit of the pump. In view of these discordant results, we have reassessed the reactivity of parietal cell autoantibodies with the two subunits of the gastric H+/K(+)-ATPase. We show here that all 26 parietal cell autoantibody-positive sera immunoblot both subunits under appropriate, but mutually exclusive, conditions. Thus, reactivity of anti-parietal cell autoantibodies with the 95 kDa alpha subunit is optimal when the SDS-PAGE is carried out with samples which are reduced but not boiled. Whereas reactivity with the 60-90 kDa beta subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the beta subunit is critically dependent on the presence of a full complement of N-linked glycans since partially deglycosylated protein, and recombinant beta subunit expressed in COS cells, bearing high mannose N-glycans, failed to bind to the autoantibody. These studies also suggest that B cell auto-epitopes are located on the lumenal domain of the beta subunit. Reactivity of parietal cell autoantibodies with a bacterial fusion protein incorporating the catalytic cytoplasmic domain of the alpha subunit suggests the presence of auto-epitopes in this region of the molecule.

Animals↗
PubMed1992

The transmembrane and flanking sequences of beta 1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization.

UDP-GlcNAc:alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnTI) is an N(in)/C(out) (type II) membrane protein, localized in the medial-Golgi, that initiates the conversion of high mannose N-glycans to complex N-glycans. Anti-rabbit GnTI antibodies were generated using a purified, enzymatically active, bacterial recombinant fusion protein as immunogen. Rabbit GnTI was effectively retained in the Golgi complex of transfected COS-1 cells and murine L cells, as assessed by indirect immunofluorescence using the species-specific anti-GnTI antibodies; no surface expression of rabbit GnTI could be detected in the transfected cells. Rabbit GnTI, stably expressed in murine L cells, was localized by immunoperoxidase electron microscopy to the medial-cisternae of the Golgi stack. The role of the transmembrane domain of GnTI in Golgi localization was examined by generation of a hybrid construct containing the amino-terminal 31 amino acids of GnTI, corresponding to the 25-residue transmembrane (signal/anchor) domain and flanking hydrophilic sequences, fused with ovalbumin; this ovalbumin/GnTI hybrid molecule was retained in the Golgi complex of transfected COS cells and stably transfected murine L cells. No surface expression of ovalbumin/GnTI was detected. In contrast, ovalbumin fused to the equivalent domains of the human transferrin receptor, a type II cell-surface protein, was efficiently expressed on the cell surface of transfected cells. The ovalbumin/GnTI hybrid molecules in the transfected L cells were N-glycosylated, indicating an N(in)/C(out) membrane orientation, and were localized by immunoperoxidase electron microscopy to one or two cisternae of the medial-Golgi (90% of stained Golgi profiles showed medial-cisternae staining). These results show that a signal contained within the transmembrane domain and flanking residues of GnTI specifies medial-Golgi localization.

Amino Acid Sequence↗
PubMed1992

Human autoantibodies as reagents to conserved Golgi components. Characterization of a peripheral, 230-kDa compartment-specific Golgi protein.

We have used a serum from a patient with Sjögren's syndrome containing high titer (100,000) anti-Golgi autoantibodies and lower titer (20,000) anti-nuclear autoantibodies to characterize the Golgi complex. The Sjögren's syndrome serum immunoprecipitated a number of components of molecular mass 35-230 kDa from detergent extracts of [35S]methionine-labeled HeLa cells; at high dilution, the serum precipitated one major 230-kDa component. Using the Sjögren's syndrome serum, cDNA clones encoding the Golgi autoantigen were isolated from a lambda gt11 HeLa cell cDNA library. Autoantibodies from the Sjögren's syndrome serum, affinity purified from a recombinant bacterial fusion protein generated from one of the cDNA clones, showed Golgi staining of human, mouse, and chicken cells by immunofluorescence. The purified autoantibodies immunoprecipitated and immunoblotted a 230-kDa component. A rabbit antiserum raised to the recombinant fusion protein specifically stained the Golgi complex by immunofluorescence and reacted with a 230-kDa protein by immunoprecipitation and immunoblotting. The 230-kDa protein was recovered in both the 100,000 x g sedimentable and soluble fractions in cell lysates and in the aqueous phase of Triton X-114 extracts. The 230-kDa autoantigen was dissociated from the Golgi complex by 15-min brefeldin A treatment, dissociation kinetics similar to that of mannosidase II. However, unlike mannosidase II, autoantigen staining was markedly reduced after drug treatment. Removal of brefeldin A resulted in reassociation of the autoantigen with the Golgi complex. The epitopes recognized by the affinity purified human and rabbit antibodies were ultrastructurally localized to the cytosolic face of one side of the Golgi stack, probably the trans-face. Taken together, the 230-kDa protein is a conserved, peripheral membrane component specifically associated with one Golgi compartment. We suggest that this peripheral Golgi protein may have a role in the compartment-specific structural organization of the Golgi or in sorting and transport of proteins.

3T3 Cells↗
PubMed1992

Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized gastric H+/K(+)-ATPase complex.

Adenosine Triphosphatases↗
PubMed1992

The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain.

The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.

Amino Acid Sequence↗
PubMed1992

The mouse gastric H,K-ATPase beta subunit. Gene structure and co-ordinate expression with the alpha subunit during ontogeny.

The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.

Adenosine Triphosphatases↗
PubMed1992

Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.

Animals↗